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The development of a solid phase screening assay for the detection of dipeptide synthesis Yue, Eliza 1995

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THE DEVELOPMENT OF A SOLID PHASE SCREENING ASSAY FOR THE DETECTION OF DIPEPTIDE SYNTHESIS by ELIZA YUE .S. (Agr.)/ The University of B r i t i s h Columbia, 1989 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Food Science) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1995 ® E l i z a Yue, 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of J? t^cB The University of British Columbia Vancouver, Canada Date DE-6 (2788) A B S T R A C T A rapid screening assay was developed to detect formation of DNP-asn-leu dipeptides on a special type of m i c r o t i t e r plate, c a l l e d the Nunc Immuno Module. Secondary amino groups, to which small molecules can be coupled, were grafted onto the surface of the plate. The carboxyl group on L-leucine molecules, activated i n the presence of 0.1 M N-hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, were covalently coupled to these amino groups; 11.04 fxq of L-Leucine was used i n the coupling reaction. The reaction mixture, consisting of eithe r commercial grade thermolysin or crude protease mixture i s o l a t e d from Bacillus subtilis culture, DNP(dinitrophenyl)-L-asparagine and pH 6 sodium acetate buffer solution, was subseguently added to the wells. During incubation at 48°C, dipeptide formation occurred between the immobilized leucine and the free DNP-L-asparagine i n the l i q u i d phase. After washing off unreacted DNP-L-asparagine, the f i n a l product was detected by the addition of an antibody-peroxidase conjugate, which reacted s p e c i f i c a l l y with the dinitrophenyl group attached to the amino group of the asparagine molecule. The amount of increase i n o p t i c a l density measured at 492 nm was proportional to the r e l a t i v e amount of dipeptides formed on the mi c r o t i t e r plate. Two-way analysis of variance showed that the e f f e c t of enzymatic treatment on the mean o p t i c a l density measured at 492 nm was s i g n i f i c a n t (P<0.01). i i T A B L E OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x INTRODUCTION J 1 LITERATURE REVIEW 5 I. Enzymatic peptide synthesis 5 a. Advantages and disadvantages 5 b. Examples of useful peptides synthesized by protease-catalysed reactions 6 c. Methods for detecting peptide formation ... 7 II . Proteases 9 a. Thermolysin 9 b. Neutral protease T 12 I I I . Immunoassay techniques 13 a. Radioimmunoassay 13 b. Advantages of enzyme linked immunosorbent assay 15 c. P r i n c i p l e of d i r e c t and i n d i r e c t ELISA .... 15 d. Choice of enzyme and substrate 16 e. M i c r o t i t e r plates 19 IV. Bacillus subtilis 21 a. Use of Bacillus subtilis as a host for production of heterologous proteins 21 b. Problems associated with using B. subtilis expression systems 2 3 MATERIALS AND METHODS 25 MATERIALS 25 METHODS 2 6 I. Thermolysin catalysed solid-phase synthesis of DNP-asn-leu dipeptide 2 6 a. Immobilization of DNP(Dinitrophenyl)-L-leucine or L-leucine to mi c r o t i t e r plates 2 6 b. Detection of immobilized amino aci d or dipeptide 29 c. Solid-phase enzymatic DNP-asn-leu dipeptide synthesis 3 0 i i i METHODS II . Enzymatic catalysed solid-phase synthesis of DNP-asn-leu dipeptide using crude enzyme mixture from Bacillus subtilis transformants 32 a . l . Transformation of B. subtilis DB428 and WB600 32 a.2. Confirmation of po s i t i v e transformants by r e s t r i c t i o n enzyme digestion of plasmid DNA 3 3 a. 3. Preparation of crude proteases from Bacillus subtilis transformants 34 b. Enzymatic solid-phase synthesis of DNP-Asn-Leu dipeptides using e i t h e r concentrated or unconcentrated crude enzyme f r a c t i o n from Bacillus subtilis (pNP22) transformants as catalysts 38 II I . Chemical assays 38 a. Protease assay using casein as a substrate . (38 b. Protease assay using azocasein as a substrate 39 c. Protein assay using BCA protein assay reagent 41 d. SDS-gel electrophoresis of thermolysin and secreted crude proteases from transformed B. subtilis DB428 and WB600 42 IV. Methods for s t a t i s t i c a l analysis 44 RESULTS AND DISCUSSIONS 45 I. Immobilization of DNP-L-leucine to the s o l i d phase 45 a. Detection of immobilized leucine molecule .. 45 b. Binding of DNP-L-leucine to conventional microtiter plate 48 c. Non-covalent binding of DNP-L-leucine 48 d. Quantification of the amount of DNP-L-leucine immobilized to the microtiter plate 49 II . Thermolysin catalysed s o l i d phase synthesis of DNP-Asn-Leu 50 a. Selection of blocking agent 50 b. Detection of the formation of DNP-Asn-Leu .. 51 c. E f f e c t of the concentration of DNP-L-asn on dipeptide formation 62 d. The e f f e c t of reaction time on dipeptide synthesis 63 e. The ef f e c t of thermolysin concentration on dipeptide synthesis 64 f. S t a t i s t i c a l Analysis 64 iv RESULTS AND DISCUSSIONS I I I . DNP-Asn-Leu reactions catalysed by crude enzyme f r a c t i o n secreted by b a c t e r i a l culture 65 a. Use of Bacillus subtilis DB 428 and WB600 to produce enzyme mixture 67 b. E f f e c t of varying the concentration of enzyme i n the reaction mixture 70 c. E f f e c t of varying the concentration of DNP-L-Asn 83 d. Comparison of peptide synthesis r a t i o f o r thermolysin and crude enzyme preparation ... 84 e. S t a t i s t i c a l analysis 85 CONCLUSIONS 91 REFERENCES 93 V LIST OF T A B L E S Table 1. The optimal concentration of DNP-L-leucine f o r coupling on s o l i d phase 46 Table 2. The e f f i c i e n c y of various blocking agents 52 Table 3. Reaction conditions for thermolysin-catalysed s o l i d -phase synthesis of DNP-Asn-Leu 53 Table 4. 2-Way analysis of variance on o p t i c a l density readings for f i v e thermolysin catalysed Leu-Asn synthesis experiments 66 Table 5. E x t r a c e l l u l a r protease a c t i v i t y from wild-type (168) and protease-deficient (DB428 and WB600) s t r a i n s of Bacillus subtilis 68 Table 6. Results of u l t r a f i l t r a t i o n of crude enzyme secreted by transformed Bacillus subtilis DB428 71 Table 7. Reaction conditions for thermolysin and crude enzyme catalysed s o l i d phase synthesis of DNP-Asn-Leu 72 Table 8. Summary of the protease a c t i v i t y of thermolysin determined by u t i l i z i n g two d i f f e r e n t substrates: Hammerstein casein and azocasein 73 Table 9. 2-Way analysis of variance on O.D.492nm i n micro-t i t e r wells from f i v e experiments of thermolysin or neutral protease T catalysed synthesis of Asn-Leu dipeptides 8 6 v i LIST OF FIGURES Figure 1. 3-Dimensional structure of thermolysin 10 Figure 2. Schematic representation of the active s i t e of thermolysin with a polypeptide substrate bound ... 10 Figure 3. Schematic chemical and physical configuration of the CovaLink NH surface 27 Figure 4. Covalent coupling of amino acid to Nunc Immuno Module 28 Figure 5a. R e s t r i c t i o n map of pNP22 3 5 Figure 5b. Analysis of pNP22 recombinant plasmid with r e s t r i c t i o n endonucleases 36 Figure 6. Standard curve for protease assay with Hammerstein casein used as a substrate 40 Figure 7. Standard curve for BCA protein assay . 43 Figure 8. Immobilization of DNP-L-leucine to Nunc Immuno Module 47 Figure 9. Exp. 1. Thermolysin catalysed synthesis of DNP-Asn-Leu . 54 Figure 10. Exp. 2. Thermolysin catalysed synthesis of DNP-Asn-Leu 55 Figure 11a. Exp. 3a. Thermolysin catalysed synthesis of DNP-Asn-Leu 56 Figure l i b . Exp. 3b. Thermolysin catalysed synthesis of DNP-Asn-Leu 57 Figure 11c. Exp. 3c. Thermolysin catalysed synthesis of DNP-Asn-Leu 58 Figure l i d . Exp. 3d. Thermolysin catalysed synthesis of DNP-Asn-Leu 59 Figure 12. Exp. 4. Thermolysin catalysed synthesis of DNP-Asn-Leu 60 Figure 13. Exp. 5. Thermolysin catalysed synthesis of DNP-Asn-Leu 61 v i i Figure 14. SDS-gel electrophoresis of thermolysin and crude proteases secreted by transformed B. subtilis DB428 and WB600 69 Figure 15. Exp. 6. Thermolysin and crude enzyme catalysed synthesis of DNP-Asn-Leu 74 Figure 16a. Exp. 7a. Crude enzyme catalysed synthesis of DNP-Asn-Leu 75 Figure 16b. Exp. 7b. Crude enzyme catalysed synthesis of DNP-Asn-Leu 76 Figure 16c. Exp. 7c. Crude enzyme catalysed synthesis of DNP-Asn-Leu 77 Figure 16d. Exp. 7d. Crude enzyme catalysed synthesis of DNP-Asn-Leu . . : 78 Figure 17. Exp. 8. Crude enzyme catalysed synthesis of DNP-Asn-Leu 79 Figure 18. Exp. 9. Thermolysin and crude enzyme catalysed synthesis of DNP-Asn-Leu 80 Figure 19. Exp. 10. Thermolysin and crude enzyme catalysed \ synthesis of DNP-Asn-Leu 81 Figure 20. Calculation of the maximum concentration of leucine immobilized on Nunc Immuno Module 88 v i i i ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. S. Nakai, who supervised t h i s study and provided advice throughout the course of t h i s t h e s i s . Appreciation i s also extended to the members of the research committee Dr. B. D i l l , Dr. B. Skura and Dr. R. MacGillivray for t h e i r guidance and constructive advice. Special thanks to Dr. E. Li-Chan and Dr. A. Matsuyama f o r t h e i r patience and for providing me with a great deal of tec h n i c a l assistance on my project. I am also p a r t i c u l a r l y grateful to my fri e n d Grace Lee, who reviewed my th e s i s f o r me, and to Shirley Joe, who helped me to type part of my written report i n spite of her very t i g h t "work" schedule. Lastly, I wish to thank my grandmother for her invaluable encouragement and unconditional support. ix I N T R O D U C T I O N Various studies have shown that enzymes can be used to catalyse peptide bond formation by reverse p r o t e o l y s i s . The enzymatic method may be preferred over conventional chemical synthesis because the reactions can be performed s t e r e o s p e c i f i c a l l y and under very mild conditions. In addition, the functional groups of amino acid side chains do not require protection since proteases show s p e c i f i c i t y for the a-carboxyl or a-amino group at the reaction s i t e (Sakina et a l . , 1988). The enzymatic method i s e s p e c i a l l y suitable for the preparation of peptides useful i n the food industry, such as aspartame, because there i s less need for hazardous chemicals. Modification of the physical and chemical properties of various enzymes i s possible by the use of genetic engineering techniques. As an example, a tremendous amount of protein engineering research has been done on s u b t i l i s i n , which i s one of the most thoroughly characterized b a c t e r i a l serine proteases secreted by a variety of Bacillus species. Some of the physical and chemical c h a r a c t e r i s t i c s of s u b t i l i s i n , including i t s thermostability, a l k a l i n e s t a b i l i t y , as well as i t s a b i l i t y to r e s i s t chemical oxidation, have been modified by using either s i t e -d irected mutagenesis or random mutagenesis techniques to introduce changes i n the nucleotide sequence encoding the enzyme ( E s t e l l et a l . , 1985; Cunningham and Wells., 1987; Takagi et a l . , 1990; Takagi, 1993; Egmond et a l . , 1994; Heringa, 1995). Hence, i t may also be possible to improve the reverse proteolysis property of an 1 INTRODUCTION / 2 enzyme by using s i m i l a r genetic engineering methods. Thermolysin, a thermostable p r o t e o l y t i c enzyme secreted by Bacillus thermoproteolyticus, has been int e n s i v e l y characterized: the primary and t e r t i a r y structures, the active s i t e and substrate-binding s i t e have been determined (Bigbee & Dahlquist, 1974; Matthews et a l . , 1972; T i t a n i et a l . , 1972). Since many papers have been published on the use of thermolysin i n promoting peptide bond formation, i t may be worthwhile to attempt to further improve the peptide bond synthesis property of t h i s enzyme by applying s i t e - d i r e c t e d or random mutagenesis techniques. One of the o r i g i n a l objectives of t h i s thesis was to randomly mutagenize targeted regions of the nprT gene, from Bacillus stearothermophilus, which encodes a thermostable neutral protease T. Since the amino acid sequence of t h i s protease i s 85% homologous to that of thermolysin, the two proteases may have s i m i l a r 3-dimensional structure as well as other physical c h a r a c t e r i s t i c s (Takagi et a l . , 1985). However, i n order to modify the reverse proteolysis property of neutral protease T, i t i s imperative to f i r s t develop a rapid and e f f i c i e n t screening system that i s capable of i d e n t i f y i n g p o s i t i v e mutants, which produce enzymes with enhanced peptide bond synthesis a c t i v i t y , from a large number of transformants. This would require a simple assay system that i s capable of detecting the presence of newly synthesized peptides. The two methods that are commonly used to detect enzymatically and chemically synthesized peptides include reverse-phase high performance l i q u i d chromatography and t h i n layer INTRODUCTION / 3 chromatography. However, neither method i s suit a b l e for rapid screening purposes. The use of microtiter plates i n an enzyme linked immunosorbent assay (ELISA) greatly f a c i l i t a t e s the handling and washing of samples. When used with automated readers and multiple well washers, these plates allow large numbers of samples to be assayed within a r e l a t i v e l y short time. Therefore, i t would be advantageous i f a si m i l a r kind of solid-phase assay system could be developed for the detection of dipeptides formed from enzyme-catalysed reverse proteolysis reactions. As i n ELISA, one of the substrates ( i . e . one of the amino acids) may be immobilized to the mi c r o t i t e r plate. The second substrate w i l l then be added to the well along with the target enzyme. The second amino acid needs to be l a b e l l e d for example, by another enzyme or by radioactive isotopes, so that i t w i l l provide a signal that i s r e a d i l y detectable. When a peptide bond i s formed between the immobilized substrate and the free amino acid i n the l i q u i d phase, the r e s u l t i n g dipeptide w i l l be bound to the well and can be e a s i l y detected by the lab e l on the second amino acid. This type of solid-phase assay could provide a rapid screening system for p o s i t i v e mutants generated from mutagenesis experiments as the use of m i c r o t i t e r plates would make routine handling of a large number of b a c t e r i a l cultures r e l a t i v e l y easy. Therefore, the objective of t h i s research was to develop a rapid and simple ELISA-like s o l i d -phase assay for the detection of dipeptides formed from enzyme catalysed reactions. L-leucine and DNP-L-asparagine would be used INTRODUCTION / 4 as the substrates for dipeptide synthesis. I n i t i a l synthesis experiments were to be catalysed by commercial grade thermolysin. Since thermolysin i s commercially available i n a concentrated form, i t s higher enzyme a c t i v i t y would l i k e l y ensure that some DNP-asn-leu would be formed i n the microtiter wells. I f the experiments using thermolysin were successful, the next step would be to use crude proteases, isolated from b a c t e r i a l cultures, as c a t a l y s t s . These data would be useful i n determining i f t h i s rapid assay i s s e n s i t i v e enough for the purpose of screening crude unpurified mutant protease T enzymes secreted by b a c t e r i a l cultures. LITERATURE REVIEW I. ENZYMATIC PEPTIDE SYNTHESIS a. Advantages and Disadvantages Proteases have been shown to be useful i n c a t a l y s i n g peptide bond synthesis by reverse proteolysis (Cheng et a l . , 1988; Miranda and Tominaga, 1991; Morihara and Oka, 1980; Morihara and Oka, 1980; Morihara, 1987; Oka and Morihara, 1978; Sakina et a l . , 1988; Steinke et a l . , 1991). Enzymatic methods may be preferred over the conventional chemical methods of synthesizing useful peptides for various reasons. F i r s t of a l l , reactions can be performed s t e r e o s p e c i f i c a l l y . Therefore, p u r i f i c a t i o n of the f i n a l product should be easier and less costly since there i s no need to remove undesired stereoisomers. Secondly, there i s l e s s need for expensive protection groups. Chemical synthesis requires the use of protecting groups on functional groups of amino acid side chains, which should not p a r t i c i p a t e i n the peptide bond formation (Bodanszky, 1988). In contrast, enzymatic synthesis of peptides can be performed without protection of these functional groups because proteases show s p e c i f i c i t y for the a-carboxyl or the a-amino group at the active s i t e (Sakina et a l . , 1988). Thirdly, product y i e l d may be higher with enzymatic synthesis mainly because p u r i f i c a t i o n of the f i n a l product should be less complicated than fo r the f i n a l product from conventional chemical production methods. Lastly, enzymatic reactions can be c a r r i e d out under very mild conditions while chemical reactions often require high temperature and high pressure. 5 LITERATURE REVIEW / 6 One of the major problems with using proteases i n catalysing peptide synthesis i s related to t h e i r narrow substrate s p e c i f i c i t y and the narrow range of conditions these enzymes can t o l e r a t e (Lars et a l . , 1991; Steinke et a l . , 1991). Often, very stringent temperature, pH, or io n i c conditions are required i n order for an enzyme to catalyse a reaction (Chen and Arnold, 1991). Another problem i s related to the fact that some enzymes are f a i r l y expensive (Morihara, 1987). However, t h i s problem may be overcome by the use of immobilized enzymes. Lastly, h y d r o l y t i c a c t i v i t i e s of proteases could lead to the formation of undesired by-products and to a decrease of product y i e l d (Wong, 1992). Since water l i m i t s y i e l d because of hydrolysis of the enzyme-substrate intermediate or of the f i n a l product, product y i e l d may be enhanced s i g n i f i c a n t l y i f the reaction i s ca r r i e d out i n the presence of mixed or pure organic solvents instead of water (Chen et a l . , 1991). However, proteases are generally unstable i n organic solvent, which quickly denatures and inactivates enzymes. For example, the h a l f - l i f e of s u b t i l i s i n BPN' i n anhydrous dimethylformamide i s only about 20 minutes, which i s hardly s u f f i c i e n t for large-scale production process (Wong, 1992). Protein engineering studies have been conducted by various research groups i n an attempt to produce proteases with enhanced s t a b i l i t y i n organic solvents (Chen and Arnold, 1991; Martinez et a l . , 1992; Wong et a l . 1990). b. Examples of Useful Peptides Synthesized by Protease-Catalysed Reactions Some proteases have been successfully used i n enzyme-assisted LITERATURE REVIEW / 7 synthesis of useful peptides and peptide hormones. For example, thermolysin has been used to prepare the precursor of aspartame, which i s then converted to aspartame by conventional chemical methods (Morihara, 1987). Paul et a l . (1988) demonstrated that aspartame could be d i r e c t l y synthesized from the coupling of L-aspa r t i c acid to L-phenylalanine methyl ester i n a reverse hydrolysis reaction catalysed by peptidase enzyme, which was is o l a t e d from a gram-positive coccus. Metenkephalin has been prepared by fragment condensation using papain (Morihara, 1987). The rS-sleep inducing peptide was made i n a s e r i e s of reactions using only papain, except i n a coupling step which was catalysed by a-chymotrypsin (Sakina et a l . , 1987). I n d u s t r i a l production of human i n s u l i n from swine i s accomplished by digestion of porcine i n s u l i n with Achromobacter protease I, and then coupling with a threonine group using trypsin or Achromobacter protease I (Morihara et a l . , 1986). This enzymatic synthesis reaction resulted i n over 90% product y i e l d while the y i e l d of t r a d i t i o n a l chemical synthesis i s much lower (about 10%). c. Methods for Detecting Peptide Formation The two methods that are commonly used for the detection and q u a n t i f i c a t i o n of enzymatically and chemically synthesized peptides are reversed phase HPLC (high performance liquid-chromatography) and t h i n layer chromatography (TLC). For example, Morihara et a l . (1986) used a reversed-phase HPLC with a column of Nu c l e o s i l 5 C i 8 to qu a n t i t a t i v e l y determine the semisynthesis of human i n s u l i n ester LITERATURE REVIEW / 8 Paul et a l . (1988) detected formation of aspartame and i t s derivatives using reverse-phase HPLC with a juBondapack C18 column. Synthesis of Cbz-Phe-Leu-NH2 was i d e n t i f i e d and product y i e l d was determined by t h i n layer chromatography (Oka and Morihara, 1978). Formation of d i - and t r i p e p t i d e esters i n reactions catalysed by thermolysin was detected by TLC using four d i f f e r e n t solvent systems for development (Miranda and Tominaga, 1991). In reversed phase HPLC, proteins are separated according to t h e i r degree of hydrophobicity by interactions between- the exposed nonpolar amino acid side chains on the protein and hydrophobic groups on the chromatographic matrix (Goheen and Stevens, 1985). Q u a l i t a t i v e analysis i s accomplished by either comparing the retention times or volumes of the synthesized peptides to the retention times of the standards, or by c o l l e c t i n g the i n d i v i d u a l components as they emerge from the chromatograph and then i d e n t i f y i n g the components by other methods (Pearse, 1980). The coupling y i e l d i s usually calculated from the peak areas or peak heights corresponding to each component. With thin-layer chromatography, the sample i s spotted at the lower end of a glass, which has been precoated with a t h i n layer of adsorbent, such as alumina or s i l i c a g e l . The solvent i s then drawn upwards by c a p i l l a r y a t t r a c t i o n . As the solvent moves past the sample the components of the sample s e l e c t i v e l y dissolve and are v e r t i c a l l y displaced on the g e l . The amount of movement of the d i f f e r e n t components on the gel i s dependent upon the solvent flow LITERATURE REVIEW / 9 rate and s e l e c t i v e retention of solutes by the stationary phase. The separated peptides and t h e i r s t a r t i n g materials are i d e n t i f i e d r e a d i l y i f colored. Otherwise, i t may be necessary to detect fluoresence which i s i n i t i a t e d with an u l t r a v i o l e t lamp or "develop" colored compounds by spraying the separated spots with a colour-forming reagent (Pearse, 1980). For example, ninhydrin i s used for producing color with amino acids containing spots on the plates. The quantity of each component can be determined by comparing the si z e of the migration spots of the separated peptides with those of the known standards. A l t e r n a t i v e l y , the colour i n t e n s i t i e s of the spots corresponding to the s t a r t i n g carboxyl components and the product can be measured using a TLC-scanner. I I . PROTEASES a. Thermolysin Thermolysin (EC 3.4.24.2) i s an e x t r a c e l l u l a r metallo-endopeptidase iso l a t e d from Bacillus thermoproteolyticus. I t i s most active at neutral pH. The enzyme has a molecular weight of 34.6 kDaltons and i s made up of a single polypeptide chain of 316 amino acid residues, lacking t h i o l groups or disulphide bonds (Fontana, 1988). Thermolysin i s one of the most well-characterized enzymes owing to extensive x-ray crystallographic and k i n e t i c studies. The 3-dimensional structure of thermolysin (Figure 1) has been elucidated and the mechanism for i t s peptide cleavage a c t i v i t y has been proposed based on crystallographic studies of various LITERATURE REVIEW / 10 Figure 2. Schematic representation of the active s i t e of thermolysin with a polypeptide substrate bound (Bigbee and Dahlquist, 1974) LITERATURE REVIEW/11 thermolysin-inhibitor complexes (Monzingo and Matthews, 1982). The metalloenzyme normally contains one zinc ion and four calcium ions per molecule. The zinc ion has been shown to be e s s e n t i a l to the c a t a l y t i c a c t i v i t y of thermolysin, and i s liganded at the active s i t e of the enzyme by His 142, His 146, and Glu 166 (Figure 2). On the other hand, the calcium ions are not involved i n c a t a l y s i s but are thought to contribute to the thermal s t a b i l i t y of the enzyme (Matthews et a l . , 1974). The presence of calcium ions i s also important i n s t a b i l i z i n g the thermolysin molecule against a u t o l y s i s (Fontana, 1988). The active s i t e of thermolysin contains at l e a s t four subsites, S 1 ( S2, S,', and S2', which p a r t i c i p a t e i n binding the extended substrate (Tran et a l . , 1991) and are responsible for substrate s p e c i f i c i t y of the enzyme. The hydrophobic pocket to the r i g h t of the zinc i s the primary recognition s i t e ( S / ) . Hydrophobic residues such as leucine, isoleucine and phenylalanine are preferred for binding at t h i s p o s i t i o n (Holden and Matthews, 1988). I t i s known that the carbonyl oxygen of the peptide bond to be cleaved i s liganded to the zinc atom, displacing the water molecule that normally acts as the fourth ligand i n the native thermolysin structure. Glu 143 and His 2 31 also appear to play a role i n hydrolysis because they are the c l o s e s t side chains to the s c i s s i l e peptide bond. Other amino aicd residues, such as Trp 115, Tyr 157, Asn 112, Ala 113 and Ala 118, i n the active s i t e region are thought to partcipate i n the p o s i t i o n i n g of the substrate backbone i n the active s i t e by hydrogen bonding to the substrate main chain. LITERATURE REVIEW / 12 Several studies have demonstrated that thermolysin can be used to promote formation of peptide synthesis v i a a reverse hydrolysis reaction. For example, the enzyme has been used i n enzymatic synthesis of asparagine-containing peptides, proline-containing t r i p e p t i d e s , cholecystokinin-octapeptide, and the precursor of aspartame (Miranda and Tominaga, 1991., Cheng et a l . , 1988, Sakina et a l . , 1988, Isowa et a l . , 1979). Although the mechanism involved i n reverse proteolysis i s not known, i t has been proposed that, i n the coupling reaction, the amino acid residues of the carboxyl and amino components used as s t a r t i n g materials occupy the same subsite i n the active s i t e of the enzyme as i n hydrolysis of the corresponding product (Morihara and Oka, 1980). b. Neutral Protease T Neutral protease T, an e x t r a c e l l u l a r enzyme with a molecular weight of 34,579 daltons, i s secreted by Bacillus stearothermophilus (Takagi et a l . , 1985). This enzyme possesses c h a r a c t e r i s t i c s s i m i l a r to thermolysin. For example, neutral protease T requires Zn 2 + for i t s p r o t e o l y t i c a c t i v i t y and Ca 2 + for i t s thermal s t a b i l i t y . In fact, t h i s enzyme has been found to be quite unstable i n the absence of Ca 2 +. For t h i s reason, i t has been suggested that the addition of Ca 2 + to the culture medium i s necessary whenever production of neutral protease i s needed (Fu j i et a l . , 1983). Neutral protease T i s a thermostable enzyme which ret a i n s about 80% of i t s a c t i v i t y even a f t e r prolonged heating at 65°C for 30 minutes. Mutant neutral protease T with enhanced LITERATURE REVIEW / 13 thermostability and altered s p e c i f i c a c t i v i t y has been obtained by recombinant DNA technology (Takagi and Imanaka, 1989). The gene fo r neutral protease T has been cloned and i t s nucleotide sequence has been determined (Takagi et a l . , 1985). The amino acid sequence of the e x t r a c e l l u l a r form of t h i s enzyme i s highly homologous (85%) to that of thermolysin. The zinc binding s i t e (His-142, His-146, and Glu-166) i n thermolysin i s also found i n neutral protease T (His-145, His-149, and Glu-169). I t i s known that at l e a s t 5 amino acid residues p a r t i c i p a t e i n the c a t a l y t i c reaction and positioning of the substrate backbone i n the active s i t e of thermolysin (Tran et a l . , 1991). These residues are found at homologous positions i n neutral protease T from B. stearothermophilus. These facts suggest that thermolysin and neutral protease T may share similar 3-dimensional structures. The h y d r o l y t i c and reverse p r o t e o l y t i c a c t i v i t i e s of these two enzymes may be a l i k e . I I I . IMMUNOASSAY TECHNIQUES a. Radioimmunoassay Infectious diseases i n humans were t r a d i t i o n a l l y i d e n t i f i e d by the c u l t i v a t i o n of the i n f e c t i n g agent i n an i n - v i t r o system or laboratory animal. However, the usefulness of t h i s technique was severely l i m i t e d by the fact that not a l l viruses that cause medically important diseases can be c u l t i v a t e d i n t i s s u e culture or animal systems (Yolken, 1982). Examples of these v i r a l agents include rotavirus, hepatitus B virus, Norwalk v i r u s , and Epstein-LITERATURE REVIEW / 14 Barr v i r u s . Another major problem associated with the c u l t i v a t i o n approach i s that some v i r a l agents require a long period of time to grow and the r e s u l t s are not useful f o r the treatment of i l l patients. These problems are largely overcome through the development of rapid assays capable of d i r e c t l y detecting i n f e c t i o u s agents i n c l i n i c a l specimens. The p r i n c i p l e of most of these assays i s based on the fact that most in f e c t i o u s agents can be detected by a s p e c i f i c antibody-antigen reaction, which can be completed and measured i n a short period of time (Kemeny and Chantler, 1988). Solid-phase radioimmunoassay used to be the most popular assay for detecting microbial antigens. Reasons for the widespread usage of radioimmunoassay are related to the f a c t that r a d i o a c t i v i t y can be detected with great s e n s i t i v i t y and that the antibody-antigen reaction can be measured objectively using generally available laboratory instrumentation (Yolken, 1982). On the other hand, the use of radioactive labels also has drawbacks. These include the short s h e l f - l i f e of radioactive labels, which i s generally l i m i t e d to 2 months, the hazards involved i n preparation and handling of radioactive isotopes, and the need fo r gamma counting instruments. Alternative non-radioactive immunoassay systems were therefore developed with the intention to overcome most of the disadvantages associated with radioimmunoassay. One of such systems that has attained wide spread usage i s the enzyme linked immunosorbent assay (ELISA). LITERATURE REVIEW / 15 b. Advantages of Enzyme Linked Immunosorbent assay ELISA methods have a number of advantages over radioimmunoassay. The l a b e l l e d reagents used are stable and can be stored f o r prolonged periods of time without loss of a c t i v i t y (Kemeny and Chantler, 1988). The use of multiwell m i c r o t i t e r plates as the solid-phase instead of tubes greatly f a c i l i t a t e s handling and washing of samples. Large numbers of samples can be assayed very rapi d l y when microtiter plates are used with automated readers and multiple well washers. A wider range of substrates and chromogens with higher s e n s i t i v i t y i s available and a v a r i e t y of enzyme-labelled antisera may now be purchased commercially which minimizes the need for preparing these reagents at the lab. F i n a l l y , ELISA has a higher pot e n t i a l s e n s i t i v i t y than radioimmunoassay because t h e o r e t i c a l l y a molecule of enzyme can generate many molecules of product. The enzyme linked immunosorbent assay (ELISA) has been widely used for the detection of antibodies and antigens i n human body f l u i d s and veterinary diseases and i n immunology (Kemeny and Chantler, 1988). Various important microbial antigens i n human f l u i d s that can be r e l i a b l y detected by ELISA include rotavirus, h e p a t i t i s B v i r u s , Haemophilus influenzae type b (Yolken, 1982). c. P r i n c i p l e of Direct and Indirect ELISA In the d i r e c t assay, an unlabelled antibody i s bound to a s o l i d phase by physical adsorption or by covalent linkage. Unbound antibody i s removed by washing. A te s t sample i s then added to the s o l i d phase along with a buffer, which serves to insure that the LITERATURE REVIEW / 16 proper pH and i o n i c strength are available for the antigen-antibody reactions. The antibody coated s o l i d phase w i l l bind s p e c i f i c antigen present i n the t e s t sample during incubation. After removal of unbound material, enzyme-labelled antibody i s added. This enzyme-antibody complex w i l l react with antigen bound to the s o l i d phase. When unbound enzyme la b e l l e d antibody i s washed away, a substrate i s added to the s o l i d phase which w i l l be converted into a coloured form by the enzyme adhered to the wel l . The amount of colour measured i s proportional to the amount of antigen present i n the t e s t sample (Yolken, 1982). In the i n d i r e c t ELISA, the t e s t sample i s added to an antibody-coated solid-phase as i n the d i r e c t system. Instead of adding an enzyme-labelled antibody to react with the bound antigen, an unlabelled antibody, which has been prepared i n a d i f f e r e n t animal species than the antibody u t i l i z e d for coating the s o l i d phase, i s added. Following removal of unreacted antibody, an enzyme-labelled antiglobulin directed against the second antibody i s added. Substrate i s then added and i s detected as described i n the previous paragraph. The main benefit for using an unlabelled second antibody from another animal source i s that a sing l e enzyme-l a b e l l e d antispecies globulin can be used for detection of a large number of d i f f e r e n t antigens while i n the d i r e c t ELISA, a d i s t i n c t antibody-enzyme conjugate i s required for each antigen to be tested (Yolken, 1982). d. Choice of enzyme and substrate The three most commonly used enzymes i n ELISA include LITERATURE REVIEW / 17 horseradish peroxidase, a l k a l i n e phosphatase, and /J-D-galactosidase. The choice of enzyme frequently i s dependent upon i t s p u rity, the s e n s i t i v i t y of i t s substrate detection, ease of conjugation of enzyme-antibody complex, and s t a b i l i t y of conjugate (Kemeny and Chantler, 1988). Horseradish peroxidase (HRP) i s a r e l a t i v e l y inexpensive and pure enzyme that also has a high turnover rate. The turnover rate of an enzyme marker greatly affects the s e n s i t i v i t y of an ELISA system. The major advantage for using t h i s enzyme i n ELISA i s that peroxidase i s a glycoprotein, whose carbohydrate portion provides a good binding s i t e for antibody with minimal interference with the f u n c t i o n a l i t y of the enzyme (Wilson and Nakane, 1978). Antibody i s linked to horseradish peroxidase by periodate oxidation, by sulphydryl-maleimide conjugation, or the two-step glutaraldehyde reaction (Kemeny and Chantler, 1988). Another advantage of horseradish peroxidase i s that various chromogens can be used with HRP substrate, hydrogen peroxide, to produce intense dark colour which can be e a s i l y measured spectrophotometrically. Common chromogens include 2,2-azino-di(3-ethylbenzothiazoline-6-sulphonate)(ABTS), orthophenylenediamine (OPD) and 3,3', 5,5'-tetramethylbenzidine hydrochloride (TMB). The l a s t two are most se n s i t i v e i n detecting low lev e l s of enzyme (Kemeny and Challacombe, 1988). There are several drawbacks associated with the use of peroxidase i n ELISA. F i r s t , the enzyme loses s e n s i t i v i t y i f i t becomes contaminated with microorganisms. O r d i n a r i l y , the addition of small amounts of antimicrobial agents LITERATURE REVIEW / 18 such as sodium azide and methanol would i n h i b i t microbial growth. However, peroxidase i s very sensitive to sodium azide and methanol. Secondly, most of the chromogens for peroxidase except TMB have been found to be carcinogenic or mutagenic. Lastly, i t has been determined that peroxidase can be inactivated by polystyrene surfaces (Berkowitz and Webert, 1981). Fortunately, t h i s problem can be overcome by pretreating polystyrene ELISA plates with Tween 20. Unlike HRP, a l k a l i n e phosphatase i s r e l a t i v e l y stable during storage and i s not s e n s i t i v e to antimicrobial agents. This enzyme has a high turnover rate and i s coupled to protein by two-step glutaraldehyde or sulphydryl/maleimide procedures. The substrate used f o r t h i s enzyme i s para-nitrophenyl phosphate, which i s non-carcinogenic. The substrate reacts with a l k a l i n e phosphatase to produce a pale yellow which again can be measured spectrophotometrically. However, colour development i s slower than with HRP substrates. S e n s i t i v i t y may be improved by using fluorescent substrates or by using a c y c l i c a l enzyme-amplified system f o r a l k a l i n e phosphatase detection (Self, 1985) . The major drawback of using alkaline phosphatase i s that i t i s expensive because enzyme of high purity needs to be obtained from an animal source such as c a l f intestine (Yolken, 1982). (8-galactosidase i s not used as often as the other two enzymes mentioned above because i t has a slower turnover rate. However, unlike HRP and a l k a l i n e phosphatase, /?-galactosidase i s not found i n plasma or other body f l u i d s and would be useful to use i n LITERATURE REVIEW / 19 sit u a t i o n s where endogenous enzyme a c t i v i t y cannot be removed. Coupling of t h i s enzyme to proteins can be accomplished by one-step glutaraldehyde and maleimide procedures (Kemeny and Chantler, 1988) . Chromogenic substrates such- as p-nitrophenyl - /3-D-galactosidase and f luorogenic substrates l i k e 4-methylumbelliferyl-/3-D-galactosidase have been used. e. M i c r o t i t e r Plates S o l i d phase supports can be c l a s s i f i e d as low and high capacity systems. High capacity supports bind appreciably more proteins than low capacity supports, and are often made of agarose, Sephadex, c e l l u l o s e , and n i t r o c e l l u l o s e . On the other hand, low capacity s o l i d phase matrices are often made from polystyrene, p o l y v i n y l c h l o r i d e (PVC), nylon, and glass. Since the choice of s o l i d phase matrix i n ELISA i s largely dependent on the ease of se t t i n g up and processing these assays, the convenience offered by mi c r o t i t e r plate format i s probably the reason for the widespread popularity of ELISA i n spite of the lim i t e d protein binding capacity of the microtiter plates. The mechanism by which proteins bind to p l a s t i c surface i s not well understood. However, i t i s believed that charge and hydrophobic interactions may be involved (Kemeny and Chantler, 1988) . Proteins can also be covalently attached to ELISA plates by pretreating proteins with glutaraldehyde (Parsons, 1981), or with carbodiimide (Rotman and Delwel, 1983). The binding capacity of d i f f e r e n t p l a s t i c surfaces varies. PVC plates have been reported LITERATURE REVIEW / 20 to have greater binding capacity (De Savigny and V o l l e r , 1980). Irradiated plates that bind more proteins than PVC plates are also a v a i l a b l e (Urbanek et a l . , 1985). Studies have shown that plates manufactured by d i f f e r e n t suppliers also have remarkably d i f f e r e n t protein binding capacities. For example, Urbanek et a l . (1985) demonstrated that Nunc Immuno-1 had higher binding capacity for bee venom phospholipase A2 than Dynatech Immunlon and Linbro m i c r o t i t e r pl a t e s . The s t a b i l i t y of protein-plate i n t e r a c t i o n i s also an important consideration because desorption of proteins from the m i c r o t i t e r plate during an assay could lead to f a l s e conclusions. The rate and extent of protein binding to m i c r o t i t e r plates are dependent upon the type of protein and i t s concentration, time, pH, and temperature of incubation (Blake and Gould, 1984) . The highest concentration of most proteins that would form a monolayer was reported to be 1 jwg/mL (Cantarero et a l . , 1980). Addition of excessive amount of protein i s wasteful as the a d d i t i o n a l protein i s loosely bound to the other proteins and would be washed off during subsequent incubation and washing steps. A high concentration of proteins could also r e s u l t i n increased non-s p e c i f i c binding of proteins to the p l a s t i c surface. Non-specific binding of proteins to the microtiter plate could lead to undesired high background noise of the assay. One way to minimize t h i s problem i s to add non-ionic detergents such as Tween 20 (Berkowitz and Webert, 1981). Blocking proteins can also be added a f t e r coating to block any vacant binding s i t e s . Common blocking proteins include g e l a t i n (0.5 - 1%) and bovine serum albumin (1%) LITERATURE REVIEW / 21 (Kemeny and Chantler, 1988). IV. BACILLUS SUBTILIS a. Use of Bacillus subtilis as a host for production of heterologous proteins Recombinant DNA technology has made i t possible 1 to commercially produce useful enzymes and proteins from microorganisms, plants, and animals v i a cloning and expression. High y i e l d s of proteins may be achieved through i n s e r t i o n of multiple copies of a s p e c i f i c gene into a desired host organism and through development of high secretion systems. The choice of host organism may also a f f e c t the production l e v e l s of the desired protein (Pitcher, 1986). As an example, secretion of c a l f rennin or chymosin i n bacteria i s d i f f i c u l t to achieve. However, the secretion of properly processed prochymosin, a precursor of c a l f rennin, has been made possible through the use of a filamentous fungus (Pitcher, 1986). T r a d i t i o n a l l y , E. coli and i t s plasmids and phages have been the main components of cloning and expression systems used i n recombinant DNA studies. However, i t i s known that laboratory s t r a i n s of E. coli are capable of exchanging genetic information with i n t e s t i n a l E. coli. In addition, the envelope of E. coli c e l l contains lipopolysaccharide, which causes endotoxin shock syndrome (Lovett and Ambulos, J r . , 1989). This makes i t necessary to rigoro u s l y p u r i f y gene products from E. coli i f these products are to be used as pharmaceuticals or i n food applications. Another problem i n using E. coli as expression system i n commercial applications i s that the organism expresses proteins LITERATURE REVIEW / 22 i n t r a c e l l u l a r l y , which complicates the process of protein recovery. As a r e s u l t , alternative cloning and expression systems have been developed which have overcome some of the problems associated with the use of E. coli. The Bacillus subtilis system has emerged as the major prokaryotic alternative to cloning i n E. coli (Lovett and Ambulos, J r . , 1989). The major reason for commercial development of expression systems i n Bacillis subtilis i s related to i t s large secretion capacity. Unlike E. coli, Bacillus subtilis as well as many other s t r a i n s of Bacillus are capable of d i r e c t l y secreting large quantities of a variety of enzymes into the growth medium. This greatly s i m p l i f i e s the recovery of the target protein since the secreted protein i s i n a r e l a t i v e l y pure and soluble form i n contrast to an insoluble i n t r a c e l l u l a r product (Mountain, 1989). In addition, the expression system i n B. subtilis allows accumulation of high l e v e l s of products i n active forms, unlike the denatured proteins usually harvested from i n c l u s i o n bodies that r e s u l t from i n t r a c e l l u l a r high-level expression and accumulation. The second reason which accounts for the increased commercial i n t e r e s t f o r making recombinant products i n B. subtilis i s rela t e d to the f a c t that well-established large scale fermentation and product recovery systems are available. Thirdly, unlike E. coli, B. subtilis i s non-pathogenic and does not produce endotoxins. In fa c t , the organism has been granted GRAS (Generally Regarded As Safe) status i n the United States. More importantly, the Food and Drug Administration gave GRAS status for the f i r s t time to an LITERATURE REVIEW / 23 heterologous enzyme (a-amylase from Bacillus stearothermophilus) produced i n B. subtilis (Mountain, 1989). b. Problems associated with using B. subtilis expression systems The commercial use of B. subtilis as an expression system for heterologous proteins suffers from several drawbacks. F i r s t l y , recombinant plasmids tend to be very unstable i n t h i s host organism. Both s t r u c t u r a l and segregational i n s t a b i l i t y of recombinant plasmids have been observed i n much greater frequency i n B. subtilis than i n E. coli (Mountain, 1989). Structural i n s t a b i l i t y r e f e r s to deletion, insertion, or rearrangement of the cloned gene within a recombinant plasmid while segregational i n s t a b i l i t y r e f e r s to the loss of the enti r e recombinant plasmid. One possible way of improving plasmid s t a b i l i t y i s to integrate multiple copies of the gene of interest into the chromosome of B. subtilis. As an example of such an approach, a B. subtilis carrying 2 copies of the a-amylase gene of B. amyloliquefaciens integrated at d i f f e r e n t locations i n the chromosome produced as much amylase as a s t r a i n carrying the gene on a recombinant plasmid at 40 copies per c e l l ( K a l l i o et a l . , 1987). Production of amylase appeared to be slower but production continued f o r a longer time f o r the integration s t r a i n . The second b a r r i e r hindering the development of e f f i c i e n t expression systems i n B. subtilis i s related to the tendency of the organism to produce high levels of e x t r a c e l l u l a r proteases which degrade the secreted foreign protein. I t i s known that B. subtilis LITERATURE REVIEW / 24 has 6 e x t r a c e l l u l a r proteases (Xu et a l . , 1991). Construction of protease d e f i c i e n t strains of host c e l l s may help to a l l e v i a t e such a problem. Construction of a double protease-deficient s t r a i n , DB 104, greatly improved the s t a b i l i t y of the secreted foreign proteins (Kawamura and Doi, 1984). A s t r a i n of Bacillus subtilis d e f i c i e n t i n s i x e x t r a c e l l u l a r proteases was recently developed (Xu et a l . , 1991). The t h i r d hurdle preventing wide application of B. subtilis as a host f o r the production of heterologous proteins i s the lack of well-characterized, strong controllable promoters that are s i m i l a r to the p_L, p_R, tr p . lac, and tac promoters which have been shown to be useful i n E. coli (Mountain, 1989). Various inducible vectors have been developed to overcome t h i s problem. Some of these vectors use either the E. coli lac system (Le Grice, 1990) or the temperature sensitive repressor from X ( B r e i t l i n g et a l . 1990) or 0 105 (Osburne et a l . , 1984) to regulate expression of target foreign proteins while others are based on the regulatory region of sacB, a sucrose-inducible gene, that encodes for the e x t r a c e l l u l a r enzyme, levansucrase (Xu et a l , 1991). M A T E R I A L S A N D M E T H O D S M A T E R I A L S Nunc Immuno Modules, CovaLink NH (cat. no. 478042), were obtained from Gibco Canada Inc., Burlington, ON. DNP (d i n i t r opheny 1) - L -leucine, L-leucine, DNP-L-asparagine, L-asparagine, thermolysin (Protease Type X from Bacillus thermoproteolyticus), l- e t h y l - 3 -(dimethyl-aminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), dimethylsulfoxide, calcium chloride, casamino acid, kanamycin monosulfate, azocasein, and o-phenylenediamine (OPD) were purchased from Sigma Chemical Co., St. Louis, MO. Rabbit anti-DNP antibody horseradish peroxidase conjugate (Ra DNP-HRP) was obtained from Dakopatts, Glostrup, Denmark. N,N-dimethylformamide (DMF), 10 N sulphuric acid, disodium hydrogen carbonate, 3 0% hydrogen peroxide, sodium bicarbonate, sodium hydroxide, sodium acetate, calcium acetate, glucose, magnesium sulphate hexahydrate, EDTA, Hammerstein casein and t r i c h l o r o a c e t i c acid were obtained from BDH Canada, Toronto, ON. Broad range molecular weight standard was purchased from Biorad, Missisauga, ON. Tryptone, yeast extract, and nu€rient agar were purchased from Difco, Detroit, MI. Carnation instant skim milk powder was purchased from a l o c a l r e t a i l store. R e s t r i c t i o n enzymes and Hind III digest fragments were puchased from Boehringer Mannheim, Quebec, Canada. Bacillus subtilis DB428 and WB600 were provided by Dr. Sui-Lam Wong (University of Calgary, Alberta, Canada). Recombinant plasmid, pNP22, was provided by Dr. T. Imanaka (Osaka University, Osaka, Japan). 25 MATERIALS AND METHODS / 26 METHODS I. THERMOLYSIN CATALYSED SOLID-PHASE SYNTHESIS OF DNP-ASN-LEU DIPEPTIDE a. Immobilization of DNP(Dinitrophenyl)-L-leucine or L-leucine to m i c r o t i t e r plate A s p e c i a l type of polystyrene microtiter plate, c a l l e d Nunc Immuno Modules, was used as the s o l i d phase on which leucine molecules were attached. These plates had s p e c i a l l i n k e r arms, c a l l e d CovaLink NH. These secondary amino groups were grafted on the polystyrene surface and served as bridgeheads for further covalent coupling. The amino groups were positioned at the end of a 2 nm long spacer arm; t h i s made the NH-groups r e a d i l y accessible for molecules present i n the l i q u i d phase (Figure 3). The l i n k e r arms permit covalent binding of even small molecules to the surface. I t has been reported that tripeptides could be detected when bound to Covalink NH whereas the same peptides were only barely detectable when bound to normal microtiter plates without l i n k e r arms (Nunc InterMed Publication). L-leucine molecules were covalently bonded to the s o l i d phase v i a the carboxyl groups using water soluble l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as coupling agent i n the presence of N-hydroxysuccinimide (NHS) (Sondergard-Andersen et a l . 1990). The mechanism of t h i s reaction i s shown i n Figure 4. The method of Sondergard-Andersen et a l . (1990) was used with a few modifications. A stock DNP-L-leucine or L-leucine s o l u t i o n was prepared by d i s s o l v i n g the amino acid e i t h e r i n dimethylsulfoxide (DMSO) and d i s t i l l e d water at 1.5 : 1 r a t i o or i n MATERIALS AND METHODS / 27 L Figure 3. Schematic chemical and physical configuration of the CovaLink NH surface (Nunc InterMed publication) MATERIALS AND METHODS / 28, Figure 4. Covalent coupling of amino acid to CovaLink NH. EDC: l-ethyl-3-(3-dimethylaminopropyl)carbodiimide NHS: N-hydroxysuccinimide MATERIALS AND METHODS / 29 d i s t i l l e d water alone. DNP-L-leucine or L-leucine s o l u t i o n was added to an equal volume of 0.1 M NHS and 0.1 M EDC, which had been pre-dissolved i n d i s t i l l e d deionized water. The mixture was thoroughly mixed and was then incubated at room temperature for 30 minutes to allow for the activation of the carboxyl groups on the DNP-L-leucine or L-leucine molecules. After incubation, the sol u t i o n was di l u t e d with ice cold 0.1 M carbonate buffer, pH 8.6. 100 fil, of the d i l u t e d mixture i n d i f f e r e n t concentrations was added to designated wells i n a Nunc Immuno Module. The plate was then incubated at 5°C for 30 minutes. A l l coupling reactions were ca r r i e d out at 5°C in t h i s thesis to minimize non-covalent binding of molecules to the CovaLink Module. After incubation, the wells were emptied and washed 3 times with 0.15 M phosphate bufferd sa l i n e , pH 7.2, containing 0.05% (v/v) Tween 20. 0.15 M phosphate buffer s a l i n e was prepared by dissolv i n g the following i n 1 L of d i s t i l l e d water: 8 g NaCl, 0.2 g KC1, 1.15 g Na2HP04.2H20, 0.2 g KH2P04; pH was adjusted to 7.2 with either 5 M hydrochloric acid or 5 M sodium hydroxide. I t should be noted that a l l buffers used i n a l l the experiments i n t h i s thesis were previously autoclaved and cooled before use. Autoclaving was used as a precautionary measure against the possible i n a c t i v a t i o n of peroxidase by contaminating microorganisms, which could be present i n buffer solutions. b. Detection of Immobilized Amino Acid or Dipeptide Rabbit anti-DNP antibody horseradish peroxidase conjugate stock (1.3 mg/mL) was di l u t e d 500 times with 0.01 M phosphate MATERIALS AND METHODS / 30 buffer (pH 7.2) containing 0.01% skim milk powder. 100 fiL of the d i l u t e d conjugate (2.6 /xg/mL) was pipetted to each m i c r o t i t e r well containing immobilized amino acid or dipeptide. The plate was incubated for 1 hour at room temperature with shaking at 110 rpm s e t t i n g i n a Psychrotherm Controlled Environment incubator shaker (New Brunswick S c i e n t i f i c Co., New Brunswick, N.J.). After incubation, the wells were emptied and were washed three times with 0.15 M phosphate bufferd saline, pH 7.2. The buffer was kept i n the wells for 15 minutes aft e r the t h i r d wash i n an attempt to remove the l a s t trace of any unreacted antibody conjugate. 100 (xl> of substrate solution (0.06% w/v o-phenylenediamine dihydrochloride, 0.015% w/v hydrogen peroxide i n 0.5 M c i t r a t e -phosphate buffer, pH 5) was then added to each well . 0.5 M citrate-phosphate buffer was prepared by d i s s o l v i n g the following i n 1 L of d i s t i l l e d water: 7.3 g c i t r i c acid, 11.86 g Na2HP04.2H20; pH was adjusted 5.0 with either 5 M hydrochloric a c i d or 5 M sodium hydroxide. The plate was l e f t at room temperature f o r 10 minutes with shaking to allow for colour development. The reaction was then stopped by addition of 100 j«L 0.1 M sulphuric acid to each well. O p t i c a l density at 492 nm was measured using an ELISA t i t e r p l a t e reader, Easy Reader EAR 400 (SLT-Labinstruments, Salzburg, A u s t r i a ) . c. Solid-phase Enzymatic DNP-Asn-Leu Dipeptide Synthesis The reaction conditions selected for DNP-asn-leu synthesis were based on the method described by Miranda and Tominaga (1991) MATERIALS AND METHODS / 31 with some modifications. The choice of substrates was l a r g e l y based on the research done by Miranda and Tominaga (1991) , who used thermolysin to catalyse the synthesis of Z-Asn-Leu-OEt. They reported that the y i e l d of the f i n a l product was 62% when Z-Asn-OH and H-LeuOEt.HCl were used at equal molar concentration. L-leucine, not l a b e l l e d with dinitrophenyl, was coupled to CovaLink module as described i n section I. a. 7.9 mg DNP-Asn was dissolved i n 3.5 mL 0.2 M sodium acetate buffer, pH 6, with 50 mM calcium acetate, was adjusted to 6 using either 5 M sodium hydroxide or 5 M hydrochloric acid. Various volumes of DNP-Asn sol u t i o n ranging from 1 to 20 JXL were pipetted to designated m i c r o t i t e r wells. One milligram of thermolysin was dissolved i n 1 mL of the same sodium acetate buffer and 1, 5 or 10 ^L of the enzyme so l u t i o n was pipetted into each of the wells. Then the f i n a l volume of each reaction mixture was made up to 100 fxh by adding sodium acetate buffer. The microtiter plate was incubated at 48 °C with shaking at 110 rpm i n a Psychrotherm Controlled Environment incubator shaker for various lengths of time with samples removed at fixed time i n t e r v a l s . A f t e r each incubation period, the peptide synthesis reaction was stopped by emptying the reactants from the wells. The wells were washed three times with 0.15 M phosphate buffer saline, pH 7.2, containing Tween 20. The plate was then blocked with 0.5% skim milk powder i n 0.1 M phosphate buffer at room temperature for 45 minutes. Immobilized dipeptides ( i . e . DNP-asp-leu) i n the m i c r o t i t e r wells were detected using anti-DNP horseradish peroxidase conjugate as described i n MATERIALS AND METHODS / 32 section I.b. I I . ENZYME CATALYSED SOLID-PHASE SYNTHESIS OF DNP-ASN-LEU DIPEPTIDE USING CRUDE ENZYME MIXTURE FROM BACILLUS SUBTILIS TRANSFORMANTS a . l . Transformation of B. subtilis DB428 and WB600 B. subtilis DB428 (He et a l . , 1992), a mutant s t r a i n d e f i c i e n t i n 4 e x t r a c e l l u l a r proteases, and B. subtilis WB600 (Xu et a l . 1991), a mutant s t r a i n d e f i c i e n t i n 6 e x t r a c e l l u l a r proteases, were used as the host organism for plasmid, pNP22 (Takagi et a l . 1985) . This plasmid c a r r i e s nprT gene (Takagi et a l . 1985) , which encodes for neutral protease T. The method used to transform DB428 and WB600 i s a modified Spizizen's procedure (Personal communication with T. Imanaka) . The procedure i s as follows: 2 0 mL TFI (Transformation I) broth was inoculated with 1 mL of overnight host culture i n a 250 mL Erlenmeyer flask. The c e l l s were allowed to grow at 37°C for 3 to 4 hours with vigorous shaking u n t i l O.D. 6 6 0 n m reached about 0.56. A Magniwhirl water bath manufactured by Blue M E l e c t r i c Company (Blue Island, IL) was used; shaker s e t t i n g was at 8. Afte r incubation, 4 mL of TFI culture was transferred into 36 mL TFII (Transformation II) broth i n a 500 mL Erlenmeyer f l a s k . The c e l l s were grown at 37°C with vigorous shaking (shaker was set at 8) f o r 1.5 hours. 5 /xL of plasmid DNA was dissolved i n 95 /zL TE buffer i n a 16mmxl50mm te s t tube. 1 mL of TFII culture was poured into t e s t tube containing plasmid DNA; the mixture was shaken (shaker speed was set at 4) at 37°C for 30 minutes. The r e s u l t i n g b a c t e r i a l c e l l s were pe l l e t e d by centrifugation at 3 000 x g for 10 minutes at MATERIALS AND METHODS / 33 room temperature. The supernatant was discarded, and 3 mL of LB broth (1% tryptone, 0.5% yeast extract, 0.5 % NaCl) was added to the t e s t tube. The culture was incubated at 37°C for at least 1 hour with gentle shaking i n a Magniwhirl water bath; shaker s e t t i n g was at 4. 100 yiL of b a c t e r i a l c e l l s was spread onto a casein agar plate containing 50 nq/mL kanamycin. The casein plate was incubated overnight at 37°C. Plasmids were i s o l a t e d from transformants that formed large halos on casein agar using the a l k a l i n e l y s i s method (Sambrook et a l . , 1989). TFI broth consisted of 2 mL 10X Spizizen's s a l t solution, 0.2 mL 2% casamino acid, 1 mL amino acid solution (1 mg/mL of each of the following amino acids: glycine, alanine, valine, isoleucine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, proline, aspartic acid, glutamic acid, h i s t i d i n e , l y s i n e , arginine, threonine), 2 mL glucose (5%), MgS04.7H20 (0.2%) , and 14.8 mL d i s t i l l e d water. 10 X Spizizen's s a l t solution was prepared by d i s s o l v i n g 20 g (NH4)2S04, 140 g K2HP04, 60 g KH2P04 and 10 g Na3C6H507.2H20 i n 1 L of water. TFII broth consisted of 3.6 mL 10X Spizizen's s a l t solution, 0.18 mL 2% casamino acid, 0.18 mL amino aci d solution, 3.6 mL glucose (5%). MgSO4.7H2O(0.2%), and 28.44 mL d i s t i l l e d water. TE buffer consisted of 10 mM T r i s (pH 8.0) and 1 mM EDTA (pH 8.0). a.2. Confirmation of postive transformants by r e s t r i c t i o n enzyme digestion of plasmid DNA P o s i t i v e transformants carrying pNP22 were confirmed by r e s t r i c t i o n enzyme digestion of plasmid DNA. The f i n a l volume of MATERIALS AND METHODS / 34 each digestion mixture was 25 JUL, which consisted of 15 /xL of the plasmid DNA, 1 /uL of 10 x r e s t r i c t i o n enzyme buffer (supplied with r e s t r i c t i o n enzyme) for each enzyme, 1 /xL of r e s t r i c t i o n enzyme(s) and s t e r i l e water. The mixture was then incubated f o r 2 hours at 37°C without shaking. Digested DNA fragments were separated by electrophoresis through 0.7 to 0.8% agarose gel using Tris-Borate-EDTA (TBE buffer) according to the procedures described by Sambrook et a l . , 1989. 25 /xL of each of the digested DNA sample was then mixed with 5 /zL of the gel loading buffer; 5 /xL of the mixture was subsequently added to each s l o t i n the agarose g e l . The r e s t r i c t i o n map and agarose gel electrophoresis pattern of pNP2 2 recombinant plasmid are presented i n Figure 5a and 5b. 10 x TBE gel loading buffer was prepared by d i s s o l v i n g 54 g T r i s , 27.5 g boric acid and 20 mL 0.5 M EDTA (pH 8.0) i n 1 L of d i s t i l l e d water. The gel loading buffer was made up of 0.25% bromophenol blue, 0.25% xylene cyanol FF and 30 % gl y c e r o l i n water. a.3. Preparation of Crude Proteases from Bacillus subtilis transformants P o s i t i v e transformants carrying pNP 22 plasmids were inoculated into 8 mL of LB broth (containing 50 itg/mL kanamycin and 5 mM CaCl 2) i n a 250 mL Erlenmeyer f l a s k . After 24 hours of incubation at 37°C with vigorous shaking i n a Magniwhirl water bath (shaker s e t t i n g was at 8) , the culture was transferred into 500 mL of fresh LB broth i n a 2 L Erlenmeyer f l a s k . The culture was then reincubated for 24 hours with shaking i n a Psychrotherm Controlled MATERIALS AND METHODS / 35 MATERIALS AND METHODS / 36 23,130 bp 9,416 bp 6,557 bp 4,3 61 bp 2,322 bp 2,027 bp 564 bp 7.1 kb 5.4 kb 4.9 kb 4.4 kb 3.2 kb Figure 5b. Analysis of pNP22 recombinant plasmid with r e s t r i c t i o n endonucleases. Lane 1. Molecular weight marker: \-HindIlI digest. Lane 2. Blank lane. Lane 3 and 5. pNP22 digested with EcoRI and P s t l . Lane 4 and 6. pNP22 digested with EcoRI and Hindlll. MATERIALS AND METHODS / 37 Environment incubator shaker at 160 rpm at 37°C. The 24 hour, 500 mL culture was centrifuged at 2900 x g for 10 minutes at 4°C to remove b a c t e r i a l c e l l s . The r e s u l t i n g supernatant containing secreted proteases was then frozen i n l i q u i d nitrogen and was stored at -16°C for l a t e r use. A l t e r n a t i v e l y , the 500 mL culture was used to inoculate a Lab P i l o t Fermentor CF3 000 (Chemap AG. , Switzerland) containing 10 L of fresh LB broth with 50 itg/mL kanamycin and 5 mM CaCl 2. The culture was incubated at 37°C with s t i r r i n g at 500 rpm. The rate of aeration was at 4 L/min; there was no pH adjustment. A f t e r 24 hours of incubation, the b a c t e r i a l c e l l s were removed by centri f u g a t i o n at 50,000 x g i n a Sharpies tubular bowl centrifuge Model T-1P (Alfa-Laval, Scarborough, ON) . The r e s u l t i n g supernatant was either d i r e c t l y used to catalyze asn-leu synthesis on CovaLink modules, or was concentrated by using a 50 mL Amicon s t i r r e d c e l l (Amicon Corp., Lexington, MA) equipped with a 10,000 mol. weight cut o f f f i l t e r membrane, or a M i l l i p o r e P e l l i c o n Cassette F i l t e r system (Millipore, Bedford, MA) using a membrane with 10,000 molecular weight cut-off. In the case of u l t r a f i l t r a t i o n , the retentate was subsequently d i a f i l t e r e d using the same u l t r a f i l t r a t i o n systems described above with 0.1 M T r i s buffer, pH 7.2 with 5 mM CaCl 2 i n order to remove the growth medium. The concentrated supernatant was then frozen using l i q u i d nitrogen and was stored at -16°C u n t i l further use. MATERIALS AND METHODS / 38 b. Enzymatic solid-phase synthesis of DNP-Asn-Leu dipeptides using either concentrated or unconcentrated crude enzyme f r a c t i o n from Bacillus subtilis (pNP22) transformants as cat a l y s t s L-leucine was immobilized to Nunc Immuno Module as described i n section I.a. DNP-L-asn solution was prepared as outlined i n section I.e. Various volumes of crude unconcentrated or concentrated enzyme mixture, prepared as described i n section II.a, ranging from 1 to 3 0 /iL, were pipetted into designated wells. The f i n a l volume per well was adjusted to 100 ^ L using sodium acetate buffer, pH 6. The plate was incubated with shaking at 110 rpm i n a Psychrotherm Controlled Environment incubator shaker at 48°C for various lengths of time with samples removed at fixe d time i n t e r v a l s . The peptide synthesis was stopped simply by emptying the reactants from the wells. After washing 3 times with 0.15 M phosphate buffer saline (pH 7.2), the presence of immobilized DNP-asn-leu was detected according to procedures described i n section I.b. I I I . CHEMICAL ASSAYS a. Protease Assay Using Casein As A Substrate Protease was assayed using a method described by F u j i et a l . (1983) with the following modifications. The enzyme preparation was d i l u t e d with 50 mM T r i s buffer (pH 7.5), which contained 5 mM calcium chloride. One mL of enzyme was then mixed with 1 mL of 50 mM T r i s buffer (pH 7.5) containing 5 mM calcium chloride and 1% casein (Hammerstein). Calcium chloride was added to prevent autodigestion of the enzyme of interest. The mixture was incubated fo r 37°C for 15 minutes. Two mL of p r e c i p i t a t i n g agent, made up MATERIALS AND METHODS / 39 with 0.1 M t r i c h l o r o a c e t i c acid, 0.22 M sodium acetate and 0.3 3 M ace t i c acid, was added to the mixture. After thorough mixing, the mixture was l e f t at room temperature for 30 minutes followed by centrifu g a t i o n at 2000 x g for 15 minutes. The supernatant was removed using a pasteur pipet. Absorbance of the supernatant at 275 nm was measured with a Shimadzu UV-Vis Recording Spectrophotometer UV-160, Kyoto, Japan. The reagent blank contained: 1 mL 50 mM T r i s buffer with 5 mM calcium chloride, 1 mL 50 mM T r i s buffer with 5 mM calcium chloride and 1% casein (Hammerstein), and 2 mL of p r e c i p i t a t i n g agent. A c a l i b r a t i o n curve was made from a 1 mg / mL stock solution of tyrosine using a range of 1 - 200 itg/mL (Figure 6) . One unit of protease was defined as the quantity required to increase the absorbance at 275 nm by an equivalent of 1 fig of tyrosine per minute at 37°C at pH 7.5. b. Protease Assay Using Azocasein as a Substrate The azocasein protease assay was modified from a procedure described by Ewings et a l . (1984). Duplicates of reaction mixture were prepared by mixing the following reagents together: . 1.25 mL of 1.5% azocasein i n 0.1 M Tris-HCl buffer (pH 7.5) with 5 mM calcium chloride; azocasein was prepared by b o i l i n g i n the above T r i s buffer for 5 minutes followed by f i l t r a t i o n through Whatman #1 f i l t e r paper . 1.25 mL 0.1 M Tris-HCl buffer (pH 7.5) with 5 mM calcium chloride . 0.25 mL of dil u t e d enzyme MATERIALS AND METHODS / 40 1.5 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 r 0 50 100 150 200 Tyrosine Content (/xg/mL) Figure 6. Standard curve for protease assay with Hammerstein casein used as a substrate. MATERIALS AND METHODS / 41 Substrate blanks and sample blanks were used as controls. Substrate blank was prepared the same way as the reaction mixture except that 0.25 mL of dil u t e d enzyme was replaced by 0.25 mL of Tris-HCl buffer. Sample blank was prepared the same way as the reaction mixture except that 2.5 mL 24% t r i c h l o r o a c e t i c acid (TCA) was added before the addition of each d i l u t e d enzyme solut i o n at time 0. A l l reaction mixtures were incubated at 48°C for 2 hours with shaking. Reaction was stopped by adding 2.5 mL of 24% TCA to each t e s t tube. The samples were vortexed immediately and were placed into an i c e bath for 15 minutes followed by centri f u g a t i o n for 15 minutes at 3 7 00 x g. The absorbance of the clear supernatant was measured at 366 nm using a Shimadzu UV-Vis Spectrophotometer. One unit of protease a c t i v i t y = the amount of enzyme required to produce an increase i n absorbance at 366 nm of 0.01 per hour. = (Reaction Mixture t 2 h r - sample blank t 0 h r - substrate blank t 2 h r) / 0.01 c. Protein Assay Using BCA Protein Assay Reagent A l l reagents used were supplied i n the BCA Protein Assay t e s t k i t (Pierce Chemical Company, Rockford, IL) . The samples and reagent blanks were pipetted into the wells of m i c r o t i t e r plates fo r convenience. Fifteen yih of enzyme or protein preparation was added to each microtiter well containing 250 yih of BCA protein assay working reagent. The samples were l e f t at room temperature for 2 hours i n order for colour development to occur. At the end MATERIALS AND METHODS / 42 of the incubation period, the o p t i c a l density of the samples was measured at 620 nm using the Easy Reader EAR 400 plate reader. A c a l i b r a t i o n curve was prepared from a 1 mg/mL thermolysin stock s o l u t i o n using a range of 0 - 1000 /zg/mL (Figure 7) . d. SDS-Gel Electrophoresis of Thermolysin and Secreted Crude Proteases from Transformed B. subtilis DB428 and WB600 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a PhastSystem™ using PhastGel Gradient 8-25 and PhastGel SDS buffer s t r i p s (Pharmacia, Uppsala, Sweden). The buffer system i n the gels was 0.112 M acetate and 0.112 M T r i s pH 6.4. The buffer system i n PhastGel SDS buffer s t r i p s was 0.20 M t r i c i n e , 0.20 M T r i s and 0.55% SDS, pH 7.5. Thermolysin sample was prepared as follows: 100 /zL of thermolysin, which was prepared by dis s o l v i n g 1 mg of enzyme i n 1 mL of 0.01 M phosphate buffer, pH 7.2, 100 /zL 0.01 M phosphate buffer, pH 7.2, 50 juL 10% SDS and 12 nL 0.05% bromophenol blue were mixed together. Samples of secreted crude proteases were prepared by combining 150 /zL of supernatants containing crude proteases, 50 /zL 0.01 M phosphate buffer, pH 7.2, 50 jtzL 10% SDS and 12 JLIL 0.05% bromophenol blue. A l l samples were heated f o r 5 minutes at 95°C i n a hot water bath. 5 /zL of each of the prepared samples was applied to the gel . When separation was completed, the gel was s i l v e r stained according to PhastSystem Development Technique F i l e NO.210 (Pharmacia). MATERIALS AND METHODS / 43 Figure 7. Standard curve for BCA protein assay (Pierce Chemical Company, Rockford, IL). MATERIALS AND METHODS / 44 IV. Methods for S t a t i s t i c a l Analysis Systat Version 5.01 (Systat Inc., Evanston, IL) was the s t a t i s t i c a l package used for data analysis . The MGLH Module was used to compute 2-way analysis of variance (ANOVA). The e f f e c t s of treatment and time were tested. A t o t a l of 3 or 4 r e p l i c a t e s were measured for each treatment and time. Linear regression analysis was performed to determine the best s t r a i g h t l i n e s for the standard curves i n the protease a c t i v i t y assay and the protein assay. The computation was c a r r i e d out using Systat Version 5.01 using the Smooth=Linear Module. R E S U L T S A N D DISCUSSIONS I. IMMOBILIZATION OF DNP-L-LEUCINE TO THE SOLID PHASE a. Detection of Immobilized Leucine Molecules Dinitrophenyl (DNP) la b e l l e d L-leucine molecules were used i n order to determine i f L-leucine molecules could be covalently coupled to the microtiter plate. Any immobilised leucine was detected by reacting with anti-DNP antibody, which was conjugated to horseradish peroxidase as described i n section I.b of Materials and Methods. When the chromogenic orthophenylenediamine (OPD) was added to the reaction mixture along with the anti-DNP antibody horseradish peroxidase substrate (hydrogen peroxide), a soluble yellow to orange product was formed i n the wells. The i n t e n s i t y of t h i s colour, which was measured at 492 nm, would be expected to be d i r e c t l y proportional to the amount of leucine covalently coupled to the mi c r o t i t e r plate. DNP-L-leucine solutions with d i f f e r e n t concentrations were prepared and were immobilized to the s o l i d phase with Covalink-NH 2 groups as outlined i n the Materials and Methods section. DNP-L-leucine was dissolved either i n d i s t i l l e d water or i n a mixture of dimethylsulf oxide and water. The experimental data indicated that maximum absorbance at 492 nm occurred when the concentration of DNP-L-leucine i n the m i c r o t i t e r well was at 25 itg/100 itL (Table 1, Figure 8) ; t h i s was equivalent to 11.04 fig L-leucine/100 tiL. In addition, absorbance was higher for DNP-L-leucine previously dissolved i n dimethylsulfoxide-d i s t i l l e d water mixture. Such difference could be explained by the 45 RESULTS AND DISCUSSIONS / 46 Table 1. The optimal concentration of DNP-L-leucine f o r coupling on s o l i d phase Cone, of DNP-leucine (/jg/100 Lit.) O p t i c a l density at 492 nm for DNP-L-leucine dissolved i n water* Op t i c a l density at 492 nm for DNP-L-leucine dissolved i n dimethyl-sulfoxide* O p t i c a l density at 492 nm for DNP-L-leuc ine dissolved i n dimethyl-sulfoxide+ Optical* density at 492 nm for non-ac t i v a t e d DNP-L-leucine i n dimethyl-sulfoxide® 0.0 0.106 ± 0.014 0.103 ± 0.004 0.033 ± 0. 003 0.026 ± 0.001 5.0 0.154 ± 0. 029 0.500 ± 0. 024 0.037 ± 0. 003 0.040 ± 0. 003 7.5 0.146 ± 0.021 0.589 ± 0.020 0.041 ± 0. 005 0.031 ± 0. 001 12.5 0.161 ± 0.016 0.531 ± 0.037 0.039 ± 0. 002 0.036 ± 0. 003 25.0 0.842 ± 0.066 1.216 ± 0.142 0.045 ± 0. 003 0.039 ± 0. 003 50. 0 0.872 ± 0.054 0.986 ± 0.056 0.048 ± 0.006 0.037 ± 0. 004 75.0 0.826 ± 0.062 0.902 ± 0.042 0.041 ± 0.005 0.042 ± 0.001 * Mean ± standard deviation of three duplicates. DNP(dinitrophenyl)-L-leucine was activated by EDC and NHS. Nunc Immuno Module was used as the s o l i d phase. + Mean ± standard deviation of two duplicates.* DNP-L-leucine was activated by EDC and NHS. Immunlon 2 polystyrene microtiter immunoassay plates without l i n k e r arms was used as the s o l i d phase. ® Mean ± standard deviation of three duplicates. DNP-L-leucine was not activated by EDC and NHS. Nunc Immuno Module was used as the s o l i d phase. RESULTS AND DISCUSSIONS / 47 E c CM CD Q b 1.50 1.20 0.90 0.60 0.30 h 0.00 0 5 7.5 12.5 25 50 75 Concentration of DNP-L-Leucine(ug/1 OOuL) Figure 8. Immobilization of DNP-L-leucine to Nunc Immuno Module. m DNP-L-leucine was dissolved i n DMSO/water and was activated by EDC and NHS. DNP-L-leucine was dissolved i n water and was activated by EDC and NHS. DNP-L-leucine was dissolved i n DMSO/water and was activated by EDC and NHS. Immunlon 2 polystyrene microtiter plate instead of Nunc Immuno Module was used. DNP-L-leucine was dissolved i n DMSO/water but was not activated by EDC and NHS. RESULTS AND DISCUSSIONS / 48 fa c t that DNP-L-leucine was more soluble i n the DMSO-water solvent than i n water alone. Since the maximum binding of DNP-L-leucine molecules occurred at 25 ug/100 /xL, which was equivalent to 11.04 itg L-leucine/100 ttL, as r e f l e c t e d by high absorbance readings, the concentration of L-leucine was kept at about 10 fig per well for a l l other experiments. b. Binding of DNP-L-leucine to Conventional M i c r o t i t e r Plate To determine whether DNP-L-leucine would bind to ordinary m i c r o t i t e r plates without any secondary amino groups, an attempt was made to attach DNP-L-leucine to Immunlon 2 polystyrene m i c r o t i t e r immunoassay plates (Dynatech Laboratories Inc., Ch a n t i l l y , VA) following the method described i n section I.a. of Materials and Methods. Figure 8 shows that only a very small amount of DNP-L-leucine would bind to a mi c r o t i t e r p l a t e without any l i n k e r arms; t h i s could be caused by the fa c t that smaller molecules do not adsorb very well to the polystyrene surface. Therefore, t h i s type of s o l i d phase was not as e f f e c t i v e i n binding small molecules as the Nunc Immuno Module. c. Noncovalent Binding of DNP-L-leucine To t e s t whether leucine molecules could bind noncovalently to the Immuno Module, d i s t i l l e d water instead of EDC-NHS was added to DNP-L-leucine mixture during the ac t i v a t i o n step. Figure 8 showed that only a very low l e v e l of noncovalent binding occurred; t h i s binding was probably due to passive adsorption of DNP-L-leucine to RESULTS AND DISCUSSIONS / 49 the l i n k e r arms. But since o p t i c a l density s i g n a l detected for EDC-NHS activated DNP-L-leucine molecules was much stronger than that detected for the unactivated molecules, i t was evident that covalent attachment of DNP-L-leucine molecules had occurred on the m i c r o t i t e r plate. ) d. Quantification of the Amount of DNP-L-leucine Immobilized to the M i c r o t i t e r Plate I t was unfortunate that the actual amount of DNP-L-leucine immobilized i n microtiter wells could not be quantified as planned. I n i t i a l l y , i t was assumed that the amount of leucine bound to the plate could be deduced from a c a l i b r a t i o n curve prepared from DNP-L-leucine solutions with a range from 0 - 100 itg/mL. In one experiment, DNP-leucine solution at various concentrations was pipetted into a normal microtiter plate so that the wells did not have any l i n k e r arms. When Ab-peroxidase and OPD substrate were subsequently added to the wells, i t was anticipated that the i n t e n s i t y of colour produced i n each well would be proportional to the amount of DNP-L-leucine present i n that well; hence, the higher the concentration, the higher the i n t e n s i t y of colour. Unfortunately, a l l of the wells ended up having the same colour i n t e n s i t y regardless of what concentration of DNP-L-leucine was present. This was because the unreacted ab-peroxidase conjugate could not be removed from the reaction mixture, and i t continued to react with the OPD substrate to produce a very intense red colour. The end r e s u l t was that every well i n the m i c r o t i t e r plate had the same reading, which were a l l off scale readings because of the RESULTS AND DISCUSSIONS / 50 intense colour, regardless of how much DNP-leucine was present i n the m i c r o t i t e r well. Although the actual amount of immobilized L-leucine could not be determined, the change i n o p t i c a l density at 492 nm was s t i l l i n d i c a t i v e of the r e l a t i v e amounts of leucine that could be coupled to the plate. Thus, the higher the o p t i c a l density reading, the greater was the amount of immobilized L-leucine. I I . THERMOLYSIN CATALYSED SOLID PHASE SYNTHESIS OF DNP-ASN-LEU For s o l i d phase dipeptide synthesis, L-leucine was the immobilized nucleophile while dinitrophenyl-L-asparagine (DNP-L-asn) was the free acyl donor. L-asparagine molecules that had been coupled with dinitrophenyl groups were used so that any dipeptide formed on the s o l i d phase could be detected using AB-peroxidase conjugate and OPD. There was another reason for using dinitrophenyl l a b e l l e d L-asparagine: l a b e l l i n g of the amino group on the L-asparagine molecule with dinitrophenyl e f f e c t i v e l y prevented formation of homopolymers of asparagine. a. Selection of Blocking Agent In order to minimize non-specific binding of Ab-peroxidase conjugate to the s o l i d phase, unreacted s i t e s l e f t on the m i c r o t i t e r plate were blocked with a blocking agent. To t e s t the effectiveness of various blocking agents on Immuno Modules, leucine molecules (without dinitrophenyl groups) were immobilized to the s o l i d phase. Unreacted li n k e r arms were blocked with blocking RESULTS AND DISCUSSIONS / 51 agents. Antibody-perioxidase conjugate was then added to the m i c r o t i t e r wells. Theoretically, the antibody-peroxidase conjugate should not bind to the plate because there was no dinitrophenyl group for the antibody to react with. Therefore, detection of absorbance at 492 nm would be caused by non-specific binding of the conjugate to the plate. The i n t e n s i t y of absorbance would be i n d i c a t i v e of the effectiveness of the blocking agent used. Various blocking agents were tested, and 0.5% skim milk powder solution was found to be the most e f f e c t i v e (Table 2) . Consequently, 0.5% skim milk powder was used as a blocking agent i n a l l of the dipeptide synthesis experiments. An ad d i t i o n a l 0.05% (w/v) skim milk powder was also added to the antibody peroxidase complex to keep non-specific binding to a minimum. b. Detection of the Formation of DNP-Asn-Leu I n i t i a l experiments were focused on synthesis reactions catalyzed by commercial grade thermolysin. Some of the experimental data are shown i n Figures 9, 10, 11 a to d, 12 and 13 with the reaction conditions of these experiments summarized i n Table 3. The data c l e a r l y showed that there was an increase i n o p t i c a l density readings at 492 nm i n those m i c r o t i t e r wells containing thermolysin when the plates were incubated f o r varying lengths of time. This suggested that some peptide synthesis a c t i v i t y had occurred i n the microtiter wells. Unfortunately, i t was not possible to quantify the amount of DNP-asn-leu formed i n the m i c r o t i t e r wells for the same reason provided e a r l i e r i n RESULTS AND DISCUSSIONS / 52 Table 2. The e f f i c i e n c y of various blocking Agents Type of blocking agent + Absorbance 4 9 2 n m 2% bovine serum albumin 0.125 ± 0.008 2% g e l a t i n 1.714 ± 0.150 0.5% skim milk powder 0.018 ± 0.001 + a l l blocking agents were dissolved i n 0.01 M phosphate buffer, pH 7.2. * Values are the mean ± standard deviation of 6 readings. RESULTS AND DISCUSSIONS / 53 Table 3. Reaction conditions 1 for thermolysin-catalysed solid-phase synthesis of DNP-Asn-Leu Thermo- Protease 2 DNP-Asn l y s i n A c t i v i t y Time of per well per well per well Incubation PH Temp. Fig . (nmol) (Mg) (units) (Hr.) (°C) 9 7.56 50.2 38.8 6 6.5 48 10 7.56 50.2 38.8 6 6.0 48 11a 7.56 50.2 38.8 6 6.0 48 l i b 37.84 50.2 38.8 6 6.0 48 l i e 75.70 50.2 38.8 6 6.0 48 l i d 151.40 50.2 38.8 6 6.0 48 12 7.56 10.0 7.8 2 6.0 48 13 7.56 10.0 7.8 2 6.0 48 76.3 nmol of L-leucine was used i n a l l of the experiments. 2 One unit of protease a c t i v i t y was defined as the quantity required to increase the absorbance of 1 /zg of tyrosine per min at 37°C. RESULTS AND DISCUSSIONS / 54 Figure 9. Exp. 1. Thermolysin catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-Teucine, 7.56 nmol DNP-L-asparagine at pH 6.5 and 50.2 /xg thermolysin with 38.8 units of protease a c t i v i t y * were added to the wells; thermolysin was not added to the control samples. Protease a c t i v i t y was defined as one unit of protease required to increase the absorbance of 1 jug of tyrosine/min at 37°C. RESULTS AND DISCUSSIONS / 55 E c CM O Q 6 0.15 0.12 h 0.09 r 0.06 0.03 W-0.00 • — thermolysin — O— control 0 1 2 3 4 5 6 Time (Hour) Figure 10. Exp. 2. Thermolysin catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6.0 and 50.2 /xg thermolysin with 38.8 units of protease a c t i v i t y * were added to the wells; thermolysin was not added to the control samples. * Protease a c t i v i t y was defined as one unit of protease required to increase the absorbance of 1 jxg of tyrosine/min at 37°C. RESULTS AND DISCUSSIONS / 56 Figure 11a. Exp. 3a. Thermolysin catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6.0 and 50.2 /zg thermolysin with 38.8 units of protease a c t i v i t y * were added to the wells; thermolysin was not added to the control samples. Protease a c t i v i t y was defined as one unit of protease required to increase the absorbance of 1 /zg of tyrosine/min at 37°C. RESULTS AND DISCUSSIONS / 57 Figure l i b . Exp. 3b. Thermolysin catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6.0 and 50.2 ixg thermolysin with 38.8 units of protease a c t i v i t y * were added to the wells; thermolysin was not added to the control samples. Protease a c t i v i t y was defined as one unit of protease required to increase the absorbance of 1 /xg of tyrosine/min at 37°C. RESULTS AND DISCUSSIONS / 58 Figure 11c. Exp. 3c. Thermolysin catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6.0 and 50.2 /xg thermolysin with 38.8 units of protease a c t i v i t y * were added to the wells; thermolysin was not added to the control samples. Protease a c t i v i t y was defined as one unit of protease required to increase the absorbance of 1 jug of tyrosine/min at 37°C. RESULTS AND DISCUSSIONS / 59 Figure l i d . Exp. 3d. Thermolysin catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6.0 and 50.2 /zg thermolysin with 38.8 units of protease a c t i v i t y * were added to the wells; thermolysin was not added to the control samples. Protease a c t i v i t y was defined as one unit of protease required to increase the absorbance of 1 jizg of tyrosine/min at 37°C. RESULTS AND DISCUSSIONS / 60 Figure 12. Exp. 4. Thermolysin catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6.0 and 10 /ig thermolysin with 7.8 units of protease a c t i v i t y * were added to the wells; thermolysin was not added to the control samples. Protease a c t i v i t y was defined as one unit of protease required to increase the absorbance of 1 u-g of tyrosine/min at 37°C. RESULTS AND DISCUSSIONS / 61 Figure 13. Exp. 5. Thermolysin catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6.0 and 10 nq thermolysin with 7.8 units of protease a c t i v i t y * were added to the wells; thermolysin was not added to the control samples. Protease a c t i v i t y was defined as one unit of protease required to increase the absorbance of 1 /Ltg of tyrosine/min at 37°C. RESULTS AND DISCUSSIONS / 62 section I.d of Results and Discussions. Nevertheless, the increase i n o p t i c a l density was ind i c a t i v e of the r e l a t i v e amount of dipetide formed on the microtiter plate; i n other words, the greater the increase, the greater was the y i e l d of DNP-asn-leu. On the other hand, the control samples with the wells containing only buffer solution and DNP-L-asparagine had r e l a t i v e l y constant o p t i c a l density readings i n most cases. The background readings observed i n these wells was probably caused by e i t h e r non-specific binding of DNP-L-asparagine, or Ab-peroxidase conjugate to the m i c r o t i t e r plates. c. E f f e c t of the Concentration of DNP-L-asn on Dipeptide Formation Figures 11 a to d show the e f f e c t of varying the concentration of DNP-L-asparagine i n the reaction mixture. The use of higher concentrations of DNP-L-asparagine did not seem to have any b e n e f i c i a l e f f e c t on dipeptide formation because there was no notable increase i n the o p t i c a l density readings i n the t e s t samples: the mean O.D.492 n m readings af t e r 4 hours of incubation were 0.106, 0.098, 0.093 and 0.094 for the t e s t groups containing 7.57 nmol, 37.84 nmol, 75.7 nmol and 151.4 nmol of DNP-L-asparagine respectively. In contrast, the use of higher concentration of DNP-L-asparagine resulted i n an increase of non-specific binding of DNP-L-asparagine to the micro- t i t e r plate as evidenced by the increased o p t i c a l density readings over time i n the control group with no thermolysin. Therefore, the amount of DNP-L-asparagine RESULTS AND DISCUSSIONS / 63 added to each well was kept at 7.56 nmol for a l l other experiments. d. The E f f e c t of Reaction Time on Dipeptide Synthesis Although Miranda and Tominaga (1991) reported that optimum reaction time for asn-leu synthesis reaction was 24 hours, experimental data with present study did not support the f a c t that longer reaction time was needed for higher y i e l d except i n experiment 3 (Figures 11a to l i d ) . In fact, when the m i c r o t i t e r plates were incubated for more than 8 hours at 48°C, evaporation of the reaction mixture actually occurred to varying degrees i n the wells i n s p i t e that each plate was already covered with a p l a s t i c l i d . Figures 9 and 10 show that o p t i c a l density measurements remained constant from 2 to 6 hours of incubation; the same observation was made i n three other s i m i l a r experiments (data not presented). Thus, most of the reaction happened within the f i r s t 2 hours. Experiment 3 (Figures 11 a to d) was the only experiment i n which the o p t i c a l density readings continued to increase over time; the reason for t h i s was not clear. Since longer reaction time did not improve the y i e l d of dipeptides, the reaction time for subsequent experiments was reduced to 2 hours. As shown i n Figures 12 and 13, the synthesis reaction proceeded r a p i d l y within the f i r s t 40 to 60 minutes, and then started to either l e v e l o f f or decline. A drop i n o p t i c a l density readings could be a t t r i b u t e d to concurrent enzymatic hydrolysis of already formed dipeptides. RESULTS AND DISCUSSIONS / 64 e. E f f e c t of Thermolysin Concentration on Dipeptide Synthesis Dipeptide formation was not adversely affected when thermolysin concentration was reduced 5 f o l d from 50.2 izg to 10 /zg per well (Figures 9 to 13) . When the o p t i c a l density readings recorded at the end of 2 hours of reaction period were compared, i the readings measured from the wells containing the lower l e v e l s of thermolysin were actually s l i g h t l y higher than those measured from m i c r o t i t e r wells containing 5 times more thermolysin: the mean o p t i c a l density measurements (after 2 hours of incubation) for figures 12 and 13 was 0.12 while the average o p t i c a l density measurements for figures 9, 10 and 11a was 0.09. I t was possible that the presence of excessive amount of enzyme a c t u a l l y promoted more hydrolysis of the formed dipeptides, which i n turn resulted i n s l i g h t l y lower o p t i c a l density measurements. f. S t a t i s t i c a l Analysis S t a t i s t i c a l analysis was performed on the data for experiments 1 to 5 i n order to determine whether or not there was a difference i n the mean o p t i c a l density measurements at 492 nm between the t e s t group with thermolysin and the control group without any enzyme. The s t a t i s t i c a l analysis chosen was a two-factor analysis of variance (or 2-way ANOVA) . The e f f e c t of time and treatments ( i . e . with and without thermolysin), and t h e i r interactions on the mean o p t i c a l density readings measured at 492 nm were tested and the RESULTS AND DISCUSSIONS / 65 r e s u l t s are summarized i n Table 4. The calculated F r a t i o s revealed that there was s i g n i f i c a n t difference (P < 0.01) i n O.D.492nm due to the d i f f e r e n t treatments i n a l l of the experiments. This c l e a r l y indicated that the mean o p t i c a l density measurements were not the same for the control groups and the t e s t groups (with enzyme treatment). Since the change i n o p t i c a l density readings at 492 nm i n the wells was related to the r e l a t i v e amount of dipeptides formed, one can therefore conclude that the e f f e c t of treatment was s i g n i f i c a n t on the amounts of DNP-asn-leu formed i n the m i c r o t i t e r wells. Hence, the experimental data and subsequent s t a t i s t i c a l analyses proved that i t was possible to use the proposed rapid screening method to detect formation DNP-asn-leu dipeptides on a solid-phase when commercial grade thermolysin was used as a c a t a l y s t . ( I I I . DNP-ASN-LEU REACTIONS CATALYSED BY CRUDE ENZYME FRACTION SECRETED BY BACTERIAL CULTURE The rapid solid-phase screening assay was developed with the intention to detect mutant enzymes with enhanced reverse p r o t e o l y s i s property. These enzymes would be produced by b a c t e r i a l c e l l s harbouring a mutagenized nprT gene. The nprT gene from Bacillus stearothermophilus encodes for a thermolysin-like neutral protease. Since any secreted enzymes from b a c t e r i a l cultures would e x i s t i n a less concentrated form than commercial grade thermolysin, i t was therefore important to determine whether such crude secreted enzyme fractions with lower enzymatic a c t i v i t y were RESULTS AND DISCUSSIONS / 66 Table 4. 2-Way analysis of variance on o p t i c a l density readings for f i v e thermolysin catalysed Leu-Asn synthesis experiments Fig. Exp. Source of Variation DF F r a t i o 9" 1 Time 3 104.4** Treatment* 1 660.7** Time x Treatment 3 73.66** Error 24 10° 2 Time 3 36.02** Treatment2 1 308.4** Time x Treatment 3 17 . 42** Error 16 l l a b 3 a Time 3 144.9** Treatment* 1 986.9** Time x Treatment 3 123.9** Error 24 l i b " 3 b Time 3 53 . 90** Treatment* 1 154.1** Time x Treatment 3 22.42** Error 24 l i e " 3 c Time 3 28.06** Treatment* 1 52.44** Time x Treatment 3 6 . 270 Error 24 . l i d " 3 d Time 3 102.0** Treatment* 1 146.4** Time x Treatment 3 22.45** Error 24 12b 4 Time 12 55.49** Treatment* 1 3125** Time x Treatment 12 44 . 52** Error 78 13" 5 Time 12 64 .71** Treatment* 1 2538** Time x Treatment 12 54.35** Error 78 a Treatment 1 = Control sample (DNP-asparagine + bu f f e r ) . Treatment 2 = Test sample (DNP-asparagine + buffer + thermolysin). b 4 wells per time-treatment combination. 0 3 wells per time-treatment combination. ** S i g n i f i c a n t at P<0.01. RESULTS AND DISCUSSIONS / 67 capable of promoting dipeptide formation on a s o l i d phase. To te s t t h i s hypothesis, crude enzyme fractions were i s o l a t e d from Bacillus subtilis DB428 and Bacillus subtilis WB600, which had been transformed with the npr T gene. a. Use of Bacillus subtilis DB428 and WB600 to Produce Enzyme Mixture The mutant stra i n s of Bacillus subtilis DB428 and WB600 were chosen as the host organisms for 2 reasons. F i r s t , Bacillus subtilis organisms produce enzyme e x t r a c e l l u l a r l y , so secreted enzyme(s) could be ea s i l y recovered from the growth medium. Second, DB428 and WB600 are protease d e f i c i e n t s t r a i n s which produce l i t t l e background enzymatic a c t i v i t y . Hence, any enzyme released into the growth medium by t h i s transformed organism would l i k e l y be the products encoded by the npr T gene. The background e x t r a c e l l u l a r protease a c t i v i t y of the wild type Bacillus subtilis ( s t r a i n 168) as well as those of the protease d e f i c i e n t DB428 and WB600 are summarized i n Table 5. Transformed Bacillus subtilis DB428(pNP22) and WB600(pNP22) were prepared as outlined i n Materials and Methods (section I I . a . l ) . The col l e c t e d supernatants with enzymatic a c t i v i t y were d i r e c t l y used to catalyse dipeptide synthesis or were subsequently concentrated by u l t r a f i l t r a t i o n and the retentate was d i a f i l t e r e d to remove low molecular weight impurities from the growth medium. Washed retentate was used to catalyse DNP-asp-leu dipeptide synthesis on the Immuno Modules. Figure 14 shows the SDS-gel electrophoresis patterns of commercial grade thermolysin as well as RESULTS AND DISCUSSIONS / 68 Table 5. E x t r a c e l l u l a r protease a c t i v i t y from wild-type (168) and protease-deficient (DB428 and WB600) s t r a i n s of Bacillus subtilis Protease A c t i v i t y 1 at 24 hr. Str a i n (unit/0.25 mL supernatant) 2 % Protease A c t i v i t y 168 DB428 WB600 772 17 11 100. 0 2 . 2 1.4 Data was the average values of the supernatant of two separate cultures, which had been incubated for 24 hours at 37°C. 1 unit of a c t i v i t y on azocasein = change i n absorbance at 366 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 69 66,200 45,000 35,000 31,000 21,000 14,400 Figure 14. SDS-gel electrophoresis of thermolysin and crude proteases secreted by transformed B. subtilis DB428 and WB600. The gel was s i l v e r stained. Lane 1. Molecular weight marker Lane 2. Commercial grade thermolysin Lane 3. B. subtilis WB600(pNP22) Lane 4. B. subtilis WB600 Lane 5. B. subtilis DB428(pNP22) Lane 6. B. subtilis DB428 RESULTS AND DISCUSSIONS / 70 of the crude proteases secreted by the transformed B. subtilis DB428 and WB600. Table 6 summarizes the re s u l t s of some of the u l t r a f i l t r a t i o n experiments which involved supernatants recovered from transformed B. subtilis DB428 cultures. The higher u l t r a f i l t r a t i o n factors obtained when M i l l i p o r e P e l l i c o n cassette f i l t e r system was used demonstrated that t h i s system was a more e f f i c i e n t concentration method that Amicon s t i r r e d c e l l system. This could be rela t e d to the f a c t that only one hour of f i l t r a t i o n was required when P e l l i c o n cassette was used. On the other hand, 4 hours of f i l t r a t i o n was needed when Amicon s t i r r e d c e l l was used. Therefore, i t was possible that autodigestion of the secreted proteases was more extensive when longer f i l t r a t i o n time was needed. b. E f f e c t of Varying the Concentration of Enzyme i n the Reaction Mixture The experimental data for crude enzyme catalysed synthesis reactions have been summarized i n Figure 15 to Figure 19 while the reaction conditions for these experiments are recorded i n Table 7. A d i f f e r e n t protease assay, which u t i l i z e d azocasein as a substrate, was used to determine the protease a c t i v i t y of both thermolysin and crude enzyme preparations because d i f f i c u l t y was experienced i n dis s o l v i n g Hammerstein casein i n the previous assay method. The re s u l t s obtained from determining the protease a c t i v i t y of thermolysin using both Hammerstein casein and azocasein as substrates are summarized i n Table 8. RESULTS AND DISCUSSIONS / 71 Table 6. Results of u l t r a f i l t r a t i o n of crude enzyme secreted by transformed Bacillus subtilis DB428 Protease Protease Protease Vo l . of Vol. of A c t i v i t y * A c t i v i t y * A c t i v i t y * Supernatant Retentate (U/0.25mL) (U/0.25mL) (U/0.25mL) U.F.1 U.F.2 of of of Factor Factor Supernatant Retentate F i l t r a t e 3 30 mL 4.5 mL 725 ' 3270 0 4.5 6.7 4 8.7 L 0.87 L 583 5960 0 10.2 10.0 * 1 unit (U) of protease a c t i v i t y = change i n absorbance at 366 nm of 0.01/2 hours (units of a c t i v i t y on azocasein). protease a c t i v i t y per 0.25 mL retentate 1 U l t r a f i l t r a t i o n (U.F.) factor = protease a c t i v i t y per 0.2 5 mL supernatant volume of retentate 2 U l t r a f i l t r a t i o n (U.F.) factor = volume of supernatant 3 Supernatant was concentrated by Amicon s t i r r e d c e l l . 4 Supernatant was concentrated by M i i l i p o r e P e l l i c o n cassette f i l t e r . RESULTS AND DISCUSSIONS / 72 Table 7. Reaction conditions 1 for thermolysin and crude enzyme catalysed s o l i d phase synthesis of DNP-Asn-Leu Fi g . Exp. DNP-Asn per well (nmol) Thermo-l y s i n per well (Mg) Crude 2 enzyme per well (ug) Protease A c t i v i t y per w e l l 3 (units) Peptide Synthesis Ratio 4 at 1 Hour of Incubation 15 7.56 50.2 5.89 7.90 3105 32 44 16a 7a 7.56 6.523 13. 045 19. 565 42 84 126 16b 7b. 16c 7c. 16d 7d. 37.84 75.70 151.40 19. 565 19.56s 19. 565 126 126 126 17 8 7.56 10.0 17. 366 621 477 3.7 x 10" 1.5 X 10J 18 7.56 10.0 17 . 366 621 477, 1.1 X IO"4 1.4 X 10"* 19 10 7.56 10.0 5.27e 26.046 621 80 715 3.4 X 10": 1.4 X 10J 7.9 x 10J 1 76.3 nmol of L-leucine was used i n a l l of the experiments. A l l dipeptide synthesis reactions were carr i e d out at pH 6. Incubation time for experiments 6 and 7 was 6 hours. Incubation time f o r experiments 8 to 10 was 2 hours. 2 Protein content was determined by BCA method. 3 1 u n i t of a c t i v i t y on azocasein = change i n absorbance at 366 nm of 0.01 during 2 hrs. at 48°C O.D. 4 9 2 n m of - O.D. 4 9 2 m n of t e s t sample control 4 Peptide synthesis r a t i o = • Units of protease a c t i v i t y per well 5 Supernatant was concentrated by Amicon s t i r r e d c e l l . RESULTS AND DISCUSSIONS / 73 Table 8. Summary of the protease a c t i v i t y of thermolysin determined by u t i l i z i n g two d i f f e r e n t substrates: Hammerstein casein and azocasein Units of Units of Amount of Protease A c t i v i t y Protease A c t i v i t y Thermolysin by Assay #1" by Assay # 2b (nmol) 0.29 7.8 ' 621 1.45 38.8 3105 a Hammerstein casein was used i n assay # 1. b Azocasein was used i n assay # 2. RESULTS AND DISCUSSIONS / 74 CM O) a 6 0.15 0.12 0.09 0.06 0.03 0.00 — • — thermolysin - O - DB428 •V — control 1 Time (Hour) Figure 15. Exp. 6. Thermolysin and crude enzyme catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6 were added to the micr o t i t e r wells. The following enzymes were added to designated wells: a. 50.2 /ug thermolysin with 3105 units of protease a c t i v i t y * b. 5.89 jug proteases secreted by B. subtilis DB428 with 32 units of protease a c t i v i t y * c. 7.89 /xg proteases secreted by B. subtilis WB600 with 44 units of protease a c t i v i t y * No enzyme was added to the control samples. 1 unit of protease a c t i v i t y on azocasein = change i n absorbance at 3 66 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 75 Figure 16a. Exp. 7a. Crude enzyme catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6 and the following amount of crude proteases, secreted by transformed B. subtilis DB428, was added to designated wells: a. 6.52 jug with 42 units of protease a c t i v i t y * b. 13.04 jug with 84 units of protease a c t i v i t y * c. 19.56 /ug with 126 units of protease a c t i v i t y * No enzyme was added to the control samples. 1 unit of protease a c t i v i t y on azocasein = change i n absorbance at 366 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 76 E c CM O) ;» Q O 0.15 0.12 0.09 r 0.06 0.03 0.00 crude enzyme — O— control 2 4 Time (Hour) Figure 16b. Exp. 7b. Crude enzyme catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 37.84 nmol DNP-L-asparagine at pH 6 and 6.52 jug of crude proteases secreted by transformed B. subtilis DB428, with 126 units of protease ac t i v i t y * , was added to designated wells; enzyme was not added to the control samples. 1 unit of protease a c t i v i t y on azocasein = change i n absorbance at 366 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 77 OJ o »3-Q 6 0.15 0.12 0.09 0.06 0.03 0.00 — crude enzyme — O— control 2 4 Time (Hour) Figure 16c. Exp. 7c. Crude enzyme catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 75.7 nmol DNP-L-asparagine at pH 6 and 6.52 ug of crude proteases secreted by transformed B. subtilis DB428, with 126 units of protease ac t i v i t y * , was added to designated wells; enzyme was not added to the control samples. 1 unit of protease a c t i v i t y on azocasein = change i n absorbance at 366 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 78 Figure 16d. Exp. 7d. Crude enzyme catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 151.4 nmol DNP-L-asparagine at pH 6 and 6.52 /zg of crude proteases secreted by transformed B. subtilis DB428, with 126 units of protease ac t i v i t y * , was added to designated wells; enzyme was not added to the control samples. 1 unit of protease a c t i v i t y on azocasein = change i n absorbance at 366 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 79 0.20 0.04 h o.oo ' — • — 1 — • — 1 — • — 1 — • — 1 — • — 1 — • — 1 0 20 40 60 80 100 120 Time (min) Figure 17. Exp. 8. Thermolysin and crude enzyme catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6 and either 10 ug thermolysin with 621 units of protease a c t i v i t y * or 17.36 ug crude proteases secreted by transformed B. subtilis DB428, with 477 units of protease activity*, was added to designated wells; enzyme was not added to the control samples. 1 unit of protease a c t i v i t y on azocasein = change i n absorbance at 366 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 80 0.03 1 • 1 • 1 • ' • 1 • 1 • 1 0 20 40 60 80 100 120 Time (min) Figure 18. Exp. 9. Thermolysin and crude enzyme catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6 and either 10 /zg thermolysin with 621 units of protease a c t i v i t y * or 17.36 jug crude proteases secreted by transformed B. subtilis DB428, with 477 units of protease activity*, was added to designated wells; enzyme was not added to the control samples. 1 unit of protease a c t i v i t y on azocasein = change i n absorbance at 366 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 81 Figure 19. Exp. 10. Thermolysin and crude enzyme catalysed synthesis of DNP-Asn-Leu. 76.3 nmol L-leucine, 7.56 nmol DNP-L-asparagine at pH 6. The following amount of thermolysin and crude proteases secreted by transformed B. subtilis DB428 were added to designated wells: a. 10 /xg thermolysin with 621 units of protease a c t i v i t y * b. 5.27 ug crude proteases with 80 units of protease a c t i v i t y * c. 26.04 ug crude proteases with 715 units of protease a c t i v i t y * No enzyme was added to the control samples. 1 unit of protease a c t i v i t y on azocasein = change i n absorbance at 3 66 nm of 0.01/2 hours RESULTS AND DISCUSSIONS / 82 Figure 15 shows that o p t i c a l density readings for samples which contained either thermolysin or crude proteases secreted by eit h e r transformed B. subtilis DB428 or WB600 increased quickly and then leveled o f f . On the other hand, the o p t i c a l density readings for the control samples, which consisted of only buffer s o l u t i o n and DNP-L-asn, remained r e l a t i v e l y constant f o r the entire incubation period. The o p t i c a l density readings measured i n m i c r o t i t e r wells containing crude proteases were notably lower than those wells containing thermolysin. This could be due to the fact that there was only a minute amount (5 to 8 fig) of crude enzyme present i n the wells, and that the enzyme had lower protease a c t i v i t y than thermolysin. Since the o p t i c a l density readings measured at 492 nm were similar i n those wells containing either proteases secreted by transformed B. subtilis DB428 or WB600, the proteases produced by only one type of transformant (B. subtilis DB428) were used to synthesize dipeptides i n subsequent experiments. Figure 16a showed that as enzyme a c t i v i t y was increased from 42 U to 126 U per well, dipeptide formation reaction was increased s l i g h t l y as well: the mean O.D.492nm readings a f t e r 4 hours of incubation was 0.093, 0.064, 0.064 when protease a c t i v i t y of crude enzyme was 12 6, 84 and 42 units, respectively. Higher o p t i c a l density readings were observed again as the protease a c t i v i t y of the crude enzyme was increased to 477 to 715 units per well (Figures 17 to 19); hence, there appeared to be a p o s i t i v e r e l a t i o n s h i p between DNP-asn-leu formation a c t i v i t y on the s o l i d phase and the protease a c t i v i t y of the crude enzymes. On the RESULTS AND DISCUSSIONS / 83 other hand, when thermolysin concentration was increased 5 f o l d to 50 jug nmole, there was an increase i n protease a c t i v i t y but the formation of dipeptides was not improved. There was probably an optimum concentration of enzyme which resulted i n maximum DNP-asn-leu formation. If the concentration of the target enzyme was increased beyond t h i s l e v e l , the unreacted or excess enzyme might a c t u a l l y i n t e r f e r e with peptide bond formation, or might increase the concurrent breakdown of the synthesized dipeptides. Aggregation of enzyme molecules could also occur when the concentration of enzyme was too high. c. E f f e c t of Varying the Concentration of DNP-L-Asn The e f f e c t of increasing the concentration of DNP-L-asn i n the reaction mixture containing crude enzyme was summarized i n Figures 16 a to d. The o p t i c a l density readings for the control samples continued to increase during the entire incubation period; s i m i l a r findings were observed i n the thermolysin catalysed reaction. The observed increase i n the o p t i c a l density readings over time i n the t e s t samples containing crude enzyme was also caused by an increase i n the non-specific binding of DNP-L-asn to the m i c r o t i t e r plate rather than by an actual increase i n DNP-asn-leu formation. This was supported by the fact that the difference i n O.D.492nm readings between the t e s t samples and the control samples at any point of incubation was si m i l a r regardless of the concentration of DNP-L-asn added to the wells. For example, the difference i n O.D.492nm at 6 hours between the t e s t group and the control group was as follows: RESULTS AND DISCUSSIONS / 84 0.051, 0.060, 0.056 and 0.059 while the concentration of DNP-L-asn used was 7.57 nmol, 37.87 nmol, 75.7 nmol and 151.4 nmol, respe c t i v e l y . d. Comparison of Peptide Synthesis Ratio for Thermolysin and Crude Enzyme Preparation A peptide synthesis r a t i o (Table 7) was calculated f o r three separate dipeptide synthesis experiments catalysed by thermolysin and crude enzyme f r a c t i o n , and was defined as the change i n o p t i c a l density readings at the end of 1 hour of incubation divided by the units of protease a c t i v i t y per well. The use of these r a t i o s permitted d i r e c t comparison of the r e l a t i v e reverse p r o t e o l y t i c e f f i c i e n c i e s of the crude enzyme preparation and commercial grade thermolysin. For experiment 9, the peptide synthesis r a t i o for thermolysin and the crude enzyme f r a c t i o n was found to be 1.1 x 10"* and 1.4 x 10"*, respectively. This showed that the crude enzyme(s) secreted by transformed Bacillus subtilis DB428 performed at l e a s t as well as, i f not better, than commercial grade thermolysin i n catalysing the formation of peptide bonds. In experiment 10, when the protease a c t i v i t y of the crude enzyme was increased from 80 units to 715 units per well, the peptide sythesis r a t i o increased about 5 f o l d (Table 7). Thermolysin performed s u r p r i s i n g l y poorly i n experiments 8 and 10 as the increase i n o p t i c a l density readings was not as much as those i n experiment 4, 5 and 9. The reason for t h i s was not clear but could be re l a t e d to increased hydrolysis of the synthesized product by thermolysin. RESULTS AND DISCUSSIONS / 85 f. S t a t i s t i c a l Analysis S t a t i s t i c a l analyses were performed for experiments 6, 7A, 8, 9 and 10 to determine i f there was a difference i n the mean o p t i c a l density measurements at 492 nm between the t e s t group with enzyme, and the control group without enzyme. Two-factor analysis of variance (or 2-way ANOVA) was again used. The e f f e c t of time, treatments ( i . e . with and without thermolysin, or with and without crude enzyme f r a c t i o n ) , and t h e i r interactions on the increase i n o p t i c a l density readings measured at 492 nm were tested. Table 9 summarizes the r e s u l t s of the s t a t i s t i c a l a nalysis. The differences i n the mean o p t i c a l density readings between a l l the control samples and the samples containing either thermolysin or crude enzyme f r a c t i o n were s i g n i f i c a n t (P < 0.01). This c l e a r l y indicated that the mean o p t i c a l density measurements were not the same fo r the control groups (with no enzyme) and for the t e s t group consisting of either commercial grade thermolysin, or crude enzyme f r a c t i o n from Bacillus subtilis DB428. Since the change i n o p t i c a l density readings at 492 nm i n the wells was re l a t e d to the r e l a t i v e amount of dipeptides formed, the treatment e f f e c t was s i g n i f i c a n t on the amounts of DNP-asn-leu formed i n the m i c r o t i t e r wells. Hence, the experimental data, and subsequent s t a t i s t i c a l analyses confirmed that the proposed rapid screening method was s e n s i t i v e enough to detect the formation of DNP-asn-leu dipeptides on a solid-phase even when crude enzyme f r a c t i o n i s o l a t e d from a c t e r i a l culture was used as a catalyst. Although the proposed rapid screening method can be used to RESULTS AND DISCUSSIONS / 86 Table 9. 2-Way analysis of variance on O.D.492nm i n m i c r o t i t e r wells from f i v e experiments of thermolysin or neutral protease T catalysed synthesis of Asn-Leu dipeptides F i g . Exp. Source of V a r i a t i o n DF F r a t i o 14d 6 Time 3 62.51** Treatment* 2 197.4** Time * Treatment• 6 22.10** Error 36 15ae 7a Time 3 407.5** Treatment" 3 365.4** Time * Treatment • 9 28.23** Error 32 16d 8 Time 12 35.14** Treatment* 2 1146** Time * Treatment 24 12.62** Error 117 17" 9 Time 12 29.94** Treatment* 2 829.8** Time * Treatment 24 6.660** Error' 117 18d 10 Time 12 48.15** Treatment' 3 668.8** Time * Treatment 36 8 . 260** Error 156 "Treatment 1 = Control sample (DNP-L-asparagine + buffer) Treatment 2 = Test sample 1 (DNP-L-asparagine + buffer + thermolysin) Treatment 3 = Test sample 2 (DNP-L-asparagine + buffer + crude enzyme fraction) bTreatment 1 = Control sample (DNP-L-asparagine + buffer) Treatment 2 = Test sample 2 (DNP-L-asparagine + buffer + 6.52 ug of crude enzyme fraction) Treatment 3 = Test sample 2 (DNP-L-asparagine + buffer + 13.04 ug of crude enzyme fraction) Treatment 4 = Test sample 2 (DNP-L-asparagine + buffer + 19.56 ug of crude enzyme fraction) treatment 1 = Control sample (DNP-L-asparagine + buffer) Treatment 2 = Test sample 1 (DNP-L-asparagine + buffer + thermolysin) Treatment 3 = Test sample 2 (DNP-L-asparagine + buffer + 5.27 ug crude enzyme fraction) Treatment 4 = Test sample 4 (DNP-L-asparagine + buffer + 26.04 ug crude enzyme fraction) d 4 wells per time-treatment combination c 3 wells per time-treatment combination ** S i g n i f i c a n t at P < 0.01. RESULTS AND DISCUSSIONS / 87 detect the formation of DNP-asn-leu dipeptides, the o p t i c a l density-readings measured at 492 nm for a l l of the experiments i n t h i s t h e s i s were r e a l l y quite low: even the highest readings were under 0.2. There are several possible explanations for the low readings. F i r s t , each well on the Nunc Immuno Module can only bind a limited number of L-leucine molecules. There are roughly 1014 secondary amino groups that are anchored to the surface of each well. This would mean that only a maximum of 21.8 ng or 0.17 nmol of leucine could bind to each well when a l l the amino groups are coupled with leucine molecules (Figure 20) . Assuming that the dipeptide synthesis reaction was 100% successful, there would only be a maximum of 0.17 nmol of L-asparagine molecules that could form peptide bond with the immobilized L-leucine molecules. Second, there was the p o s s i b i l i t y that a portion of the added DNP-L-asparagine molecules were unable to form a bond with the immobilized L-leucine molecules either because of s t e r i c hindrance or of t h e i r i n a b i l i t y to assume the conformation for proper binding. Third, some of the antibody peroxidase conjugate molecules might not have been able to complex with the dinitrophenol group on L-asparagine for the same reasons given above. Fourth, a l l of the synthesis reactions took place i n an aqueous environment, which could have s i g n i f i c a n t l y lowered the y i e l d of dipeptides as water l i m i t s y i e l d because of hydrolysis of the enzyme-substrate intermediate, or the f i n a l product. I t has been reported that product y i e l d could be enhanced s i g n i f i c a n t l y i f the peptide synthesis reaction i s carried out i n the presence of RESULTS AND DISCUSSIONS / 88 Approximate surface density of secondary amino groups on the surface of Covalink module = 10 1 4 /cm2 Approximate surface area of each microtiter well = 1 cm2 Avogadro number = 6 xlO 2 3 molecules per mole If every secondary amino group i n each well i s coupled with a leucine molecule, then there should be 10 1 4 molecules leucine per well. 1014 molecules leucine x 1 mole x 131 g 6 x IO23 molecules mole = 0.17 nmol leucine or 21.8 ng leucine per well Figure 20. Calculation of the Maximum Concentration of Leucine Immobilized on Nunc Immuno Module RESULTS AND DISCUSSIONS / 89 mixed or pure organic solvents instead of water (Chen et a l . , 1991) . Organic solvents were not used i n a l l of the experiments i n t h i s t h e s i s because of concerns over the p o s s i b i l i t y that organic solvent might damage the polystyrene surface, which would r e s u l t i n problems i n measuring the o p t i c a l density readings at 492 nm. Moreover, most enzymes are usually not very stable i n organic solvents. Lastly, the reaction conditions for synthesis of dipeptides on microtiter plates had not been completely optimized. Miranda and Tominaga (1991) reported that product y i e l d for the synthesis of Z-Asn-Leu-OEt was highest when the following experimental conditions were used: 1.0 mmol Z-Asn-OH, 1.0 mmole H-Leu-OEt.HCl, 21 nmol thermolysin, 2 g ammonium su l f a t e , 0.2 M sodium acetate pH 6, incubation temperature at 48°C. The recommended concentration of the various reactants could not be used i n the solid-phase synthesis of DNP-asn-leu because there were r e s t r i c t i o n s on how much one can add to a m i c r o t i t e r w e l l . The use of ammonium su l f a t e to improve product y i e l d by p r e c i p i t a t i n g the formed peptides was c l e a r l y not applicable on a m i c r o t i t e r plate. More work can s t i l l be done to determine the pH and temperature conditions that are best suited for peptide synthesis reactions on m i c r o t i t e r plates. The concentration of enzyme can be lowered even more i n the case of thermolysin catalysed reactions while the opposite i s true for the crude enzyme f r a c t i o n . Future investigation should be focused on increasing the s e n s i t i v i t y of t h i s rapid screening assay. One possible solution involves the use of fluorescent substrates for peroxidase enzymes. RESULTS AND DISCUSSIONS / 90 The use of other enzymes with higher substrate turnover rates i s another p o s s i b i l i t y . An example of such an enzyme would be catalase. Catalase can react with 30,000 /zmol of H202/min per mg of enzyme, which i s 30-fold greater than that of other enzymes used i n enzyme immunoassays (Yolken, 1982). CONCLUSIONS A rapid screening assay was developed to detect the formation of DNP-asparagine-leucine dipeptides on m i c r o t i t e r plates, which had secondary amino groups grafted on the surface. Conventional detection methods including reverse-phase high performance l i q u i d chromatography and t h i n layer chromatography are not suitable methods for rapid screening purpose as they are much more time consuming and labour intensive. Commercial grade thermolysin was f i r s t used as the catalyst for the synthesis reactions. The best reaction conditions for thermolysin catalysed synthesis were as follows: 76.3 nmol L-leucine, 10 ug thermolysin with 7.8 units of protease a c t i v i t y (using Hammerstein casein as a substrate), 7.56 nmol DNP-L-asparagine i n 0.2 M sodium acetate buffer, pH 6, with 50 mM calcium acetate, at 48°C for 2 hours. The presence of the f i n a l products was detected by the addition of anti-DNP antibody horseradish peroxidase conjugate, which reacted with the dinitrophenyl group on the asparagine molecule to produce a colour that could be measured at 492 nm. The e f f e c t of the enzyme treatment on the mean o p t i c a l density measurements was found to be s i g n i f i c a n t (P < 0.01). Crude enzymes secreted by transformed Bacillus subtilis DB428 and WB600 was then used to catalyse DNP-asparagine-leucine synthesis. The reaction conditions employed were s i m i l a r to those described for thermolsyin catalysed reactions except that 5 to 2 6 fig of crude enzyme with protease a c t i v i t y i n the range of 32 to 715 units (using azocasein as a substrate) was used. The e f f e c t of the 91 CONCLUSIONS / 92 enzyme treatment on the mean o p t i c a l density measurements at 492 nm was found to be s i g n i f i c a n t (P < 0.01). For future research studies, more work should be done on improving the s e n s i t i v i t y of the rapid screening assay. At the same time, random mutagenesis experiments should be c a r r i e d out on targeted regions of the gene (nprT) which encodes for neutral protease T from Bacillus stearothermophilus i n an attempt to produce mutant enzymes with enhanced reverse p r o t e o l y s i s a c t i v i t y . The developed rapid screening assay could then be used to i d e n t i f y those p o s i t i v e mutants. 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