UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Stabilization and mechanistic studies of soluble processing alpha-glucosidase I from saccharomyces cerevisiae Li, Patrick Ying Xia

Abstract

Processing α-glucosidase I (E.C.3.2.1.106) is a key enzyme in the biosynthesis of asparagine-linked oligosaccharides catalyzing the first processing event after the en bloc transfer of Glc₃Man₉GlcNAc₂ to proteins. The stability and mechanism of this enzyme were investigated in this research. The enzyme was isolated from dry yeast cells and was further purified using affinity chromatography with an enzyme inhibitor, N-methyl-N-(5- carboxypentyl)-l-deoxynojirimycin, as the ligand, and Concanavalin A-Sepharose chromatography. To improve the long-term stability of the enzyme, various additives were added into the phosphate buffer used for extraction and isolation. These additives were different protease inhibitors, including protease inhibitor cocktail [Sigma, product No. P8215, containing 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A, transepoxysuccinyl- L-leucylamido(4-guanidino)butane(E-64), and 1,10-phenanthroline] (1%) and phenylmethylsulfonyl fluoride (PMSF) (100 uM); reducing agent - dithiothreitol (DTT) (0.5 mM); chelating agent - ethylenediaminetetraacetic acid (EDTA) (0.2 mg/ml); polyol - glycerol (10%); and protective protein solution - bovine serum albumin (BSA) (1 mg/ml). The stability of the isolated enzyme was studied when kept at 4°C, -25°C, -80°C, and after freeze drying. Kinetic evaluation of several synthesized substrate analogues and chemical modification of the active site of the enzyme were also attempted. It was showed in this research that processing α -glucosidase I could be further purified using Concanavalin ASepharose chromatography. The stability of the enzyme could be much improved by the addition of glycerol, EDTA, BSA, PMSF and protease inhibitors during isolation. Extremely low freezing temperature (-25°C and -80°C) could help to retain enzyme activity during storage. Processing α -glucosidase I was an inverting enzyme which catalyzes hydrolysis with inversion of anomeric configuration. Synthetic substrate analogues 2'Fluoro- αGlc(l-2)αGlc-0-grease and 2'N3-αGlc(l-2)αGlc-0-grease were competitive inhibitors of the enzyme and at least one acidic amino acid might be present at the active site of the enzyme. It is believed that results from this research will provide useful information in the design of appropriate enzyme inhibitors that could be beneficial to human health.

Item Media

Item Citations and Data

Rights

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

Usage Statistics