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Exploring protein structure-function relationships in xanthanases from glycoside hydrolase family 9

University of British Columbia

The hydrolysis of xanthan is a commercially relevant reaction. This is due to the widespread use of this polysaccharide as a rheology modifier in many industries. Therefore, investigations into the structure and function of enzymes capable of altering xanthan properties, including endo-xanthanases, is warranted. This thesis describes investigations into the structure-function relationship of PspXan9, a bacterial xanthanase from glycoside hydrolase family 9 (GH9). To do this, enzyme kinetic assays were performed to determine preferred biochemical conditions, substrate specificity and Michaelis-Menten kinetics. These revealed that PspXan9 was highly specific for xanthan following a pretreatment with xanthan lyase, and notably, required calcium for increased activity and stability. The appearance of only two products over the course of a reaction, as monitored by high-performance size exclusion chromatography coupled to a UV-detector (HPSEC-UV), demonstrated that hydrolysis of lyase-treated xanthan with PspXan9 occurred in a processive fashion. However, the identity of these products as lyase-treated xanthan tetrasaccharides and octasaccharides was only realized following analysis with liquid chromatography coupled to mass spectrometry (LC-MS), tandem mass spectrometry (MS/MS) and 1D- and 2D-Nuclear Magnetic Resonance (NMR). Analysis of the PspXan9 x-ray crystal structure was used to reveal structural insights that guided targeted mutations to perform subsite mapping. Changes resulting from each mutation were tested for activity, and degree of processivity through viscometric and HPSEC-UV analysis. Lastly, protein similarity networks were utilized to find a putative GH9 xanthanase subgroup as well as a possible progenitor GH9 whose protein expression was attempted.

Text

2019-12-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/72844

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/