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Title:	Production of α-mannosidase i and its mannosynthase variants from the cold-adapted yeast glaciozyma antarctica for oligosaccharide synthesis
Authors:	Dharshini Elangovan (P78680)
Supervisor:	Muhammad Mukram Mohamed Mackeen, Prof. Dr.
Keywords:	Oligosaccharides Oligosaccharide synthesis Yeast glaciozyma antarctica Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia
Issue Date:	18-Jan-2019
Description:	Oligosaccharides are becoming more important due to their involvement in biological recognition processes, signaling mechanisms and therapeutic applications. This creates the demand for effective methods of oligosaccharide preparation that are stereo- and regioselective. Oligosaccharides can be synthesised chemically or enzymatically, however chemical synthesis requires multiple protection, activation, and deprotection steps. Regio- and stereo-selectivity of oligosaccharides can be achieved through enzymatic synthesis using glycosyl transferases (GTs) or glycosidases (GHs) without the need for protecting steps. The limited availability of GTs and high cost of their activated sugar substrates, makes the use of GHs more desirable. Oligosaccharide synthesis using GHs can be controlled by thermodynamic (reverse hydrolysis) and kinetic factors (transglycosylation). Low yields from synthetic reactions catalysed by GHs restricts their use for the large-scale preparation of oligosaccharides. The use of glycosynthases, a nucleophilic mutant of GHs, is becoming the preferred approach for the synthesis of oligosaccharides because of its high yielding potential. This study reports the production of an N-glycosylation-related endoplasmic reticulum (ER) α-1,2-mannosidase (GAManI) from G. antarctica, the preparation of α-1,2-mannosidase variants (E108G, D241G and E412G) as α-mannosynthases and the enzymatic synthesis of oligosaccharides using these enzymes. GAManI and mannosynthase variants were expressed (~60 kDa) in P. pastoris at 25 °C with 1.0% methanol induction. The protein was purified by IEX chromatography and identified by Western blot and LC-MS/MS. GAManI showed optimal activity at pH 6 and 37 °C. The D241G and E412G mutants showed reduced hydrolytic activity by 20% and 99%, respectively, compared to the GAManI. The reverse hydrolysis approach using natural sugars did not show product formation. The transglycosylation reaction by GAManI towards pnitrophenyl-D-glucopyranoside (pNPG) as donor and acceptor molecules showed the production of two pNP-derived peaks. These products were observed by HPLC-UV (Peak 1, Rt 6.0 min and Peak 2, Rt 7.0 min). Optimum conditions for synthesis via transglycosylation using the GAManI were 37 °C, pH 6, ratio 1:3 (donor: acceptor) for 12 hours of incubation, with the D241G mutant providing a slightly higher yield (~8%) than wild-type. These two product peaks were identified as pNP-disaccharides using DI-ESI-MS/MS. Enzymatic characterisation of the products using β-glucanase (hydrolysis: +ve) and α-glucosidase (hydrolysis: -ve) suggested that the products formed are most likely β-1,3 or β-1,6 pNP diglucosaccharides. Transglycosylation with pNPG (donor) and pNP-α-mannopyranoside (acceptor) also showed formation of a disaccharide pNP peak as shown by HPLC-UV and DI-ESI-MS. Monosaccharide composition analysis confirmed the product formed was a gluco-manno-pNP disaccharide. These disaccharide products are also most probably formed by residual activity of β-glucosidase (host protein) instead of GAManI. The final approach of oligosaccharide synthesis by GAManI and its variants was carried using the β-mannopyranosyl azide (β-Man-N3) as donor and pNP-glycosides/natural sugars (acceptors), respectively. The β-Man-N3 was prepared by Zemplen deacetylation of 2,3,4,6-tetra-O-acetyl-β-mannopyranosyl azide. The identity of the deprotected product was confirmed by DI-ESI-MS and NMR. However, the glycosynthase reactions did not show product formation. In conclusion, all the enzyme and oligosaccharide yields obtained were low (when the synthesis worked). The glycosynthase approach was unsuccessful with the azide donors (β-Man-N3 and β-Glc-N3).,'Certification of Master's/Doctoral Thesis' is not available,Ph.D.
Pages:	262
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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