Ivan N Zorov
Federal Research Center of Biotechnology- RAS, Russia
Title: Î’-glucosidases from penicillium and Aspergillus expression, purifi cation and catalytic properties
Biography
Biography: Ivan N Zorov
Abstract
The enzymatic saccharifi cation of polysaccharides from lignocellulose biomass is a crucial stage in production of second-generation biofuels (ethanol, butanol, etc.), bifunctional organic acids (lactic, fumaric) for biopolymer synthesis. Cellulose is converted to glucose under the synergistic action of at least three types of glycoside hydrolases (endoglucanases, exo-cellobiohydrolases and β-glucosidases). β-Glucosidase (BGL) is the key enzyme that completes the saccharifi cation process, converting cellobiose to glucose. A novel bgl1 gene, encoding GH3 family β-glucosidase from Penicillium verruculosum (PvBGL) was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain. BGL from Aspergillus niger (rAnBGL) was expressed in the same recipient host. Both BGLs were desalted and purifi ed from cultural broth by AEX, HIC, and GF chromatography using columns packed with Bio-Gel P4, Source 15Q, Source15ISO, and Superdex75 for fi nal polishing. SDS-PAGE followed by MALDI-TOF/TOF analysis of tripsin digests were used for purity control and proteins identifi cation. Temperature and pH-optima for both ezymes were the same 65oC and pH value 4, 5-4, 6 respectively. Th e half-life time of rPvBGL and rAnBGL was 10 and 5 min respectively at 70oC. Th e activity of both enzymes decreases with the increase of degree of cellooligosaccharides polymerization (DP). Kinetic studies revieled diff erences between PvBGL and AnBGL. Th e catalytic effi ciency of AnBGL was about 35% higher to compare PvBGL in p-NP-β-D-glucoside and cellobiose hydrolysis, while remains the same for 1,4-β-oligosaccharides with higher DP. AnBGL was more specifi c in hydrolysis of 1,4-β-cellooligosaccharides, while PvBGL was much more effi cient in hydrolysis of 1,3-β- and mixed 1,3-β-/1,4-β-oligosaccharides.