High Expression Of Aspergillus Aculeatus β-glucosidaseⅠ In Pichia Pastoris And Research Of FN3Domain

Aspergillus aculeatus β-glucosidase I (ABGL) is one of the important β-glucosidase inGH3Family. Its excellent hydrolytic ability, can be used to hydrolyze a variety of substratescontained β-1,4glycoside, such as pNPG, esculin and cellobiose. ABGL can be used to assistthe cellulase in the cellulose degradation by reducing the content of cellobiose, so as to unlockinhibitions on cellulase activity to produce more glucose, brings a bright future in degradationof cellulose like bagasse. FN3region is located in the C terminal of the glycoside hydrolase.And it is a special unit which function is not clear yet, it may play an important role in substrateshydrolysis. Further reported show that ABGL can catalyze glucose and different chain lengthfatty alcohol to synthesize alkyl glycoside, the yield of the alky glycoside is reducing as thesubstrate is getting longer. This study attempts to improve the expression of ABGL in PichiaPastoris by the methods of in vitro multi-copy technique and methanol-glycerol mixed feedhigh density fermentation. And ABGL will be used in the research of synergistic hydrolysis ofbagasse with cellulose, which can provide solutions of reducing the cost of cellulosedegradation in industry. In the same time, we will take homology modeling method,moleculardocking method and biological experiments to research the relativity between FN3region andhydrolystic function, and the substrate specificity in synthesis of alkyl glycoside. It has greatsignificance to learn about the hydrolytic properties of ABGL and define the screening methodthrough molecular simulation. The specific research content is listed below:1) Improve the expression by in vitro Multi-copy technique, high density fermentation,methanol-glycerol mixed feed high density fermentationpHKA-(ABGL)3pastoris containing three copies of ABGL gene has been constructed, and5transformed yeast GS115/pHKA-(ABGL)3with higher activity have been screening throughesculin filter plate. The fifth day of the activity is83.15U/mL which is3.35fold higher thanpreviously report in the flask fermentaion by added2%methanol.Glycerol-methanol mixed feed express more β-glucosidase than methanol feed when add2%methanol and0.05%glycerol in inducing period in shake flask fermentation. The optimalvolume ratio between methanol and glycerol is40:1and it is applied in10L fermntor. Glycerol- methanol feed (methanol: glycerol=40:1) display higher production of β-glucosidase, reach1013U/mL at fifth inducing day, so as to increase14.5%compared with methanol feeding;Pichia pastoris fermentation show that GS115/pHKA-(ABGL)3can reach979U/mL atfifth inducing day, and it is2.94fold higher than control GS115/pPIC9K-ABGL in50Lfermentor.2) ABGL were applied in synergistic hydrolysis of bagasse with cellulaseThe research of synergistic hydrolysis with cellulase show that the optimal ratio betweenABGL amount and cellulose amount is2.5:1in hydrolyzing bagasse, and the glucose yieldincrease13.91%compared with control group.3) Initial function analysis of ABGL FN3domainAlong with the length of the substrate glycoside increases, the strength of the protontransport chain in catalytic pocket gradually retricted when analyse dockings among ABGL andbutyl, hexyl, molecular octyl, decyl and dodecyl β-D-glucoside ligand. Meanwhile, EntropicEnergy display a higher score, the score of Jain and the number of negative binding energy poseare decreasing, indicate that theoretic docking intensity decrease in line with the experimentalresult. Biological experiment tells us a720mutant remain lower hydrolystic ability than ABGLagainst pNPG, esculin and cellobiose, while other mutants have no hydrolysis activity, In themolecular docking study, the distance beween-OH in catalytic residues and αC of ABGL isshorter than mutants, and the score of binding poses is higher than most mutants, these twoaspects are quite important for predicting binding affinity. All the result shows that FN3regionplays an important role in hydrolyzing substrate, and the lack of FN3region will decrease thehydrolytic activity.