Overexpression Of β-glucosidase Gene And Basic Study On Its Application

β-Glucosidase is an important constituent of cellulase complex.However,theβ-glucosidase activity in the cellulase system of Trichoderma reesei,the conventional cellulase producer and the main source of commercial cellulase,is extremely low,which affects cellulases work synergistically to achieve complete hydrolysis of cellulose.In this thesis,Aspergillus niger β-glucosidase gene(bgl)was cloned and codon optimized,and heterologous expressed in Pichia pastoris and Trichoderm reesei.The obtained BGL was then used in the synthesis of cellulase soluble inducer and enzymatic hydrolysis of cellulose.Major results in this thesis are as follows.bgl gene was codon-optimized according to codon preference of P.pastoris.The resulting P.pastoris expression plasmid pPIC9K-bgl was then transformed into P.pastoris using electroporation and putative transformants were selected.BGL production performance of transformant was studied under 5L bio-reactor.An optimized mixed-feed strategy was established,in which glycerol/methanol/corn steep powder were fed and methanol concentration was maintained 0.7%(v/v).Recombinant β-glucosidase activity(BGA)reached 129 IU/mL after 96 h of fermentation.This result could be meaningful in promoting large-scale BGL production.The cellulase soluble inducer sophorose was produced using glucose solution as substrate via recombinant β-glucosidase catalyzed transglycosylation reaction.After reaction process optimization,the sophorose concentration reached 70.85 g/L.Sophorose has been acknowledged as the most potent soluble inducer for cellulase production.However,its high price limits its industrial application in cellulase production.The results in this study offer a new approach to economical production of cellulase soluble inducer and are of great application value.Also,targeted integration and heterologous expression of A.niger bgl in T.reesei was studied.The foreign bgl was codon optimized and integrated at acel,encoding transcriptional repressor,site in T.reesei,which achieved the overexpression of foreign bgl and in-situ disruption of ACE1.Fermentation under shake flask showed the resulting transformants revealed a significant increase in both BGA and filter paper activity(FPA).The BGA increased to 25.13 IU/mL,167-fold higher than that of the host strain.Meanwhile,the ratio of BGA to FPA in the obtained cellulase system was improved from 0.05 to 1.25,leading to a better synergistic effect between cellulase components.Furthermore,submerged fermentation of recombinant T.reesei was carried out in 50 m3 under continuous fed-batch fermentation using in-house sugar mixture GSM containing sophorose as carbon source and inducer.An automated fed process was established according to the properties of the strain and GSM,which allowed for the limited growth of the strain and continuous boost of BGA.The resulting FPA and BGA reached 126.6 IU/mL and 157.8 IU/mL respectively in 50 m3 fermenter after 144 h of fermentation.This result reaches an advanced international level.Enzymatic hydrolysis of corn stover was carried out using the cellulase produced by recombinant T.reesei.Compared to the performance of host strain,recombinant cellulase with improved BGA achieved higher hydrolysis yield.Moreover,enhancement in BGA/FPA value led to significant improvement in hydrolysis yield.When BGA/FPA value was up to 15 with an addition of 300 IU/g substrate xylanase,the enzymatic hydrolysis yield of corn stover increased significantly to 93.5%.This result laid a solid foundation for the development of lignocellulosic biomass-degrading enzymes.