Identification Of Intracellular ?-glucosidase In Aspergillus Niger And Its Role In The Regulation Of Lignocellulose-degrading Enzyme Synthesis

Aspergillus niger is a proficient plant cell wall degrader,and it produces a wide spectrum of cellulases and hemicellulases.Therefore,it has great value in crop straw feed and fertilizer utilization.However,the regulation mechanism of the lignocellulose-degrading enzyme system in A.niger remains unclear.On the other hand,although A.niger is featured with its copious amount of extracellular ?-glucosidase,which is generally used to balance the cellulolytic enzyme cocktails for lignocellulose saccharification,the possibility of producing intracellular ?-glucosidase is unknown.In this study,fifteen putative ?-glucosidase genes(bgls)in A.niger were annotated based on A.niger CBS 513.88 genome,and five of them were proposed as intracellular BGLs due to the lack of signal peptide.Here,the function and protein localization of these five putative intracellular bgls were carefully characterized.Moreover,the role of the intracellular BGL in the synthesis of A.n.iger lignocellulosedegrading enzymes was also investigated.The main results of this study were as follows.(1)Identification of the putative intracellular bgls in A.niger.By testing the growth of the recombinant Saccharomyces cerevisiae strains,the function of the putative bgls can be easily identified.When cellobiose was used as the sole carbon source,only recombinant yeast carrying the An03g03740 gene(designated as bgl1B)could grow,while recombinant yeast carrying An03g05330,An06g02040,An15g01890,or An17g00520 genes could not grow.These results indicated that BGL1B is a ?-glucosidase,while the other 4 putative bgl genes may be non-functional pseudogenes.The mass spectrometry analysis of the total intracellular and extracellular proteins of A.niger under various carbon sources was used to further confirmed the conclusion,which showed that only BGL 1B was detected in the cultures.(2)Characterization of BGL 1B.The BGL1B was purified from the recombinant S.cerevisiae harboring bgl1B,and p-nitrophenyl-?-D-glucopyranoside(pNPGlc)was used as the substrate to characterize the enzyme.The optimal reaction conditions for BGL 1B were 40? and pH 5.6.The kinetic parameters Km of BGL1B towardspNPGlc is 0.233 ± 0.058 mM,and Ki towards glucose is 119.8±4.35 mM,indicating it has the ability to withstand high concentrations of glucose.Moreover,high transglycosidic activity of BGL 1B was detected when it was used to hydrolyze cellodextrins.Sophorose,laminaribiose,and cellotriose were produced when cellobiose was used as the substrate,and sophorose and laminaribiose were formed when cellotriose was used as the substrate.The results showed that BGL1B had higher hydrolysis activity on its transglycosylation products(laminaribiose,cellooligosaccharide,and sophorose)than that ofpNPGlc,but it did not have the catalytic activity toward gentiobiose and lactose.Thus,it is reasonable to infer that BGL1B has the catalytic activity on ?-1,2,?-1,3,and ?-1,4-glucoside bonds,but not ?-1,6-glucoside bonds and ?-1,4-galactoside bonds.(3)Investigation of BGL1B localization in A.niger.Both the intracellular and extracellular total protein samples of A.niger that grew on various carbon sources were prepared,and then these protein samples were analyzed by western blot against BGL1B antibody.The results showed that A.niger had the highest intracellular BGL1B expression when the fungus grew on wheat straw or steam-exploded bagasse.However,it had deficient intracellular BGL1B expression when it grew on cellobiose or glucose.On the other hand,no BGL1B protein was detected in all the extracellular protein samples.To further confirm the BGL1B localization in A.niger,all these protein samples were also analyzed by mass spectrometry.The results showed that 22 and 1 peptides of BGL1B were detected in the intracellular protein samples of A.niger that grew on wheat straw and cellobiose,respectively.Again,no BGL1B peptides appeared in all the extracellular protein samples.(4)BGL1B participated in the regulation of the synthesis of lignocellulose-degrading enzymes in A.niger.Both the BGL1B overexpression strain(OE::bgl1B)and strain harboring empty plasmid(CK strain)were built,by using the efficient protoplast-PEG-mediated transformation method optimized in this work.By compared the phenotypic difference between the OE::bgl1B and CK strains,the results indicated that the overexpression of intracellular BGL1B promoted the production of most of lignocellulose-degrading enzymes in A.niger.Furthermore,the inducing effect of BGLIB transglycosidic products on the synthesis of A.niger lignocellulose-degrading enzymes was investigatd.The results showed that in the presence of the ?-glucosidase inhibitor nojirimycin,laminaribiose and cellobiose can independently induce the lignocellulose-degrading enzymes in A,niger,including endoglucanase,exoglucanase,?-glucosidase,xylanase and ?-L-arabinofuranosidase,etc.And laminaribiose is better than cellobiose in inducing the lignocellulose-degrading enzymes;Sophorose does not have such inducing ability.All the above results showed that the intracellular BGL1B participated in the regulation of the synthesis of lignocellulose-degrading enzymes in A.niger,and the regulation mechanism was proposed as follows:cellulose was firstly degraded into cellooligosacchrides or monosaccharides by the small amount of secreted cellulases when A.niger grew on cellulose.The monosaccharides are directly utilized by the fungus,while parts of the cellooligosacchrides were transferred into the cells.And then,parts of the intracellular cellooligosaccharides were transferred into laminaribiose through the transglycosidation by intracellular BGL.Laminaribiose and the undegraded cellobiose could promote the expression of the main lignocellulose-degrading enzymes as inducers.At the same time,parts of the intracellular cellooligosaccharides were hydrolyzed into glucose by intracellular BGL.Glucose resulted the expression of the repressor creA,which could inhibit the expression of lignocellulose-degrading enzymes to some extent.This dual effect of BGL1B on the expression of lignocellulose-degrading enzymes is generally displayed as the induction of synthesis of enzymes in A.niger.In summary,all the 5 putative intracellular bgls were carefully analyzed,and only bgl1B was identified as a functional intracellular ?-glucosidase gene.The further characterization of BGL1B found that it had a unique transglycosidic activity.Moreover,BGL1B could promote the production of lignocellulose-degrading enzymes by its transglycosidic product laminaribiose in A.niger.Thus,the role of intracellular ?-glucosidase in the regulation of the synthesis of lignocellulose-degrading enzymes was firstly confirmed in A.niger.This work will offer guidance to understand the regulation mechanism of cellulases production in A.niger,and it also will lay the foundations for creating more productive A.niger strains.