Characterization Of Recombinant Thermophilic β-glucosidase And Its Synergistic Catalysis With Other Cellulases

β-glucosidase, as an important member of the cellulase system, plays significantrole in biomass conversion, and is a rate limiting factor during enzymatic hydrolysis ofcellulose. Accumulation of cellooligosaccharides during cellulose degration results inthe inhibition of activities of the endoglucanase and exoglucanase. This inhibiton canbe removed by adding adequate β-glucosidases, which can turn cellooligosaccharides,especially cellobiose, into glucose. Characterization of novel β-glucosidases fromthermophilic bacteria is necessary, due to its high thermostability, glucose tolerance,compatibility with other enzymes, and resistance to tough circumstances in the biofuelindustry.The thermophilic microorganism Caldicellulosiruptor bescii DSM6725wasisolated from hot springs in the Valley of Geysers，it grows at temperatures of up to90℃, and secrets a number of glycohydrolases, among which we study theβ-glucosidase (CbBgl1A）with the gene code ACM59590.1in this article. Aftersuccessively constructing recombinant plasmid using pET-21b(+), we purified ourenzyme with the Ni-NTA affinity chromatography, and found that CbBgl1A has beenoverexpressed in E. coli. Sequence alignment and zymogram suggested thatconserved Glu163and Glu361are catalytic acid/base and nucleophile, respectively.CbBgl1A is specific for β-linked substrates, and could cleave a range ofcellooligosaccharides and aryl-β-glycosides. CbBgl1A showed highest catalyticactivity of76.4U/mg on4-nitrophenyl-β-d-glucopyranoside (pNPGlc), at85℃, pH6.8, and the half-life at70℃was128hours, displaying high thermostability.Catalytic kinetic assay showed that CbBgl1A has a substrate preference for pNPGlcwith a kcat/Kmvalue of84.0s1mM1, while the kcat/Kmvalue for cellobiose is8.35s1mM1. The difference of catalytic efficiency on cellobiose from pNPGlc wasmainly caused by the high Kmvalue and the weak binding ability. Substrate inhibition was not observed, and the enzyme could tolerate a high concentration of glucose witha Kivalue of113.8mM.In the biofuel industry, the production of cellooligosaccharides causes inhibitionof the activities of both the endoglucanase and exoglucanase, adding sufficientβ-glucosidase can remove this inhibition. For judging enzyme’s ability to eliminatethe inhibition and increase the activity, we detected synergistic effect of CbBgl1Awith several endo-or exo-glucanases. CbBgl1A showed different synergy value withFnCel5A, CbCel9A, CbCbh48A on RAC. The highest synergy value of2.6wasobtained for the hydrolysis of regenerated amorphous cellulose using a combinationof cellobiohydrolase CbCbh48A from C. bescii and CbBgl1A at a molar ratio of1:5,where the complete conversion of oligosaccharides to glucose was obtained in2h.The high synergistic efficiency with exogluconase brings a huge potential in thebiomass conversion and industry application.To reveal the three dimensional structure, catalysis mechanism, and substratespecificity mechanism, we performed molecular modeling and docking. Choosing theβ-glucosidase from C. cellulovorans（PDB ID:3AHX）as the model, the model ofCbBgl1A showed classical (β/α)8-TIM barrel fold, typical of a GH family1enzyme,The catalytic acid/base of CbBgl1A is Glu163, and catalytic nucleophile of CbBgl1Ais Glu361. The distance between the closest atoms of the catalytic side-chains ofGlu163and Glu361is3.7, which is typical of a retaining enzyme and allows for theformation of a glycosyl-enzyme intermediate. Molecular docking revealed whyCbBgl1A has lower catalytic activity on its natural substrate cellobiose than pNPGlc.When the best substrate pNPGlc was docked into the binding pocket, both the glyconegroup and paranitrophenol lay perfectly in the substrate cleft of the active site, thedistance between Glu361and C1atom is3.7, which is suitable for initiatingnucleophilic attack. The glycone group was well organized through four hydrogenbonds and some hydrophobic interactions. When cellobiose was docked into theactive site of CbBgl1A, the glycone had a180flip, and the distance between the C1of cellobiose and the OE1of Glu361was4.3, a little further for the nucleophilicattack compared to pNPGlc. The glycone just formed three hydrogen bonds withsurrounding amino acids. All these led the lower affinity, and worse catalysis towardcellobiose by the enzyme, compared with the substrate of pNPGlc.We also did crystallography on CbBgl1A to obtain the crystal structure, which is the basis of further molecular engineering. We found the crystals of CbBgl1A in theIndex37solvent, at14℃after two days, and ultimately got big dense crystals in theoptimized condition of18%PEG1500. This could be the first step for analyzing thestructure and function of the enzyme.