Cloning, Expression And Researches Of Enzyme Properties Of β-Glucosidases Screened From Uncultured Bacteria

β-glucosidase is an important component of the cellulase system.β-glucosidase canhydrolyze cellubiose and degrade the cellulose completely. Thus, we constructgenetically engineered bacteria that containing high level expression plasmid ofβ-glucosidase gene to accelerate the use of cellulose.It's an effective way to discover noval genes by constucting metagenomic library ofuncultured bacterium. In this research, we got3β-glucosidase positive clones whichwere named pGXAG142, pGXAG313 and pGXAG805 after screening the alkalinecontaminant metagenomic library AL01. These clones produced predictβ-glucosidase tohydrolyze Esculin on the special plates. The bioinformatics analysis indicated that theseclones' foreign DNA fragments contained ORFs which encoded 281, 170, 481 aminoacids respectively, and their respective pI/Mw were 4.88/29079.25 kDa, 6.28/18513.32kDa,5.16/57383.4 kDa. After analyzed by BLAST software, we found that there were noDNA or amino acids identities with the knownβ-glucosidase genes in the Genbank. Wepredicted that they might be the novelβ-glucosidase genes. Then the ORFs weresubcloned into pETBlue-2 vector and transformed into Rosetta(DE3)plysS. Therecombinant expression clones could expressβ-glucosidase on the special plate clearly.The enzyme data of pGXAG142G/Rosetta(DE3)plysS showed that its optimum pHvalue and temperature wre 5.5-8.0 and 55℃respectively, Km=0.238 mmol·L^-1,Vmax=10.6U.min^-1·ml^-1,and its enzyme activity under the optimum condition were 24.8U·ml^-1. Different metal ions showed different effects on this enzyme's activities, K⁺,Mg²⁺,Zn²⁺will enhance its activity, while Ca²⁺,Co²⁺,Cu²⁺,Ba²⁺will cause obvious inhibition. The results of HPLC demonstrates that pGXAG142G encodesβ-glucosidase.Coulture-independent method is the effective way on discovering novel genes, ourresearches on novalβ-glucosidase prove this theory again.