Screening And Preliminary Characterization Of A Novel β-glucosidase Gene

β-glucosidase（β-D-Glucosidase, EC3.2.1.21）, it belongs to cellulase enzymes, it is one of the three important component cellulolytic enzymes and capable of hydrolysising non-reducing β-D-glucose keys to release β-D-glucose and the corresponding ligands.β-glucosidase has many applications in industry. For example, it can be used to degrade cellulose to produce bioethanol, improve food flavor, transform soybean isoflavone glycoside compounds, produce Gentiooligosaccharide, control pests and diseases, et al. But the current industrial β-glycosidases are most derived from Trichoderma spp and other fungus. The reaction conditions（such as temperature, pH） of β-glycosidases have relatively narrow ranges and the β-glycosidases have low enzyme activity, which results in high economic costs and limiting its wide applications.To obtain the β-glucosidase which has good enzymatic properties and high enzyme activity, a high quality metagenomic library was successfully constructed using microbial communities of straw-back soil from Heilongjiang province and a novel β-glucosidase gene bgl2238 was screened by function-driven screening method from the library, the full-length of the bgl2238 was 2238 bp, which encoded a protein of 745 amino acids.Bgl2238 amino acid sequence analysis revealed that the predicted molecular weight of Bgl2238 was 80.67 kDa, pI value of 4.95 and the Bgl2238 was a acidic protein with good stable structure, highly hydrophobicity and no signal peptide sequence. The sequence homology analysis of Bgl2238 showed a highest similarity of 58 % with the β-glucosidase from Bacteroides thetaiotaomicron（GenBank: WP₀48691945.1）, and it belonged to theenzyme of GH3 and GH3 C superfamily.The bgl2238 gene was amplified by PCR technology, and was successfully cloned to pET-32a（+） vector, and it obtained high expression in E. coli BL21（DE3）. The results of optimizing expression conditions of recombinant Bgl2238 showed that the highest expression level（10.42 U/ml） was obtained when the cells were induced at OD600=0.8 and25 ℃ for 11 hours using 1.0 mM IPTG. Then the study on the enzymatic properties of the recombinant Bgl2238 showed that its optimal pH and temperature were 6.10 and 44 ℃,respectively, and the maximum specific activity of recombinant Bgl2238 reached 29.1U/mg. In the range of 4 50 ℃, the enzyme activity of Bgl2238 remained more than 75 %after 4 h incubation, which indicated Bgl2238 had good thermostability. Moreover, the Km and Vmax values of recombinant Bgl2238 were 0.296 mM and 576 uM/min, using4-nitrophenyl-β-D-glucopyranoside（pNPG） as a substrate; 0.543 m M and 572 uM/min using cellobiose as a substrate. Different concentrations of K+, Na+, Fe²⁺, Mg²⁺, Mn²⁺, Cu²⁺,Ca²⁺, Co²⁺, Zn²⁺ and Ni²⁺ could strongly increased the enzymatic activity, and the Fe²⁺ had the most promotion to the enzymatic activity. 10 mM Fe²⁺ could promote enzymatic activity to 260 %. Low concentrations of 1 mM SDS, EDTA and imidazole had weak inhibition to the enzymatic activity, but at 100 mM of them strongly inhibited the enzymatic activity. Urea and guanidine hydrochloride and guanidine thiocyanate promoted the enzymatic activity when the concentration of them was 1 mM, moreover, at high concentrations（100 mM）, Bgl2238 enzyme activity remained 97 %, 52 % and 48 %,respectively. In addition, the enzyme activity of Bgl2238 was promoted by DMSO,methanol, ethanol, isopropanol, Triton X-100 at 1 mM. Interestingly, when the concentration of DMSO was up to 100 mM, the enzyme activity still remained more than85 %, and the concentration of Triton X-100 increased to 100 mM could promote the enzyme activity, which indicated that Bgl2238 had strong tolerance of organic solvents,and will have a good prospect in industrial applications. Glucose as a product of enzymatic reaction, it has a certain feedback inhibition to the enzyme activity, however, when the concentration of glucose was up to 1.0 M, the residual activity of Bgl2238 remained about56 %, indicating that Bgl2238 had a certain resistance to glucose.The bgl2238 gene was also cloned to yeast expression vector, and the recombinant protein was high soluble expresstion in saccharomyces cerevisiae INVSC1, The amount of Bgl2238 protein expression wss 17.1 U/ml and enzyme specific activity was 38.7 U/mg.In this study, a high-quality metagenomic library was successfully constructed using microbial communities of straw-back soil from Heilongjiang and a novel β-glucosidase gene bgl2238 was screened by function-driven screening method from the library.Furthermore, this gene was heterologously overexpressed in prokaryotic expression system and the enzymatic properties of the recombinant enzyme were also investigated. And the Bgl2238 expression in eukaryotic expression system was explored. The study will not only broaden β-glucosidase kinds and research field to β-glucosidase, but also enrich cellulase source and has a certain value on strengthening cellulase applied research of uncultured microbial.