Gene Cloning, Overexpression And Characterization Of A Novel β-Glucosidase Derived From Metagenomic Library Of Mangrove Soil

β-Glucosidase is a kind of three cellulases, which catalyze the hydrolysis ofvarious oligosaccharides and cellobiose to form glucose and corresponding aglycone. Ithas great application value in hydrolyzing of cellulose, improving food’s flavor, aswell as prevention and control of disease and pest, etc. Currently, most commercialβ-glucosidases are of fungal origin, and have some universal drawbacks, includinglow activity, narrow application range of temperature and pH. Thus, it leads high costof β-glucosidase in the production and application. It has become the bottleneck of thebroad application of β-glucosidase in industry.To obtain β-glucosidase with high activity and good properties, metagenomiclibraries with high quality were constructed using soil samples collected frommangrove soil samples of Qiao island, Zhuhai city, and a novel β-glucosidase genenamed wz-2was identified successfully by function-driven screening from ametagenomic library. Sequence analysis revealed that the length of this gene was1392bp, which encoded a protein of463amino acids. Moreover, wz-2showed ahighest similarity of84%with the β-glucosidase from Enterobacter mori LMG25706when aligned with other β-glucosidases through BLAST online. The gene wz-2was amplified via PCR and cloned to pET-32a vector successfully,and then the expression vector was transformed into Escherichia coli BL21（DE3）. Toobtain the highest expression level of recombinant enzyme, the induction conditionswere further optimized. The results revealed that the highest expression level wasobtained when the cells were induced at OD600=0.8and30℃for14h using1.1mMIPTG.The properties of recombinant enzyme were investigated, and the results showedthat its optimal temperature and pH were51℃and6.4, respectively. Moreover, itsKmand Vmaxvalues were0.408and444μM/min, using p-NPG as substrate. Inaddition, different1mM metal ions had different impacts to enzymatic activity ofWZ-2. Fe²⁺, Mg²⁺, Mn²⁺, and Na+increased its activity, and Mg²⁺had the highestenhancement to its activity. However, Zn²⁺, Ca²⁺, K+, and Cu²⁺of1mM inhibited itsactivity, and the inhibition of Zn²⁺and Cu²⁺of1mM was very significant, and1mMHg²⁺inhibited its activity completely. In addition, Li+and Co²⁺had little effects onenzymatic activity. Five chemical regents including EDTA, acetone, SDS, carbamideand imidazole were used to investigate the effect on enzymatic activity, the resultsrevealed that EDTA and SDS inhibited the activity of recombinant enzyme no matterthe concentrations of EDTA and SDS were1mM or100mM, and EDTA displayedhigher inhibition to the enzymatic activity. Moreover, when1mM carbamide orimidazole existed, the enzymatic activity was obviously improved. However,100mMcarbamide lost its enhancement to the enzymatic activity, and100mM imidazoleshowed its inhibition to the enzymatic activity. In addition, acetone （1mM or100mM）had little effects on enzymatic activity, which suggested the enzyme was high tolerantto acetone.In summary, a gene encoding a novel β-glucosidase was identified throughactivity-based functional screening of a metagenomic library with high quality frommangrove soil. Furthermore, this gene was overexpressed heterologously in E. coli,and the enzymatic properties of recombinant enzyme were also investigated. Thisstudy will enrich the source of cellulase, and widen the research field of cellulose, andthus has great significance to strengthen the theoretical and applied research of cellulases from unculturable microbes.