Gene Cloning And Directed Evolution Of A Novel β-glucosidase Derived From Metagenomic Library Of Guts Inhabiting Microbes Of Coptotermes Fonnosanus

β-glucosidase, one of the three cellulases, plays a key role in the degradation ofcellulose. It can catalyze the hydrolysis of various oligosaccharides and cellobiose toform glucose and corresponding aglycone, and thus has great application value inimproving food’s flavor, developing of healthcare products, hydrolyzing of cellulose,as well as detection of the disease and the study of drugs, etc. Currently, mostcommercial β-glucosidases are from Trichoderma fungi, they have some universaldrawbacks, including low activity, narrow application range of temperature and pH.Therefore, it leads high cost of β-glucosidase in the production and application. It hasbecome the bottleneck of the broad application of β-glucosidase in industry.In order to obtain β-glucosidase with high activity and good properties,metagenomic libraries with high quality were constructed using FormosaSubterranean Termite hindgut microflora, and a clone with high activity wasidentified successfully by function-driven screening from the metagenomic library.Sequence analysis revealed that the length of this insert clones was5415bp, whichincluded two β-glucosidase genes （bg11923and bg11392）. The length of twoβ-glucosidase genes was3311bp, which contained a four-nucleotide overlap. Theresult of recombinant expression indicated that the recombinant protein from bg11923or bg11392had no activity. However, the fusion protein exhibited high β-glucosidase activity when bg11923and bg11392were expressed as a whole gene （bg13311）. Toour best knowledge, it is the first report of exhibiting β-glucosidase activity throughthe interaction of two β-glucosidase subunits or domains. The finding has importantimplications for the study of molecular structure of β-glucosidase and interactionamong protein moleculars from different domains. Moreover, Bgl1923showed ahighest similarity of84%with the β-glucosidase from Enterobacter cloacae subsp.dissolvens SDM, and Bgl1392showed a similarity of70%with the β-glucosidasefrom the hindgut unknown microbial enrichment culture of Reticulitermes santonensitermite when they aligned with other β-glucosidases in GeneBank through BLASTonline tool.The gene bg13311was amplified via high-fidelity PCR and cloned to pET-32a（+）vector successfully, and then the expression vector was transformed into Escherichiacoli BL21（DE3）. To obtain the best expression of bg13311in E. coli, the inductionconditions of bg13311were optimized. The results revealed that the highestexpression level （8.55U/ml） was obtained when the cells were induced at OD600=0.8and25oC for16hours using1.0mM IPTG. Next, the properties of recombinantenzyme were investigated, and the results showed that its optimal temperature and pHwere51℃and6.8, respectively. Moreover, the Kmand Vmaxvalues of recombinantenzyme were0.290mM and573μM/min, using pNPG as substrate;0.134mM and816μM/min, using cellobiose as substrate. In addition,1mM and10mM differentmetal ions had different impacts to enzymatic activity of Bg13311. Li+, Na+and K+increased its activity. Unexpectedly, Mn²⁺、Cu²⁺、Mg²⁺、Ni²⁺、Zn²⁺、Fe²⁺and Fe3+,which often lies in the active center, did not stimulate the activity of the enzyme. Onthe contrary, they inhibited the enzyme activity followed with the enhancement of,and Mg²⁺displayed a particularly significant inhibition to the enzymatic activity. Also,heavy metal ions including Ag⁺and Hg²⁺had strong inhibition to the activity ofBg13311, Ca²⁺and Co²⁺displayed high inhibition to the enzymatic activity. As atypical protein denaturant, SDS and EDTA had strong inhibitory effect on enzymeactivity. High concentration of guanidine hydrochloride and guandine throcyanate （GITC） showed the inhibition to the enzyme activity. Low concentration of ureahas promoting effect on enzyme activity, but it`s promoting effect disappeared whenadded at high concentration. In addition, Bgl3311shows strong tolerance of acetone,imidazole, and urea. As a product of the enzymatic reaction, glucose has a majoreffect on the activity of enzyme. The results showed that the activity of Bgl3311wasreduced sharply followed with the enhancement of glucose concentration.But whenglucose concentration reached up to1.5M, the enzymatic activity still retained64.5%of the highest activity, which suggested a high tolerance on glucose. Interestingly,when glucose concentration was less than1.0M, the presence of glucose couldimprove the enzymatic thermostability significantly. When the concentration of NaClwas less than0.8M, it increased the enzymatic activity obviously.However, theenzymatic activity began to be inhibited when NaCl concentration was more than0.8M, and the inhibition was sharply increased along with the enhancement of NaClconcentration. When the concentration of NaCl reached to3M, Bgl3311almost lostits full activity.Because the activity of Bgl3311was not high enough, directed evolution methodwas used to modify the enzyme. Random mutagenesis libraries were constructed viaerror-prone PCR, and screening for mutant enzyme with higher activity wasperformed with pNPG as substrate. Finally, a mutant enzyme Bglm0326withimproved activity was identified from the library. The sequencing analysis indicatedthat this mutant had two missense mutations （S556A and H860L） and a synonymousmutation （T446T）, and its activity （22.24U/ml） for pNPG was increased to2.6-foldhigher than that of the wild type （8.55U/ml）, which suggested it is an effectivemethod to improve the activity of-glucosidase using error-prone PCR.In summary, a metagenomic library with high quality was constructedsuccessfully using Formosa Subterranean Termite hindgut microflora in this study,and a β-glucosidase gene （bgl3311） with unique mechanism was identified byfunction-driven screening from the metagenomic library. Then, prokaryoticoverexpression of bgl3311was carried out, and the enzymatic properties ofrecombinant enzyme were determined. Finally, error-prone PCR technology was used to explore the directed evolution of Bgl3311.The results of this study has importantimplications for enriching cellulose source,developing enzymatic applications, andaccelerating the basic research and the application research of cellulase fromuncultured microorganisms.