Cloning, Characterization And Expression Of The Novel Cellulase Genes From Uncultured Microorganisms

Modern biomass energy as a new class of renewable energy, resource-rich, low pollution, high calorific value and other characteristics, mainly through a series of conversion technologies to produce high-grade energy and replace fossil fuel, foremost among which is the fuel ethanol production, it has been widespread concern and attention of the world. The first generation of fuel ethanol is mainly take advantage of starch and sugar substances (corn, soybeans, sugar cane and food crops and so on) for the raw materials, not only the yield is limited, the production cost subject to the changes in of the food prices, but also caused a the non-performing competition of the food and energy. Therefore, the researchers turned to the second generation of fuel ethanol production in order to improve the sustainability of fuel ethanol, the main use of lignocellulosic raw materials (such as crop straw, energy plants), the hydrolysis of carbohydrate fermentation production of bio-ethanol.At present, the second generation biofuel research mainly focus on two aspects to reduce the cost, one is to look for new cellulase genes or build high-efficient cellulase engineering strains, the other is to develop the high cellulose content of energy plants. Our laboratory built Miscanthus domesticated cattle rumen microbial metagenomic library and termite gut metagenomic library was screened with the functional genes of cellulase activity2-C5and1-B6In this experiment, the material.2-C5was screened with a beta-glucosidase and CMC activity from cattle rumen microbial library, functional gene is1414bp, ORF length of657bp (is named unbifCS) encoding219amino acids.1-B6strain was screened with beta-glucosidase activity from the termite gut microbes library, functional gene is1021bp ORF length of825bp (is named ungluB6) encoding275amino acids. The functional genes was cloned based on sequence information by design PCR primers, prokaryotic expression system in Escherichia coli BL21(DE3) and pET28a (+) expression vector was recombinated, The functional strains pET-unbifC5/BL21was successfully constructed. After0.1mM IPTG induction at37℃for6h, functional proteins unbifC5expressed, and purified by Ni-NTA His· Bind Resins analysis more. In unbifC5function protein expression studies, we found that it still has a beta-glucosidase activity and CMC enzyme activity is lost, may be a full-length gene encoding multiple open reading frame; the optimum reaction temperature and pH of unbifC5were37℃,6.4; Under acid condition the enzyme activity is stable but reduced after heating. The beta-glucosidase activity reached65.49U/mL with PNPG as determination substrate. When the low concentrations of glucose presence, the beta-glucosidase enzyme activity of functional protein increased, and in the concentration of nearly400mM glucose the beta-glucosidase enzyme activity still retains60%, the others characteristics of unbifC5need further study. The purpose of this paper is to tap the wealth of uncultured microorganisms cellulase resources, looking for novel cellulase gene, laid a theoretical foundation for the production of bio-ethanol fuel.