Isolation And Identification Of Cellulase Bacteria From Termites And Vector Construction And Prokaryotic Expression Of Cellulase Genes

Lignin(18-35%),cellulose(20-50%)and hemicellulose(15-35%)is the main component of lignocellulose.Among them,cellulose is a linear polysaccharide formed by linking D-glucopyranosyl groups through ?-1,4 glycosidic bonds.It is also the most important constituent of plants and accounts for about 20%-40%of the total weight,although cellulose is the most abundant renewable polymer,but its polymer form can't be directly used by animal organisms.However,termites have a large number of microorganisms that degrade cellulose,and they ofen use wood that is rich in lignocellulosic.This study collected termite samples from Foping County of Shaanxi Province,screening strains which producing high activity cellulase from termites,cloning the cellulase gene,and expression it in Escherichia coli.This aimed to provide theoretical technology and basic materials for the artificial recombinant strains;After through the construction of Lactobacillus,the cellulase gene expressed and secreted in Lactobacillus reuteri.Finally,the degradation effect of recombinant strains to coarse fodder can be detected through the fermentation experiment.The main results of this study are as follows:1.The samples collected from the termites were identified by morphology and 16S rRNA,and they were determined to be Reticulitermes chinensis.Four strains which producing cellulase were screened out from termites by the medium of sodium carboxymethylcellulose and Congo red staining.They are identified as Bacillus subtilis,Enterobacter asburiae,Bacillus thuringiensis and Bacillus amyloliquefaciens.The cellulase activity of Bacillus subtilis is the highest.The optimum growth temperature and pH of the strain are 42? and 6.5,respectively.The optimum temperature and pH of enzymatic reaction are 70? and 4.5,respectively.Under these conditions,the cellulase activity of the strain is 2.36 U/mL.2.Primers were designed by related sequences of NCBI(sequence number:CP018173.1;KT992142.1).Two cellulase genes were amplified from Bacillus subtilis:endo-1,4-?-D-glucanase gene Cbs and ?-1,4-glucosidase gene BaG.The protein which is expressed by BaG gene expression in Escherichia coli has no cellulase activity.And the cellulase activity of the protein which is expressed by Cbs gene in Escherichia coli was up to 4.14 U/mL at 60 ? and pH 5.0.The cellulase gene Cbs and BaG were fused expressed in Escherichia coli.The molecular weight of the fused expressed protein was about 35 kDa,and the cellulase activity was 4.57 U/mL under the optimum condition 70 ? and pH 4.5.This indicates the gene BaG without cellulase activity and the Cbs gene fused expression to a new protein with higher cellulase activity.3.Gene Cbs 2 and Lactobacillus constitutive promoter(P32),signal peptide(SPlpo373)gene were connected to the pLEM-415 vector by double enzymes restriction,adapter ligation.Then we get the vector which can express and secret endoglucanase.The results that the vector express in Lactobacillus reuteri showed:The optimum growth time and temperature of the recombination strain are 21 h and 37 ?,respectively.Under the best conditions,the cellulase activity reaches 3.0 U/mL,the molecular weight of the enzyme is 55 kDa.The optimum temperature and pH of enzymatic reaction are 60-70 ? and 5.5.The enzyme has the best stability in the pH 5.5 and temperature 50-60 ?.Mg2+ can promot the cellulase activity;while the facor of Zn2+,Fe2+,Ba2+ and EDTA can inhibit it.4.Using recombinant strains to ferment the crude feed,the results showed that the corn straw,wheat straw,alfalfa hay had certain degradation.Compared with the wild strain,three kinds of forage crude fiber content decreased by 12.02± 1.23%,5.66±1.23%,11.75±0.90%.In this study,four strains which producing cellulase were screened from the Reticulitermes chinensis.And the cellulase activity(2.36 U/mL)of Bacillus subtilis is the highest.Two cellulase genes were amplified from Bacillus subtilis.The highest activity of endoglucanase is 4.14 U/mL,and the activity of ?-glucosidase is inactive.Two cellulase genes fused to produce a protein with higher cellulase activity.The CMCase activity of recombinant Lactobacillus is 3.0 U/mL,which have a certain degradation effect on the coarse feed.