Expression And Activities Of Recombinant CBHII Gene In Escherichia Coli And Lactobacillus

Currently research of cellulase mainly on glucan endonuclease,but exonuclease is the only can act on the crystalline cellulose components in the hydrolysis of cellulose,CBHâ…¡gene is one of the exonucleases,and it is the key enzyme of the whole hydrolysis process of cellulose.Data proved that the low pH value prevent the hydrolytic efficiency of cellulose through prevent microorganisms growth(the lowest pH value that bacteria and fungi growth is 5.9).Using genetic engineering introducing the function of hydrolysis cellulose into the bacterium of rumen microorganism maybe is the efficient approach to increase the digestibility of cellulose. The research and development of cellulase molecular biology and genetic engineering will accelerate the application of cellulase in industrial and agricultural process,it have practical significance for mankind to solve the energy crisis,food shortages,and environmental pollution problems.This study is to obtain the transgenic engineering bacteria through recombinant vector,recombinant vector transformed the accept bacteria which were Escherichia coli and Lactobacillus,finally checking the acid cellulase activity to validate the transgenic bacteria in Escherichia coli and Lactobacillus.First,the pW425t,a prokaryotic expression shuttle vector between Escherichia coli and Lactobacillus, and plasmid pMD18-T-CBHâ…¡were digested with restricted endonuclease Smaâ… and Sphâ… ,reclaimed and ligated the aim segments with T4 DNA ligase,the purified CBHâ…¡gene was subcloned into the expression vector pW425t,sequencing results showed CBHâ…¡gene order and GeneBank published is 99.79%,through sequencing to validate the ligation site and correct reading frame shift,thus the recombinant vector pW425t-CBHâ…¡was constructed.Secondly,recombinant vector pW425t-CBHâ…¡was transformed into thyAgene -mutant sensitive accepter Escherichia coli and Lactobacillus with CaCl₂ and the electroporation method respectively.Thirdly,the transformed bacteria were incubated on LB and MRS media,through isolation of the plasmid DNA,restriction enzyme analysis,PCR,growth function remedy to identification the positive clone. The results of 1%electrophoretic indicate that CBHâ…¡gene may integrate into Escherichia coli and Lactobacillus,the PCR productions' molecular weight is 1.4kb respectively.Finally,the positive clones were cultivated in LB and MRS liquid media for the cellulase activity analysis.Analysis through SDS-PAGE of the expressed product in cell showed that an anticipated protein of about 49.6kd had been produced. CBHâ…¡transformants can produce clear hydrolysis halos on the Congo-Red-CMC plate,this proved that the both transgenic Escherichia coli and Lactobacillus have the ability of secreting acid cellulase.This study uses the CMC and pNPC method to check the cellulase activity,Optimum enzyme activity was at 50℃and pH5.0.