Cloning, Expression And Molecular Modification Of The End-β-1, 4-Glucanase Gene From Coptotermes Formosanus

Cellulose(Cellulose)is the most organic material and renewable resources,which is the main product of photosynthesis.There are more than 10 billion tons of biomass plants on Earth,Over half of which is cellulose and hemicellulose.However,the cellulose has not been used fully.As the shortage of petrochemical energy on the Earth,people start to use cellulose to fermente ethanol.But now the activity of cellulose are low and fermentation are very slow,so searching for new cellulases,or mutating the existing cellulase gene to get higher cellulase become a hot research.In this research,we extracted Coptotermesformosanus' total RNA,and obtained the cDNA by reverse transcription.The gene encoding endo-β-1,4-glucanase was isolated by PCR,and then it was ligated to an Ecoli vector and a yeast vector to construct the cellulases-producing gene engineering strains.The endo-β-1,4-glucanase of recombinant Ecoli strains was purified by nickel affinity chromatography.The results showed that the optimal tempreture and pH of purified endoglucanase were 42℃/6.5 respectively;The Vmax and Km for endo-β-1,4-glucanase were shown to be 0.071 umol/mL~*min /80.271mg/mL/13.544U/mg.In this research,we determined three endo-β-1,4-glucanase amino acid mutation sites(C53,E53 and C56)by three-dimensional model of endo-β-1,4-glucanase and ProSA2003 software(knowledge-based potentials).The optimum temperature,optimum pH,Km,Vmax and activity of mut-C53 were shown to be 42℃/6.5/37.683mg/mL/0.0334umol/mL~*min/9.912 U/mg respectively; The optimum temperature,optimum pH,Km,Vmax and activity of mut-E53 were shown to be 37℃/6.5/93.819mg/mL/0.124umol/mL~*min/16.421U/mg respectively;The optimum temperature,optimum pH,Km,Vmax and activity of mut-C56 were shown to be 42℃/8.0/26.809mg/mL/ 0.0324umol/mL~*min/16.470U/mg respectively.