Direction Evolution Of High Temperature,low PH Cellulase

Cellulose is the most abundant and widely distributed renewable biopolymers on the word.The transformation of cellulose has very important significance for the solution of energy crisis problems.Cellulose enzyme can effectively degrade cellulose,the use of cellulose enzyme degradation of pollution-free and efficient,but the cellulose enzyme used in industrial production has a lot of inadaptability,people also through various methods to improve cellulase enzyme activity or raise the enzymology characteristics of it.In this test,a strain named JC-1 from the soil which can produce cellulase was screened.It is a bacterium through gram staining and the colony morphology observation and 16 SrDNA molecular biology identification.Fortunately,the strain was identified a G+ bacterium and could produce spores.Eventually,the strain JC-1was prove to be Bacillus amyloliquefaciens strain by constructing phylogenetic tree.In order to determine the optimal bacterial growth and enzyme production condition,experiments were at temperature 37?,pH7.0,quantity 3% and 36 h cultivation.The optimum carbon source was CMC-Na,while the nitrogen source was peptone.The best enzyme activity conditions were at temperature 50?,pH5-6.A novel mutation approach,the atmospheric room temperature plasma(ARTP),was used to treat Bacillus amyloliquefaciens strain JC-1 for the breeding of high cellulose productivity.The mutant with high cellulose productivity was quickly screened out based on the ratio of transparent circle on congo red screening plate and the cellulose productivity on 96-well plate.The obtained mutant T-16 exhibited good genetic stability and cellulose productivity reached 1.759U/mL,which was 41.8% higher than that of original strain.The fermentation characteristics of the mutant strain T-16 were studied and the results showed that the cellulose production time advanced 8 hours compared with the original strain.In order to further transform the enzyme molecule,the endoglucanase gene of Bacillus amyloliquefaciens T-16 and T-8 were cloned.The method is established by optimizing the time of DNase I digestion and the quantity of DNA template of primerless PCR and cycle number primer PCR.The glucanase gene was connected to the expression vector was transformed into E.coli BL 21(DE3).Finally the enzyme was successfully expressed.Eleven mutant strains were screened by screening and re-screening.The enzymatic properties of endoglucanase CY049 and CY173 expressed by DNA shuffling were researched.The temperature and pH value of CY049 the enzyme activity was65 ?and 4.5,respectively.Under 85 heat preservation 60 min,enzyme activity to keep ?more than 50%.The temperature and pH value of CY173 the enzyme activity was 55 ?and 4.5,respectively?...