The Random Mutation And UV Mutation On Two Components Of Cellulase Gene

In order to further enhance the expression ability ofβ-glucosidase and endoglucanase from the strain of genetic engineering and provide technical guidance for further research and application on two kinds of enzymes,we explored the feasibility and methods on directed evolution of two kinds of enzymes and mutation breeding of the strain of genetic engineering,the main results are as follows:(1)β-glucosidase was directedly evolved in vitro by error-prone PCR,then it was ligated with pTRC99A vector and transferred into E.coli BL21 CodonPlus(DE3)-RIL.The results show that the gene ofβ-glucosidase was unable to express in E.coli BL21 CodonPlus(DE3)-RIL, the protein had formed into inclusion body.(2)The strain of genetic engineering GS115-bgl1-38 was mutated by ultraviolet light,two high production strain was obtained:GS115-bgl1-9 mutation and GS115-bgl1-37 mutation,their enzyme production capacities increased by 2 times and 1.5 times separately,two mutants kept stable after 10 times subculture.We also have studied on characters of two mutants,compared with the contrast,mutations's optimal reaction temperature had little change,but the optimal pH had expanding tendency, metal ions had most impact on ability.(3)By single and orthogonal test,we studied the culture medium for the expression ofβ-glucosidase from GS115-bgl1-9 mutation. The results indicated that the appropriate conditions were initial pH5.0 of BMGY,adding 0.5% histidine, inoculumed 4% with additive amount of methanol 0.5% every 24h. In addition, adding 0.2% Tween80 can enhanceβ-glucosidase activity.(4)Endoglucanase gene from B.thermoliquefaciens-NL was directedly evolved in vitro by error-prone PCR,then it was ligated with pTRC99A vector and transferred into E.coli BL21 CodonPlus(DE3)-RIL,the mutant library had been construsted.Four lower production strains were obtained ,they were named: pTRC99A-Cel5A-1,pTRC99A-Cel5A-2,pTRC99A-Cel5A-3 and pTRC99A-Cel5A-4,the ability of 4 mutative strains were: 0.050 IU/ml,0.050 IU/ml,0.048 IU/ml,0.043 IU/ml. Compared with the contrast, characters of 4 mutations were changed a little, and through sequencing we knew the bases of genes of 4 mutations were changed a little , so we thought that the change of bases from gene may lead to the change of enzymes'character.