The Reserch Of Improving Activity Of Keratinase Gene From Bacillus Subtilis By Directed Evolution

Keratinases are a group of proteolytic enzymes that can hydrolyze insoluble keratins,and it can be secreted by many kinds of microbes.Due to the keratin after degradation contains many amino acids and trace elements, growth factors.With the degradation of keratinase, we can make the abandoned feather keratin into the animal feed additive, and add more nutrients for animal feeding. The degradation of keratinase is under a mild reaction condition, and won’t produce the problem of environmental polIution.Through protein engineering techniques to transform the Keratinase gene is an effective way to achieve higher scientific and practical value of keratinases.In this study, directed evolution technology has been used to transform the Keratinase gene, the error-prone PCR technology is a mature means in the lab.After the exploration of the error-prone PCR conditions:adding the manganese ions in the system, and increasing the concentration of magnesium ions and adjusting the ratio of dNTP.Random mutation is introduced to construct genetic diversity mutant library.Use high-throughput screening methods for screening, won a corner of the production plant is highly active protease mutations in the gene engineering bacteria GS, A maximum keratinase yield can be achieved 30U/mL, compared with the original strain DS (20U/mL) increased 50%, and the special activity is 95.43U/mg,compared with the original strain DS (59.17 U/mg) increased 61%.The optimal reaction temperature for the enzyme is 60℃, the optimum reaction pH is 7.5.After DNA sequencing we found that 10 bases have changed in mutant sequence.These mutations caused 4 amino acid changes, that is His126Asp, Cys165Gly, Thr215Ser, Gly300Ser. After protein secondary structure prediction we found that the mutant Keratinase GS has the similar physicochemical property and isoelectric point compare with original strain.After protein three-dimensional structure prediction we found that the mutant keratinase GS has the same backbone and 3D structure with the original strain, and the active center has no change.The mutation of 4 amino acid may lead to micro-variation on protein structure and increase the flexibility of enzyme constitution,that may result in the inceasing of enzyme activity.In summary, the mutant keratinase with better catalytic efficiency was generated. What is more, structure analysis of mutant and natural keratinase explored the relationship between structure and function and the synergy effect of mutant sites for keratinase properties, which could provide the experimental basis for the further application of protein engineering techniques for improving the physico-chemical properties of the keratinase. Also, the development of these keratinase variants will enhance the industrialization application.