Expression And Fermentation Optimization Of Stenotrophomonas Maltophilia Keratinase In P. Pastoris

Keratinases are proteinases, which have special activity of degrading keratin substrates,, and they have broad application prospects on fodder, cosmetics, leather industry and environmental protection. However, because of the low level of expression of keratinase and pathogenicity of the strains, there are few original strains utilized in commercial application.In previous studies, we screened a Stenotrophomonas maltophilia BBE11-1, which secrets one kind of keratinase degraded wool. Based on the advantages of Pichia pastoris expression system, this study successfully constructed P. pastoris SMD1168-p PIC9k-ker D, which has high expression of keratinase, then analyzed characterization of the recombinant keratinase. Furthermore, keratinase fermentation process was optimized to achieve high production of keratinase in 3 L fermentor. The major results are presented as follows:1. Stenotrophomonas maltophilia BBE11-1 keratinase gene ker D was optimized according to Pichia codon, and then it was connected to p PIC9 k, transferred to E. coli JM109. The p PIC9k-ker D was digested and transformed to P. pastoris SMD1168. Followed by plate screening and enzyme activity detection, a best recombinant strain was screened, whose keratinase yield was 256 U· m L-1. The optimal recombinant strain was named as P. pastoris SMD1168-p PIC9k-ker D.2. The keratinase was purified by ammonium sulfate precipitation, filtration, and gel purification sequentially. Ultimately pure keratinase was obtained. Characterization of the recombinant keratinase was investigated. We found that optimum reaction p H of recombinant keratinase is 10 and the optimum reaction temperature is 60 oC. Placed in 50 oC for 100 min, the recombinant keratinase still has more than 60% residual activity; under p H 5 to 9, the recombinant keratinase has good stability. In addition, studies on effects of different chemicals on keratinase activity suggest that Zn2+, Mn2+, Fe3+, Ca2+ can promote keratinase activity, Mg2+ has little effect, while Cu2+ inhibits keratinase activity. PMSF can inhibit all the keratinase activity. SDS and EDTA can significantly inhibit keratinase activity, and weak reducing agent Na2SO3 and DTT can promote keratinase activity. Results of substrate suggested that keratinase has strong activity on insoluble wool and feather. SEM picture of wool handled by keratinase showed that keratinase could destroy scale layer of wool fibers, which indicates keratinase can reduce felting shrinkage of wool.3. In the fed-batch phase, glycerol fed-batch utilized exponential feeding. Influence of mixed carbon feeding strategy on keratinase production were studied, the results showed that feeding strategy, sorbitol and mannitol mixed with methanol in suitable proportions, can promote yield of keratinase. When methanol and mannitol mix ratio was 20:0.5(w/w), keratinase yield reached 2048 U· m L-1, which was improved 87.2% compared with the control. In fed-batch phase, maintained culture system p H 5.5; p H in induction phase was optimized. The result suggested that keratinase production increases along with p H increase in certain range; under p H 7.5, keratinase yield reached 2432 U· m L-1, the enhancements was 18.3% compared with production under p H 5.5. Furthermore, effects of NH4+ concentration on the yield of the recombinant keratinase were explored. Ultimately, the optimum N H4+ concentration was determined as 500 mmol· L-1, and the keratinase yield reached 3175 U· m L-1 at 156 h, which is at the leading level and provides a reference for the keratinase industrial production.