关键词: 角蛋白酶, 异源表达, 信号肽, 芽孢杆菌

Abstract: This work is objected to screen and construct a strain of efficiently degrading feather keratin for increasing the secretory expression of keratinase. Soil samples were collected from pigs and sheep pens of five different regions in China,and milk medium was used in preliminary screening,then feathers as the sole carbon and nitrogen source were used to rescreen strain producing keratinase. The strain was classified and determined based on the observed morphology and 16S rDNA sequence Analysis. In order to further improve the fermentation activity of the strain,5 signal peptide sequences（KerK,YoaW,DacB,NprE,and SacB）were screened and optimized,and recombinant plasmid was constructed one the basis of pWB980,which was transformed into Bacillus subtilis WB600 to express keratinase. The strain that efficiently degraded feather was identified as Bacillus sp. At 37℃ and after 48 h fermentation,the keratinase activity of the initial strain M was 21.98 U/mL. The recombinant strain R3-DacB containing signal peptide DacB were obtained via screening and optimizing the signal peptides,and its keratinase activity reached the highest by 226.34 U/mL,which was 10 folds of that by the initial strain M. In conclusion,the recombinant strain R3-DacB has promising effect on the degradation of feather waste,which is of great significance to the industrial production of keratinase.

Key words: keratinase, heterologous expression, signal peptide, Bacillus sp.