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A keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 was grown in a 3-L batch fermenter containing wool waste as the main medium and cell growth rate was determined as the key factor to affect keratinase yield. Three strategies of temperature-shift procedure, two-stage DO control and fed-batch process were used to change growth rate. And a 62.2% improvement of keratinase yield was achieved. With the glucose fed-batch procedure in 30-L fermenter, keratinase production was significantly improved up to 117.7% (1728 U/ml) as compared with initial data (793.8 U/ml) in a 3-L fermenter and with much shortened fermentation time within 18 h. Significant structure changes and high levels of free amino acids from wool decomposition indicated the possible applications for wool waste management and fertilizer industry. The remarkable digestion of wool cuticle also suggested its potential utilization in textile industry.

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Keratinases are proteolytic enzymes capable of degrading insoluble keratins. The importance of these enzymes is being increasingly recognized in fields as diverse as animal feed production, textile processing, detergent formulation, leather manufacture, and medicine. To enhance the thermostability of Bacillus licheniformis BBE11-1 keratinase, the PoPMuSiC algorithm was applied to predict the folding free energy change (ΔΔG) of amino acid substitutions. Use of the algorithm in combination with molecular modification of homologous subtilisin allowed the introduction of four amino acid substitutions (N122Y, N217S, A193P, N160C) into the enzyme by site-directed mutagenesis, and the mutant genes were expressed in Bacillus subtilis WB600. The quadruple mutant displayed synergistic or additive effects with an 8.6-fold increase in the t 1/2 value at 60 °C. The N122Y substitution also led to an approximately 5.6-fold increase in catalytic efficiency compared to that of the wild-type keratinase. These results provide further insight into the thermostability of keratinase and suggest further potential industrial applications.