Isolation Of Silkworm Sericin And Feather Degragation Bacteria And Optimization Of Keratinase Fermentation Medium

The samples were obtained from the reeling mill waste water in Yi shui county, Linyi and Taian. Silkworm sericin and feather degradation bacteria were isolated through selective medium. REP -PCR was used to fingerprint different silkworm sericin degradation bacterial isolates. Silkworm sericin and feather degradation bacteria were identified by means of phenotypic characteristics and phylogenetic analysis of 16S rRNA sequences. Keratinase activity of silkworm sericin and feather degradation bacteria was measured. We found the optimal concentrations of components of S59 strain that had highest enzyme activity through the cultivation in shake-flask fermentation.The main results were as follows:Thirty-nine bacterial isolates that had sericin degradation activities were obtained from the reeling mill waste water by Coomassie Brilliant Blue G-250 method. The silkworm sericin degradation activities of these isolates were between 2.69% and 28.86%. REP -PCR was used to fingerprint different silkworm sericin degradation bacterial isolates. The main results were as follows: 39 silkworm sericin degradation bacteria were divided into 12 clusters at the dissimilarity of 0.9 with REP -PCR fingerprints, respectively.10 strains that had sericin degradation activities also had feather degradation activities. And their degradation activities were between 7.06 U～13.49 U. We found that 10 strains belonged to Bacillus sp. by means of phenotypic characteristics and phylogenetic analysis of 16S rRNA sequences. Their 16S rRNA sequences had been registered at Genbank.Through the cultivation in shake-flask fermentation, the effects of nutrition on production of keratinase produced by S59 were studied. The optimum carbon, nitrogen, C/N, inorganic salt were confirmed by one-factor-at-a-time. In the optimization of medium, the influences of glucose, powder of corn, feather and CaCO3 on keratinase production were first evaluated using a fractional factorial design. Powder of corn and feather tested had significant effect on keratinase production. And they both affected keratinase production negatively. CaCO3 and glucose had nonsignificant effect on keratinase production, so select low level. In the last step, the optimal concentrations of components were determined by central composite design and response surface analysis.The components of optimum medium for production of keratinase were: glucose 5.00 g, powder of corn 32.83 g, feather 10.67 g, CaCO3 3.00 g, ddH2O 1000 mL. The enzyme activity of optimized medium and fermentation conditions got to 41.04 U, 204.23% higher than that of basal medium.