拟无枝酸菌属羽毛降解菌株BJA-103的诱变育种

Mutagenesis breeding of feather degrading strain Amycolatopsis.sp BJA-103

【目的】对具有羽毛降解能力的BJA-103菌株进行硫酸二乙酯(DES)诱变,筛选羽毛降解速率更快、降解程度更高的突变株,并对突变株产生的角蛋白酶性质进行初步探究,为其进一步扩大发酵及生产应用提供理论依据。【方法】使用DES对BJA-103菌株进行诱变,涂布解除氮代谢分解调节筛选培养基为第1次初筛,点植法接种牛奶平板为第2次初筛,羽毛摇瓶降解为复筛。得到理想突变株后,对该突变株进行羽毛降解试验,初步探究降解时间、温度、pH对角蛋白酶活性的影响。【结果】筛选获得解除氮代谢分解调节突变株库(1253株),牛奶平板筛选水解圈宽度为8.5~12 mm的突变株5株(RT、BM′、CV′、FX′、NK′),平均水解圈大小为野生型的2.5倍。羽毛降解复筛得到突变株NK′,降解试验中,其48 h内的羽毛降解率为65.29%,远大于野生型(12.82%);其最终降解率为90.74%(降解144 h),大于野生型的降解率79.24%。突变株NK′产生的角蛋白酶最适发酵时间为72 h,最适反应温度为70℃,最适反应pH为11。【结论】筛选出1株羽毛降解速率快、降解程度高的突变株NK′,其所生产的角蛋白酶可耐高温、高碱环境。

【Objective】Diethyl sulfate(DES)was applied for mutagenesis of a feather degrading strain,BJA-103 to obtain mutant with higher feather-degrading rate and keratinase from the mutant was preliminarily investigated to provide basis for further fermentation and application.【Method】After DES was use for mutagenesis,first screening was carried out by nitrogen catabolite repression medium followed by the second screening of smaller hydrolysis halos in skim milk plate medium.The final mutant was obtained by feather degrading screening.Then,effects of degrading time,temperature and pH on degradation of the mutant were analyzed.【Result】The mutant library of 1253 mutants with freed nitrogen catabolite repression was obtained through first screening,and 5 mutants(RT,BM′,CV′,FX′and NK′)with hydrolysis halos(8.5-12 mm)of 2.5 times larger than wild type were screened in the second step.Finally,mutant NK′was selected in feather degrading screening.Its feather-degrading rate of 65.29%in 48 h was much larger than that of wild type(12.82%).Its final feather-degrading rate of 90.74%was also larger than that of wild-type(79.24%).The optimal conditions for keratinase from NK′were fermentation time of 72 h,temperature of 70℃and pH of 11.【Conclusion】One mutant NK′with high and fast feather-degrading rate was screened and its keratinase was resistant to high temperature and high alkalinity.