Radha S, Gunasekaran P
Department of Genetics, Center for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India.
Bioresour Technol. 2008 Sep;99(13):5528-37. doi: 10.1016/j.biortech.2007.10.052. Epub 2007 Dec 18.
The ker gene encoding pre-pro keratinase of Bacillus licheniformis MKU3 was cloned with xylose inducible promoter (PxylA) or alpha-amylase promoter (PamyL) or both in Escherichia coli-Bacillus shuttle vector, pWH1520 generating recombinant plasmids pWHK3, pWAK3 and pWXAK3 respectively. Compared with Bacillius megaterium MS941 (pWXAK3) expressing ker gene with PxylA-PamyL promoters, B. megaterium MS941 (pWAK3) with PamyL displayed higher keratinase yield (168.6 U/ml) and specific activity (14.59 U/mg) after 36 h of growth in LB medium, however the keratinase yield decreased in the culture grown in LB medium supplemented with starch or xylose or both. A maximum yield of 186.3 U/ml with specific activity of 17.25 U/mg was obtained from xylose induced keratinase expression in B. megaterium MS941 (pWHK3) grown for 24h. The recombinant plasmids were stably maintained with sustained expression of keratinase for about 60 generations in B. megaterium MS941 rather than in B. megaterium 1,4945.
在大肠杆菌-芽孢杆菌穿梭载体pWH1520中，利用木糖诱导型启动子（PxylA）或α-淀粉酶启动子（PamyL）或二者，克隆了地衣芽孢杆菌MKU3编码前原角蛋白酶的ker基因，分别构建了重组质粒pWHK3、pWAK3和pWXAK3。与用PxylA-PamyL启动子表达ker基因的巨大芽孢杆菌MS941（pWXAK3）相比，在LB培养基中生长36小时后，带有PamyL的巨大芽孢杆菌MS941（pWAK3）显示出更高的角蛋白酶产量（168.6 U/ml）和比活性（14.59 U/mg），然而，在添加淀粉或木糖或二者的LB培养基中培养时，角蛋白酶产量下降。在巨大芽孢杆菌MS941（pWHK3）中，木糖诱导的角蛋白酶表达在培养24小时后，获得了最大产量186.3 U/ml，比活性为17.25 U/mg。重组质粒在巨大芽孢杆菌MS941中稳定维持，角蛋白酶持续表达约60代，而在巨大芽孢杆菌14945中则不然。
