Radha Selvaraj, Gunasekaran Paramasamy
Department of Genetics, Center for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India.
Protein Expr Purif. 2009 Mar;64(1):24-31. doi: 10.1016/j.pep.2008.10.008. Epub 2008 Oct 25.
The keratinase gene from Bacillus licheniformis MKU3 was cloned and successfully expressed in Bacillus megaterium MS941 as well as in Pichia pastoris X33. Compared with parent strain, the recombinant B. megaterium produced 3-fold increased level of keratinase while the recombinant P. pastoris strain had produced 2.9-fold increased level of keratinase. The keratinases from recombinant P. pastoris (pPZK3) and B. megaterium MS941 (pWAK3) were purified to 67.7- and 85.1-folds, respectively, through affinity chromatography. The purified keratinases had the specific activity of 365.7 and 1277.7 U/mg, respectively. Recombinant keratinase from B. megaterium was a monomeric protein with an apparent molecular mass of 30 kDa which was appropriately glycosylated in P. pastoris to have a molecular mass of 39 kDa. The keratinases from both recombinant strains had similar properties such as temperature and pH optimum for activity, and sensitivity to various metal ions, additives and inhibitors. There was considerable enzyme stability due to its glycosylation in yeast system. At pH 11 the glycosylated keratinase retained 95% of activity and 75% of its activity at 80 degrees C. The purified keratinase hydrolyzed a broad range of substrates and displayed effective degradation of keratin substrates. The K(m) and V(max) of the keratinase for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA was found to be 0.201 mM and 61.09 U/s, respectively. Stability in the presence of detergents, surfactants, metal ions and solvents make this keratinase suitable for industrial processes.
地衣芽孢杆菌MKU3的角蛋白酶基因被克隆，并成功在巨大芽孢杆菌MS941以及毕赤酵母X33中表达。与亲本菌株相比，重组巨大芽孢杆菌产生的角蛋白酶水平提高了3倍，而重组毕赤酵母菌株产生的角蛋白酶水平提高了2.9倍。通过亲和层析，分别将重组毕赤酵母（pPZK3）和巨大芽孢杆菌MS941（pWAK3）产生的角蛋白酶纯化了67.7倍和85.1倍。纯化后的角蛋白酶比活性分别为365.7和1277.7 U/mg。巨大芽孢杆菌的重组角蛋白酶是一种单体蛋白，表观分子量为30 kDa，在毕赤酵母中适当糖基化后分子量为39 kDa。两种重组菌株产生的角蛋白酶具有相似的性质，如活性的最适温度和pH值，以及对各种金属离子、添加剂和抑制剂的敏感性。由于其在酵母系统中的糖基化，角蛋白酶具有相当高的稳定性。在pH 11时，糖基化角蛋白酶保留了95%的活性，在80℃时保留了75%的活性。纯化后的角蛋白酶能水解多种底物，并对角蛋白底物有有效的降解作用。该角蛋白酶对底物N-琥珀酰-Ala-Ala-Pro-Phe-pNA的K(m)和V(max)分别为0.201 mM和61.09 U/s。在洗涤剂、表面活性剂、金属离子和溶剂存在下的稳定性使得这种角蛋白酶适用于工业生产过程。
