Swapping of pro-sequences between keratinases of Bacillus licheniformis and Bacillus pumilus: Altered substrate specificity and thermostability
2012
Rajput, Rinky | Tiwary, Ekta | Sharma, Richa | Gupta, Rani
Pro-sequences were swapped in cis between keratinases from Bacillus licheniformis (Ker BL) and Bacillus pumilus (Ker BP) to construct Ker ProBP–BL and Ker ProBL–BP, respectively. Expression of these keratinases was carried out constitutively by E. coli HB101-pEZZ18 system. They were characterized with respect to their parent enzymes, Ker BL and Ker BP, respectively. Ker ProBP–BL became more thermostable with a t₁/₂ of 45min at 80°C contrary to Ker BL which was not stable beyond 60°C. Similarly, the activity of Ker ProBP–BL on keratin and casein substrate, i.e. K:C ratio increased to 1.2 in comparison to 0.1 for Ker BL. Hydrolysis of insulin B-chain revealed that the cleavage sites increased to six from four in case of Ker ProBP–BL in comparison to Ker BL. However, cleavage sites decreased from seven to four in case of Ker ProBL–BP in comparison to the parent keratinase, Ker BP. Likewise, Ker ProBL–BP revealed altered pH and temperature kinetics with optima at pH 10 and 60°C in comparison to Ker BP which had optima at pH 9 and 70°C. It also cleaved soluble substrates with better efficiency in comparison to Ker BP with K:C ratio of 1.6. Pro-sequence mediated conformational changes were also observed in trans and were almost similar to the features acquired by the chimeras constructed in cis by swapping the pro-sequence region.
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