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Swapping of pro-sequences between keratinases of Bacillus licheniformis and Bacillus pumilus: Altered substrate specificity and thermostability

Paper ID Volume ID Publish Year Pages File Format Full-Text
17433 42668 2012 8 PDF Available
Title
Swapping of pro-sequences between keratinases of Bacillus licheniformis and Bacillus pumilus: Altered substrate specificity and thermostability
Abstract

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 45 min 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.

► Ker ProBP–BL and Ker ProBL–BP were generated by pro-sequence swapping. ► Variants were generated in trans/vitro by folding in the presence of pro-sequence. ► Changes acquired by variants were similar to the source of pro-sequence. ► Pro-sequence plays a major role in determining the final properties of mature enzyme.

Keywords
Bacillus licheniformis; Bacillus pumilus; Keratinase; Pro-sequence
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Swapping of pro-sequences between keratinases of Bacillus licheniformis and Bacillus pumilus: Altered substrate specificity and thermostability
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 51, Issue 3, 10 August 2012, Pages 131–138
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us