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 t(1/2) 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.
authors: Department of Microbiology, University of Delhi, South Campus, New Delhi 110021, India.
authors: Rinky
authors: R
authors: Rajput
authors: Ekta
authors: E
authors: Tiwary
authors: Richa
authors: R
authors: Sharma
authors: Rani
authors: R
authors: Gupta
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004792:Enzyme Precursors; D010447:Peptide Hydrolases; C035855:keratinase