Point mutation Gln121-Arg increased temperature optima of Bacillus lipase (1.4 subfamily) by fifteen degrees

Goomber, Shelly and Kumar, Rakesh and Singh, Ranvir and Mishra, Neelima and Kaur, Jagdeep (2016) Point mutation Gln121-Arg increased temperature optima of Bacillus lipase (1.4 subfamily) by fifteen degrees. In: INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 88 . pp. 507-514.

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Abstract

Small molecular weight Bacillus lipases are industrially attractive because of its alkaline optimum pH, broad substrate specificity and production in high yield by overexpression both in Escherichia coli and Bacillus subtilis. Its major limitation of being mesophilic in nature is constantly targeted by laboratory evolution studies. Herein metagenomically isolated Bacillus LipJ was randomly evolved by error prone PCR and library of variants were screened for enhanced thermostability. Point mutant Gln121Arg was extensively characterized and it showed dramatic shift of Temp. opt to 50 degrees C compared to 37 degrees C for parent enzyme. Thermostability studies at 45 degrees C and 50 degrees C determined six fold increase in half life for point variant Gln121Arg compared to LipJ. Circular dichroism (CD) and tryptophan fluorescence study established enhanced thermostability of Gln121Arg. Specific activity of point variant Gln121Arg was comparable to wild type with increased substrate affinity (Km reduced). Reduced kcat for variant Gln121Arg infer that kinetic and catalytic efficiency of mutant was compromised. Structural implications by homolog modelling predicted Gln121 to be placed within longest loop of the structure at surface. Localization of loop due to additional polar interactions by Arg121 to protein core defines molecular basis of enhanced thermostability of random point variant Gln121Arg. (C) 2016 Elsevier B.V. All rights reserved.

Item Type:	Journal Article
Publication:	INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Publisher:	ELSEVIER SCIENCE BV
Additional Information:	Copy right for this article belongs to the ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS
Keywords:	Directed evolution; Lipase; Error prone PCR; Thermostability; Homolog modelling
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	30 Jun 2016 05:43
Last Modified:	30 Jun 2016 05:43
URI:	http://eprints.iisc.ac.in/id/eprint/54078

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