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Designing of nitrile hydratase from alkaline protease using quanidine hydrochloride and cobalt metal ion

Paper ID Volume ID Publish Year Pages File Format Full-Text
55225 47043 2012 6 PDF Available
Title
Designing of nitrile hydratase from alkaline protease using quanidine hydrochloride and cobalt metal ion
Abstract

Alkaline protease (22.5 kDa) from Bacillus licheniformis was denatured using 3–6 M Guanidine hydrochloride followed by renaturation with cobalt metal ion in buffer solution (pH 7.5) in order to design cobalt dependent nitrile hydratase AP-xMGdn-Co, where x = 3, 4, 5 and 6. The native alkaline protease showed a 20% residual molar ellipticity at 212 nm while on treatment with Gdn-HCl, it got reduced to 12% due to nearly complete collapse of the globular structure of the protein. The observed circular dichroism spectra of the renatured proteases with Co metal ion are different from the native enzyme, because those molecules contain many metal chelating sites through its coordinating amino acid residues. The introduction of cobalt centered new active site was evidenced by the fluorescence spectroscopy (appearance of a new peak at 360 nm) and ICP analysis (0.0476–0.0553 mg/L). The specific activities of the modified enzymes were measured using the selective catalytic hydrolysis of 3-cyanopyridine to nicotinamide. The highest specific activity of renatured enzymes (AP-xMGdn-Co) were obtained at optimum pH of 7.5 and temperature of 50 °C, due to the proper co-ordination of amino acid residue with cobalt metal ion and the appropriate arrangement of α-helix, β-strand, turn and unordered segment in the globular structure.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (335 K)Download as PowerPoint slideHighlights► Alkaline protease was modified into nitrile hydratase by reversible denaturation. ► Guanidium hydrochloride denatured enzyme was renatured in Co ion solution. ► Activity of renatured enzyme was measured for nitrile to amide conversion. ► Determined specific activity and kinetic parameters with 3-cyanopyridine substrate.

Keywords
Alkaline protease; Guanidine hydrochloride; Reversible denaturation; Cobalt metal ion; Renaturation; Nitrile hydratase activity
First Page Preview
Designing of nitrile hydratase from alkaline protease using quanidine hydrochloride and cobalt metal ion
Publisher
Database: Elsevier - ScienceDirect
Journal: Catalysis Today - Volume 198, Issue 1, 30 December 2012, Pages 353–358
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Catalysis