The Open Microbiology Journal


ISSN: 1874-2858 ― Volume 10, 2016

Purification of Protease from Pseudomonas thermaerum GW1 Isolated from Poultry Waste Site



Smriti Gaur, Sarita Agrahari , Neeraj Wadhwa*
Department of Biotechnology, Jaypee Institute of Information Technology (Deemed University) A-10, sec 62, Noida, Uttar Pradesh, India

Abstract

An extracellular protease was purified from Pseudomonas thermaerum GW1 a new strain identified by morphological, biochemical and 16S rDNA sequencing. It was isolated from soil of Poultry waste site at Ghazipur near Ghaziabad, Delhi. The strain produces extra cellular protease in the culture media that was maintained at 37°C, 140 rpm. The media was harvested for protease after 48 hrs of incubation at 37°C in basal media supplemented with 1% casein. We report 6.08 fold purification of enzyme following ammonium sulphate precipitation and DEAE-cellulose chromatography. The molecular weight of the enzyme was estimated to be approximately 43,000 daltons as shown by casein zymography studies. The optimum pH for the proteolytic activity was pH 8.0 and enzyme remained stable between pH 5 -11 at 60°C. Interestingly Mn2+ (5mM) activated enzyme activity by 5 fold, while Cu2+, Mg2+and Ca2+ moderately activated enzyme activity, where as Zn2+, Fe2+ and Hg2+ inhibited enzyme activity. The protease produced was stable in presence of 50 % (v/v) ethylacetate and acetone. Isopropanol, methanol and benzene increased protease activity by 2.7, 1.3 and 1.1 fold respectively but was inhibited in presence of glycerol and DMSO. This organic solvent-stable protease could be used as a biocatalyst for enzymatic peptide synthesis

Keywords: Protease, Pseudomonas, Casein zymography.


Article Information


Identifiers and Pagination:

Year: 2010
Volume: 4
First Page: 67
Last Page: 74
Publisher Id: TOMICROJ-4-67
DOI: 10.2174/1874285801004010067

Article History:

Received Date: 7/6/2010
Revision Received Date: 22/6/2010
Acceptance Date: 25/6/2010
Electronic publication date: 13/8/2010
Collection year: 2010

© Gaur et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the Department of Biotechnology, Jaypee Institute of Information Technology (Deemed University) A-10, sec 62, Noida, Uttar Pradesh, India; Tel: + (91)-120-2400973-976; Fax: + (91)-120-2400986; E-mail: neeraj.wadhwa@jiit.ac.in


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