Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus.

Singh, J and Vohra, R M and Sahoo, Debendra K (2001) Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus. Journal of industrial microbiology & biotechnology, 26 (6). pp. 387-93. ISSN 1367-5435

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Abstract

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M(r)) of 28.7 kDa, whereas protease B, with a M(r) of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K(m) values of these two proteases on SAAPF-pNa were higher than that for alpha-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH(2)-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family.

Item Type:	Article
Additional Information:	Copyright of this article belongs to Springer Science.
Uncontrolled Keywords:	alkalophilic Bacillus; serine protease; properties; substrate specificity
Subjects:	Q Science > QR Microbiology
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	04 Jan 2012 14:54
Last Modified:	07 Jan 2015 06:16
URI:	http://crdd.osdd.net/open/id/eprint/287

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