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Purification and Characterization of Serine Proteinase from Alkaliphilic Bacillus sp. RS1
Publisher(s)
University of the Thai Chamber of Commerce
Date Issued
2007
Other Contributor(s)
University of the Thai Chamber of Commerce. School of Science and Technology
Abstract
Alkaliphilic Bacillus sp. RS1, isolated from a waste water treatment plant of a pulp and paper manufacturer in Kanjanaburi provine Thailand, produced an extracellular serine proteinase and was identified as Bacillus halodurans DSM 497 through 16S rDNA analysis. Maximum enzyme activity was achieved when the bacterium was grown on 2% deoiled soybean meal with shaking at 250 rpm, pH 11, 370C and 60 h incubation period. The enzyme has been purified by ammonium sulfate precipitation and a combination of DEAE ion-exchange chromatography, Mono Q ion-exchange chromatography and Gel-filtration Sepracryl S-100 chromatography. Purified enzyme activity was strongly inhibited by phenylmethyl sulphonylfluoride. The purified enzyme was thus a serine proteinase. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 11(possible the serine residuce present at the active site). The optimum temperature of serine was 60 0C. It hydrolyzed natural substrate AZCL-casein and caseine. The molecular weight of the purified serine proteinase were determined to be 44.7 and 30.2 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). In investigating the Bacillus halodurans DSM 497, serine proteinase can catalyzed hydrolysis of Gyl-Arg-p-nitroanilide dihydroxychloride but not catalyzed Ala-Ala-Phe p-nitroanilide, Val-Ala-p-nitroanilide acetate, N-succinyl-Gly-Gly-Phe-p-nitroanilide, N-succinyl-L-Phenylalanine-p-nitroanilide and N-Benzoyl-L-Tyrosine-p- nitroanilide indicated that P1 subsite of serine proteinase specific for arginine.
Sponsorship
Research Support Office, UTCC
Subject(s)
Degree Grantor
University of the Thai Chamber of Commerce
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public
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Rights Holder(s)
University of the Thai Chamber of Commerce
Bibliographic Citation
Ratchanee Saiprajong, Surapong Pinitglang, Khanok Ratanakhanokchai (2007) Purification and Characterization of Serine Proteinase from Alkaliphilic Bacillus sp. RS1.
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