Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.
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ABSTRACT:
SUBMITTER: Jorgensen PL
PROVIDER: S-EPMC91903 | biostudies-literature | 2000 Feb
REPOSITORIES: biostudies-literature
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Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.
Jørgensen P L PL Tangney M M Pedersen P E PE Hastrup S S Diderichsen B B Jørgensen S T ST
Applied and environmental microbiology 20000201 2
A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique. ...[more]
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Project description:This paper presents the results of the study on the production of protease by Bacillus luteus H11 isolated from an alkaline soda lime. B. luteus H11 was identified as an alkalohalophilic bacterium, and its extracellular serine endoprotease also showed an extreme alkali- and halotolerance. It was remarkably stable in the presence of NaCl up to 5 M. The enzyme was active in a broad range of pH values and temperatures, with an optimum pH of 10.5 and a temperature of 45 °C. It had a molecular mass of about 37 kDa and showed activity against azocasein and a synthetic substrate for the subtilisin-like protease, N-succinyl-l-phenylalanine-p-nitroanilide. The halo-alkaline protease produced by B. luteus H11 seems to be significant from an industrial perspective because of its tolerance towards high salinity and alkalinity as well as its stability against some organic solvents, surfactants and oxidants. These properties make the protease suitable for applications in food, detergent and pharmaceutical industries, and also in environmental bioremediation.
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Project description:We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.
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