A novel neutral protease from Thermoactinomyces species 27a: sequencing of the gene, purification, and characterization of the enzyme

The nucleotide sequence of the previously cloned (Zabolotskaya, M. V., Nosovskaya, E. A., Kaplun, M. A., and Akimkina, T. V. (2001). Mol. Gen. Mikrobiol. Virusol. No 1, 32-34) DNA fragment from Thermoactinomyces sp. 27a (GenBank Accession No. AY280367) containing the metalloproteinase gene was deter...

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Bibliographic Details
Published in:The Protein Journal 2004-10, Vol.23 (7), p.483-492
Main Authors: Zabolotskaya, Maria V, Demidyuk, Ilya V, Akimkina, Tatiana V, Kostrov, Sergey V
Format: Article
Language:English
Subjects:
Bacillus subtilis
Bacteria
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Base Sequence
Cloning, Molecular
Enzyme Precursors - chemistry
Enzyme Precursors - genetics
Enzymes
Gene Expression
Micromonosporaceae - enzymology
Micromonosporaceae - genetics
Molecular Sequence Data
Peptide Hydrolases - chemistry
Peptide Hydrolases - genetics
Peptide Hydrolases - isolation & purification
Thermoactinomyces
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Summary:The nucleotide sequence of the previously cloned (Zabolotskaya, M. V., Nosovskaya, E. A., Kaplun, M. A., and Akimkina, T. V. (2001). Mol. Gen. Mikrobiol. Virusol. No 1, 32-34) DNA fragment from Thermoactinomyces sp. 27a (GenBank Accession No. AY280367) containing the metalloproteinase gene was determined. A continuous open reading frame encoding a polypeptide of 673 aa was revealed. Analysis of this sequence demonstrated that the metalloproteinase from Thermoactinomyces sp. 27a is synthesized as a preproprotein and includes a leader peptide (26 aa), N-terminal propeptide (215 aa), mature region (317 aa), and additional C-terminal domain (115 aa). The recombinant enzyme from Thermoactinomyces sp. 27a was expressed in Bacillus subtilis AJ73 cells and purified by anion exchange chromatography to an electrophoretically homogeneous state. The determined N-terminal amino acid sequence of the mature protein was identical to that deduced from the gene. The obtained data suggest that the mature protein should include 432 aa and have a calculated molecular weight of 46,262 Da. However, the molecular weight of the mature protein determined by mass spectrometry was 34,190+/-70 Da indicating a C-terminal processing. The proteinase was not inhibited by phenylmethyl sulfonyl fluoride but was inhibited by o-phenanthroline and ethylenediaminetetraacetic acid. The enzyme had maximum activity by azocasein hydrolysis at 55 degrees C and pH 6.5-7.5; it was stable at pH 7.5-8.5 and remained stable at 50 degrees C for several hours. The k(cat)/Km for 3-(2-furyl)acryloyl-glycyl-L-leucine amide hydrolysis was (2.8+/-0.1) x 10(3) M(-1) x s(-1).
ISSN:1572-3887
1875-8355
1573-4943
DOI:10.1007/s10930-004-5225-y