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 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).
Zabolotskaya
Maria V
MV
Laboratory of Protein Engineering, Institute of Molecular Genetics of Russian Academy of Sciences, pl Akad Kurchatova 2, 123182 Moscow, Russia. zabolotskaya@img.ras.ru
Demidyuk
Ilya V
IV
Akimkina
Tatiana V
TV
Kostrov
Sergey V
SV
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D008848:Micromonosporaceae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D010447:Peptide Hydrolases