Molecular and biochemical characterization of an extracellular serine-protease from Vibrio metschnikovii J1.
A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30 degrees C in media containing casein as carbon source (14,000 U ml(-1)). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60 degrees C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K (m) and K (cat) of the purified enzyme using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide were 0.158 mM and 1.14 x 10(5) min(-1), respectively. The catalytic efficiency (K (cat) /K (m)) was 7.23 x 10(8) min(-1) M(-1). The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.
Jellouli
Kemel
K
Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d'Ingénieurs de Sfax, B.P. W 3038, Sfax, Tunisia. kemel.j@gmail.com
Bougatef
Ali
A
Manni
Laila
L
Agrebi
Rym
R
Siala
Rayda
R
Younes
Islem
I
Nasri
Moncef
M
D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D014871:Water Microbiology
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004791:Enzyme Inhibitors; D012336:RNA, Ribosomal, 16S; D010664:Phenylmethylsulfonyl Fluoride; D012697:Serine Endopeptidases