Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce.
A gram-positive thermotolerant bacterium, designated strain RKK-04, was isolated from a fermented Thai fish sauce broth as it demonstrated high proteolytic activity. A phylogenetic analysis based on comparisons of 16S rRNA gene sequences showed that strain RKK-04 is Bacillus licheniformis. The proteolytic enzyme, which was purified 80-fold with 18% yield, has a molecular mass of 31 kDa and an isoelectric point higher than 9.3. The optimum pH and temperature of the enzyme activity were found to be 10.0 and 50 degrees C, respectively. The addition of diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride completely inhibited enzymatic activity. These results showed that the enzyme is a subtilisin-like alkaline serine proteinase. On the other hand, the enzyme exhibited unique cleavage sites in oxidized insulin B-chain that differed from those of other subtilisin-like proteases. High enzymatic activity was also retained under high salt conditions (30% NaCl). The myosin heavy chain of fish protein was completely digested by reaction with this enzyme. Thus the halotolerant proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation.
Toyokawa
Yoichi
Y
Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa, 903-0213, Japan.
Takahara
Hiroaki
H
Reungsang
Alissara
A
Fukuta
Masakazu
M
Hachimine
Yuki
Y
Tachibana
Shinjiro
S
Yasuda
Masaaki
M
D000205:Actomyosin / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003212:Condiments / Q000382:microbiology*; D005285:Fermentation; D005396:Fish Products / Q000382:microbiology*; D029941:Fish Proteins / Q000378:metabolism; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D029941:Fish Proteins; D007328:Insulin; D008670:Metals; D012965:Sodium Chloride; D010664:Phenylmethylsulfonyl Fluoride; D000205:Actomyosin; D057057:Serine Proteases; D020860:Subtilisin