Purification and Characterization of Bacillus Subtilis Jm-3 Protease From Anchovy Sauce

Abstract

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its Km and Vmaxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine protease.

Isolation, Purification, and Optimization of Thermophilic and Alkaliphilc Protease Originating From Hot Water Spring Bacteria

“…11). As per previous results regarding protease purification, the values of specific activities observed for different microbial species such as B. licheniformis, B. subtilis, and B. megaterium were 13.33-159381 U/mg of protein with 4.25-200 purification fold [28,29]. …”

“…Wide-ranging results of protease purification (4.25-200-fold) with various specific activities (13.34 U/mg of protein) and % recovery (2-21%) have been described for different microbial species such as Bacillus licheniformis, Bacillus subtilis, and Bacillus megaterium (Fig. 10) [28][29][30].…”

Response surface analysis on the effect of temperature and pH on Growth and proteolytic activity of thermophilic Bacillus sp

“…were characterized by Murao et al (1988) and Toogood et al (1995). Kim (2002) characterized the acid protease produced by B. subtilis JM-3, and reported that optimal pH and temperature were 5.5 and 60 o C, respectively. The high values of biomass on the level 33.8 mg/ml were observed in large range of temperature and pH (Fig.…”

Salt-Tolerant Acid Proteases: Purification, Identification, Enzyme Characteristics, and Applications for Soybean Paste and Sauce Industry

“…Kim and S.M. Kim, 2005;Fu et al, 2008;Chung, 1984;Hwang et al, 2010). The specific activity of the R. japonicus protease II was the highest among these five proteases, and that of the B. megaterium KLP-98 was the lowest.…”

“…However, the protease produced by B. Kim, 2005;Fu et al, 2008). Thus, these proteases were salt-tolerant proteases which can be used in soybean paste and soy sauce productions.…”