Fibrinolytic proteases from bacteria isolated from fermented foods

Abstract

Fibrinolytic protease-producing bacteria were isolated from 20 kinds of fermented food. Initially, 163 isolates were screened for their fibrinolytic activities by fibrin plate assay, and 21 isolates with strong activity were selected and subjected to identification. They were identified as Bacillus sp. and divided into 4 groups by internal transcribed spacers-polymerase chain reaction (ITS-PCR) fingerprinting. Group I (12 isolates) was closely related to B. subtilis, Group II (4 isolates) was related to B. vallismortis and Group III (4 isolates) was related to B. amyloliquefaciens. However, Group IV (2 isolates) showed different pattern from type strains and unidentified. Next, the enzymatic pattern was performed by fibrin-zymogram technique, which divided the fibrinolytic protease-producing bacteria into 6 groups based on their activity patterns. Then fibrinolytic activity of all isolates was evaluated by the hydrolysis of fibrin clot assay. Six representative isolates that showed the highest activity in each group of enzyme pattern , THY-C1, PD-Al0, K-A7, K-B16, TJW-A9 and TISTR 651, were chosen and subjected to 16S rRNA gene sequencing. They were closely related to the type strains of B. licheniformis (98.9 - 99.8%), B. subtilis (98.9 – 99.5%), B. vallismortis (98.9 – 99.4%) and B. amyloliquefaciens (97.6 – 98.3%) base on 16S rRNA gene sequence similarity. Moreover, THY-C1, PD-A10 and K-A7 were differentiated from related Bacillus species based on the phenotypic characteristics, DNA G + C content, fatty acid profile, rep- PCR fingerprinting and low DNA-DNA relatedness (<60%). Therefore, they presented the novel species of genus Bacillus, namely Bacillus thailandensis sp. nov., Bacillus siamensis sp. nov. and Bacillus kapii sp. nov., respectively. The optimal condition for fibrinolytic protease production by THY-C1 was evaluated. The enzyme production increased about 10 folds (101.2 unit/ml) when the bacterium was inoculated at 5% (v/v) into a medium containing 0.6% (w/v) yeast extract and 1.5% (w/v) sucrose at pH 9.0 with fibrin supplementation and incubated for 30 h at 37℃. The enzyme was optimally active at pH 7-8 and stable over a broad pH range from 6-11. Its optimum temperature was around 50℃. Enzyme was stable up to 50℃ and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), a specific inhibitor of serine protease. Compared to commercial fibrinolytic proteases, the cell-free supernatant of THY-C1 showed stronger activity than the commercial Nattokinase 1 and the commercial Nattokinase 2, but lower than the commercial Nattokinase 3. Moreover, fibrinolytic protease showed it stability to pancreatine and bile salt but lost it activity after incubated with pepsin in in vitro digestive model. Lastly, fibrinolytic protease produced by PD-A10, that showed the highest enzyme production in the optimal condition among 6 representative bacteria, was purified to homogeneity by column chromatography on Resource Q. The enzyme was purified 12.2 fold, with a yield of 51.4%. The molecular weight of the purified enzyme was estimated as 371.5 kDa. The enzyme exhibited a higher affinity toward H-D-Val-Leu-Arg-pNA with Vmax and Km values of 0.295 mM/ml/min and 0.28 mM, respectively. The enzyme was optimally active at pH 7.0, and its optimum temperature was at 50℃. The enzyme activity was relatively stable at pH 7-9 and maximum temperature at 50℃. The activity was inhibited by chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), indicating that the chymotrypsin–like serine protease. The purified enzyme could completely hydrolyze a fibrin and fibrinogen substrate in vitro within 2 h and 1 h respectively. Moreover, the purified enzyme possessed its fibrinolytic activity and fibrinogenolytic activity rather than thrombinlike activity.