• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.72-79
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• 2005

A new Bacillus peptidase, bacillopeptidase DJ-2 (bpDJ-2), with molecular mass of 42 kDa and isoelectric point (pI) of 3.5- 3.7, was purified to homogeneity from Bacillus sp. DJ-2 isolated from Doen-Jang, a traditional Korean soybean fermented food. The enzyme was identified as an extracellular serine fibrinolytic protease. The optimal conditions for the reaction were pH 9.0 and $60^{\circ}C$. The first 18 amino acid residues of the N-terminal amino acid sequence of bpDJ-2 were TDGVEWNVDQIDAPKAW, which is identical to that of bacillopeptidase F (bpf). However, based on their Nterminal amino acid sequence, molecular size, and pI, it is different from that of bpf and extracellular 90 kDa. The whole (2,541 bp, full-bpDJ-2) and mature (1,956 bp, mature-bpDJ-2) genes were cloned, and its nucleotide sequence and deduced amino acid sequence were determined. The expressed proteins, full-bpDJ-2 and mature-bpDJ-2, were detected on SDSPAGE at expected sizes of 92 and 68 kDa, respectively.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.515-518
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• 2011

A gene encoding bacillopeptidase F, bpr86-1, was cloned from B. amyloliquefaciens CH86-1 isolated from cheonggukjang. This gene could encode a preproenzyme of 1,431 amino acids. When bpr86-1 was introduced into B. subtilis WB600 via pHY300PLK, an E. coli-Bacillus shuttle vector, the transformant showed fibrinolytic activity. During growth on LB, the fibrinolytic activity of cells increased sharply when they entered the stationary phase. The highest activity (761.4 mU/mg protein) was observed at 96 h of cultivation.

• 생명과학회지
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• 제19권3호
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• pp.397-402
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• 2009

청국장에서 혈전 용해능이 우수한 균주를 분리하기 위해 짚을 이용하여 직접 청국장을 제조하였으며 이로부터 1, 000여종의 균주를 1차적으로 분리하였다. 2차적으로 skim milk가 첨가된 배지 및 fibrin 배지의 단백질분해 실험을 통해 혈전 용해능이 우수한 균주를 분리하였다. 이 과정을 통해 선발된 균주는 J-1, J-2, J-3, J-4, J-5로 명명된 5종 균주이었으며 그 중 Bacillus 계통의 J-4 균주가 가장 활성이 높은 균주로 선별되었다. Bacillus subtilis J-4 균주가 생산하는 protease를 DEAE-sepharose column을 사용하여 부분 정제 분리한 결과 SDS-PAGE Gel 상에서 45.0 kDa의 질량이었다. 이 단백질을 MALDI-TOF 및 PMF(Peptide Mass Fingerprinting)를 사용하여 분석한 결과 neutral protease와 bacillopeptidase F가 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1469-1476
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• 2007

Jeot-gal is a traditional Korean fermented seafood and has long been used for seasoning. We isolated 188 strains from shrimp, anchovy, and yellow corvina Jeot-gal, and screened sixteen strains that showed strong fibrinolytic activities on a fibrin plate. Among those strains, the strain that had the largest halo zone was chosen and identified as Bacillus licheniformis by using 16S rDNA sequencing and an API CHB kit. The fibrinolytic activity of Bacillus licheniformis was characterized and designated as bpKJ-31. The active component of bpKJ-31 was identified as a 37 kDa protein, designated bacillopeptidase F, by internal peptide mapping and N-terminal sequencing. The optimum activity of bpKJ-31 was shown at pH 9 and $40^{\circ}C$, with a chromogenic substrate for plasmin. It had high degrading activity for the $B{\beta}$-chain and $A{\alpha}$-chain of fibrin(ogen), and also acted on thrombin, but not skim milk and casein. The amidolytic activity of bpKJ-31 was inhibited by 1 mM phenylmethanesulfonyl fluoride, but 1 mM EDTA did not affect the enzyme activity, indicating that bpKJ-31 is an alkaline serine protease, like a plasmin. The bpKJ-31 showed approximately 14.3% higher fibrinolytic activity than the plasmin. These features of bpKJ-31 make it attractive as a health-promoting biomaterial.