• 한국식품영양과학회지
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• 제33권1호
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• pp.41-46
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• 2004

전통된장과 공장산 된장 제품, 메주와 Koji, 그리고 된장발효공정에서의 혈전용해효소 활성과 microflora의 상관관계를 조사하였다. 전통된장의 혈전용해 활성은 평균 2.42 unit/g으로 공장산 된장의 평균 1.58 unit/g보다 1 unit 이상 높은 분포를 나타내었으며 전통된장에서는 Bacillus의 밀도가 높았고 acid producing bacteria, 곰팡이, 효모는 모두 공장산 된장에서 높은 밀도를 나타내었다. 된장의 Bacillus group 분포와 혈전용해효소활성은 양의 상관관계를 나타내었으며 fungal group 분포와 혈전용해효소활성은 음의 상관관계를 나타내었다. 메주의 혈전용해 활성은 평균 6.54 unit/g수준으로 Koji의 1.46 unit/g보다 5 unit 이상 높은 분포를 나타내었으며 Bacillus의 서식밀도와 높은 상관관계를 보였다. 된장 발효과정 중 Bacillus와 곰팡이는 휴지 상태를 유지하였으며 혈전용해효소 활성도 담금 초기의 수준을 유지하였다. 결과적으로 된장의 혈전용해활성은 된장 담금 이전의 원료구성에 따라 결정되며, 메주나 Koji에서 유래된 혈전용해 효소는 된장의 발효과정에서도 계속 활성을 유지하는 것으로 확인되었다. 본 실험 결과, 혈전용해효소 활성이 높은 된장을 제조하기 위해서는 메주나 Koji의 제조공정에서 효소활성을 유도하여 된장 담금 시 첨가하는 공정의 설정이 유효할 것으로 제안하였다.

• 생명과학회지
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• 제11권4호
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• pp.304-310
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• 2001

In order to produce a high quality and functional black bean Chungkugjang, a bacterium which has potent enzyme activities(protease:124.8 U/$m\ell$, $\alpha$-amylase: 78.2U/$m\ell$, glucoamylase; 13.9U/$m\ell$, ), was isolated by using the halo zone method and identified by morphological, cultural and biochemical properties, and then finally confirmed by GP-microplate identification system as Bacillus megatrium SMY-212. The isolated SMY-212 system was also high in the formation of mucoid material and fibrinolytic activity.

• BMB Reports
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• 제28권5호
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• pp.398-401
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• 1995

Two fibrinolytic enzymes from the autolysate of Lumbricus rubellus were purified in homogeneous form. Their molecular sizes were 31,000 (Enz1) and 35,000 (Enz2) Da. respectively. However, the N-Terminal amino acid sequences of Enz1 and Enz2 were exactly the same: Ile-Val-Gly-Gly-Ile-Glu-Ala-Arg-Pro-Tyr-Glu-Phe-Pro-Trp-Gln-. These results indicate that Enz1 is a shortened form of Enz2 formed during autolysis. When a synthetic substrate, Ile-Pro-Arg-pNA, was used, the catalytic activity were observed in the pH range of 5-10 and the kinetic parameters including $K_m$ (1.6 ${\mu}m$) and $V_{max}$ (40 nmol/jmin/mg) were almost identical between the two enzymes. However, the fibrinolytic activity of Enz2 was at least 1.25 times higher than that of Enz1, suggesting that the C-terminal region of Enz2 is important in fibrinolysis but not in amidolysis. Furtheimore. fibrinolytic activity of the enzymes was increased by the addition of the lipid extracted from L. rubellus in the presence of $MgCl_2$ or $CaCl_2$. The stimulatary effect of lipid on Enz2 was higher compared to Enz1.

• 한국미생물·생명공학회지
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• 제49권3호
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• pp.289-297
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• 2021

Bacillus velezensis CJ1, showing significant fibrinolytic activity, was isolated from Myeolchi Jeotgal, a popular Korean fermented seafood. When B. velezensis CJ1 was grown on four different culture media, the culture on the Luria-Bertani (LB) broth showed the highest fibrinolytic activity (102.94 mU/μl) at 48 h. LB was also the best medium for growth. SDS-PAGE of culture supernatant showed four major bands, 38, 35, 27, and 22 kDa in size. Fibrin zymography showed four active bands, 50, 47, 40, and 30 kDa in size. A gene homologous to aprE of the Bacillus species was cloned by PCR. DNA sequencing showed that aprECJ1 can encode a protease consisting of 382 amino acids. The translated amino acid sequence of AprECJ1 showed high identity values with those of B. velezensis strains and other Bacillus species. The aprECJ1 gene was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, and overexpressed. A 27 kDa band corresponding to the mature form of AprECJ1 was produced and confirmed by SDS-PAGE and fibrin zymography. B. subtilis WB600 [pHYaprECJ1] showed 1.8-fold higher fibrinolytic activity than B. velezensis CJ1 at 48 h.

• 생명과학회지
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• 제16권2호
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• pp.274-281
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• 2006

흑두청국으로부터 분리된 혈전용해력이 우수한 Bacillus subtilis BB-1(KFCC 11344P)으로부터 혈전용해효소 유전자를 PCR법에 의해 크로닝하였고 이를 BCF-1으로 명명하였다. BCF-1의 DNA 염기서열결정 결과 1,145 bp 크기의 혈전용해 효소로, 일본의 natto로부터 분리된 nattokinase 유전자와 99%의 상동성을 보임을 확인하였다. 혈전용해효소 유전자의 발현을 위하여 Bacillus 발현계인 Bacillus-E. coli의 shuttle vector인 pEB vector에 크로닝 하고 host로서 B. subtilis 168에 형질전환시켜 대량 발현시켰다. 생산된 혈전용해효소의 최 적활성 pH와 온도는 7.0과 $35^{\circ}C$로 확인되었다, 기질에 대한 분해양상을 조사한 결과 fibrin에서만 특이적으로 강한 분해가 일어났으며, skim milk에서 아주 약한 분해능을 보였으나 blood agar, gelatin, casein에서는 전혀 분해능을 보이지 않았다. 특히 blood agar plate에서 분해능이 없는 것으로 보아 혈액 내에서의 적혈구 파괴현상과 같은 부작용에 대한 위험을 배제할 수 있을 것으로 사료된다. BCF-1에 의해 생산된 혈전용해효소는 fibrin 특이적으로 활성을 나타냄을 확인할 수 있으며, 이는 임상적이나 산업적으로 적용하였을 때 부작용에 대한 위험적인 문제는 배제될 수 있으리라 생각된다.

• 미생물학회지
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• 제45권4호
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• pp.377-384
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• 2009

전통 메주로부터 plasmin의 혈전용해 활성보다 약 58% 더 높은 활성을 나타내는 MJ-226을 분리하여 동정한 결과, Bacillus subtilis와 유사한 형태학적, 생화학적 및 당 발효능을 나타내었다. B. subtilis MJ-226은 Tryptic Soy Broth (TSB) 배지 상에서 최대의 혈전용해효소 활성을 나타내었고, $37^{\circ}C$에서 24~26시간 배양했을 때 가장 높은 활성을 나타내었고, TSB 배지 내에 glucose와 fructose 2.0%와 peptone 및 yeast extract 1.0% 각각을 첨가한 경우 활성이 증가되었다. 하지만 lactose, sucrose, beef extract, casein 및 tryptophan 등에 의해선 활성이 오히려 감소되었다. B. subtilis MJ-226이 생산하는 혈전용해효소는 pH 6.0~8.0 및 온도 $35\sim40^{\circ}C$에서 매우 안정하였으며, 또한 $MnSO_4$, $CaCl_2$, KCl 및 NaCl 5 mM 농도의 금속이온에 대해서도 비교적 안정함을 유지하였다. 그러나 $CuSO_4$, $MgSO_4$, $ZnSO_4$, $FeSO_4$와 $BaCl_2$에 등의 금속이온과 iodoacetic acid, leupeptin, phenylmethanesulphonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), thiourea, trans-1,2-diaminocyclohexane-N,N,N',N'- tetraacetic acid (CDTA) 및 ethylenediaminetetraacetic acid (EDTA) 등의 저해제들과 반응한 경우에는 매우 불안정한 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제23권7호
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• pp.974-983
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• 2013

Bacillus amyloliquefaciens CB1 was isolated from cheonggukjang, a Korean fermented soy food. B. amyloliquefaciens CB1 secretes proteases with fibrinolytic activities. A gene homologous to aprE of Bacillus subtilis, aprECB1, was cloned from B. amyloliquefaciens CB1, and DNA sequencing showed that aprECB1 can encode a prepro-type serine protease consisting of 382 amino acids. When aprECB1 was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, transformants showed fibrinolytic activity and produced a 28 kDa protein, the size expected for the mature enzyme. The 28 kDa fibrinolytic enzyme was purified from the culture supernatant of B. subtilis WB600 transformant. AprECB1 was completely inhibited by phenylmethylsulfonyl fluoride and almost completely inhibited by EDTA and EGTA, indicating that it is a serine metalloprotease. AprECB1 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, a known substrate for ${\alpha}$-chymotrypsin. $A{\alpha}$ and $B{\beta}$ chains of fibrinogen were quickly degraded by AprECB1, but the ${\gamma}$-chain was resistant.

• Journal of Microbiology and Biotechnology
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• 제20권2호
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• pp.370-374
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• 2010

A gene encoding the major secreted fibrinolytic protein of Bacillus amyloliquefaciens CH86-1 was cloned from genomic DNAs. DNA sequencing showed that the gene, aprE86-1, could direct the synthesis of a mature protein 275 amino acids in length after processing. When aprE86-1 was introduced into B. subtilis, a mature 27-kDa protein was produced as expected. The fibrinolytic activity of the B. subtilis transformant (TF) was higher than that of B. amyloliquefaciens CH86-1, showing the possibility of increasing the fibrinolytic activity of Bacillus strains through genetic engineering.

• BMB Reports
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• 제34권2호
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• pp.134-138
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• 2001

By adding sodium chloride (2.5%) into a Bacillus amyloliquefaciens DJ-4 culture broth, two serine-type fibrinolytic proteases with a molecular weight of 29 (subtilisin DJ-4) and 38-kDa were stimulated on the SDS-fibrin zymogram or inhibitor gels. B. amyloliquefaciens DJ-4 showed the highest proteolytic activity (5.52 plasmin NIH unit/ml) on the fibrin plate based on the molar ratio when cells were subjected to the 2.5% NaCl. Using a fibrin plate, the secreted protease from this strain in the presence of 5% NaCl showed that about 49% of the enzyme's activity remained after incubation at $60^{\circ}C$ for 30 min, but as the salt concentration was increased (10% NaCl) the activity nearly disappeared (0.14 plasmin NIH unit/ml). However, through a fibrin zymography assay, three fibrinolytic enzymes (38, 53 and 80-kDa) from the cells in the presence of 10% NaCl were detected. Also, two salt-activated serine-type fibrinolytic professes (29 and 38kDa) showed thermostability from 65 to $70^{\circ}C$ for 30 min. Furthermore, these professes also showed stability, pH 6-11. In particular, 29-kDa (subtilisin DJ-4) was very stable in the pH range of 4-11 at $4^{\circ}C$ for 48 h.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1863-1870
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• 2015

Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy-, Spec^S) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner.