Purification and Characterization of Subtilisin DJ-4 Secreted by<i>Bacillus</i>sp. Strain DJ-4 Screened from<i>Doen-Jang</i>

Kim, Seung Ho; Choi, Nack Shick

doi:10.1271/bbb.64.1722

Cited by 136 publications

(104 citation statements)

References 11 publications

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“…It is dissolved by Wbrinolytic enzyme, plasmin (EC 3.4.21.7), which maintains blood Xow at vascular injury sites and is an important component of the normal hemostatic response. When clots are not lysed, they accumulate in blood vessels, and cause thrombosis leading to myocardial infarction and other cardiovascular diseases [1,2]. The major thrombolytic agents are classiWed into two types.…”

Section: Introductionmentioning

confidence: 99%

“…Many bacterial Wbrinolytic enzymes were discovered from fermented foods, such as natto [3,[5][6][7], shiokara [8] in Japanese food, Chungkook-Jang [1], Doen-Jang [2], and Jeot-Gal [9] in Korean food, douchi [10] in Chinese food, and Tempeh [11] in Indonesian food. Of them Shiokara (Japan) and Jeot-Gal (Korea) are Wsh-fermented foods, which are used as important additive for improving the taste of other foods as well as being foods themselves [12].…”

Section: Introductionmentioning

confidence: 99%

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Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Choi

Song

Chung

et al. 2008

J Ind Microbiol Biotechnol

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A novel Wbrinolytic enzyme (AJ) was puriWed from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and Wbrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85°C, respectively. AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K m ) and K cat values of AJ towards -casein were 0.38 mM and 19.73 s ¡1 , respectively. AJ cleaved the A -chain of Wbrinogen but did not aVect the B -and -chains, indicating that it is an -Wbrinogenase. The Wbrinolytic activity was inhibited by diisopropyl Xuorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other Wbrinolytic enzymes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Choi

Song

Chung

et al. 2008

J Ind Microbiol Biotechnol

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“…Therefore, microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. [7][8][9] Fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus from traditional fermented foods. These bacterial strains have been found to effectively produce extracellular and intracellular proteases.…”

Section: Introductionmentioning

confidence: 99%

Partial Purification of Alkaline Protease as Thrombolytic Agent from Mutant Strain Bacillus licheniformis EMS250-O-1

Zaman

Mamun

Khan

et al. 2017

Dhaka Univ. J. Pharm. Sci

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Thrombosis leads to myocardial infarction, stroke and other cardiovascular complications. Microbial thrombolytic agents such as urokinase, streptokinase etc. are used to treat complications related to thrombosis. To search for new microbial enzymes as thrombolytics having better efficacy and specificity, Bacillus licheniformis EMS-O-1 mutant strain was cultured in modified urea-molasses media followed by purification using ammonium sulphate precipitation and ultrafiltration through centricon tube of 100 MWCO value. The yield of crude enzyme was 11129.14 U/mg and after purification 40180.46 U/mg. Purification process increased the specific activity of purified enzyme to 12.28 fold with a recovery of 17.79%. The purified enzyme was a serine protease with molecular weight of 25.5 kDa as confirmed by irreversible inhibition of activity with phenylmethylsulfonyl fluoride (PMSF) followed by SDS-PAGE gel image and by LC-MS analyses. In vitro clot lysis assay of the purified enzyme exhibited 38.30% thrombolytic activity. The crude enzymes from the mutant strain EMS-O-1 were found to be stable up to 50 o C and showed maximum stability between pH range 7.5 to 8.5. These findings signify that proteases produced by B. licheniformis mutant have the potential to be developed as a viable thrombolytic agent.

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“…Bacillus strains are well-known for secreting various extracellular proteases, which contain wide range of proteins with molecular weights that range from 15 kDa to 100 kDa (Burg et al, 1990;Kobayashi et al, 1999;Kim and Choi, 2000;jeong and Han, 2001). In order to detect these Bacillus proteases, zymographic techniques are widely used (Heussen and Dowdle, 1980;Choi and Kim, 1999;Kim and Choi, 1999).…”

Section: Introductionmentioning

confidence: 99%

Relationship Between Acrylamide Concentration and Enzymatic Activity in An Improved Single Fibrin Zymogram Gel System

Choi¹,

Kim²,

Lee³

et al. 2002

BMB Reports

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Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDSfibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10 kDa to over 100 kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.

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Purification and Characterization of Subtilisin DJ-4 Secreted byBacillussp. Strain DJ-4 Screened fromDoen-Jang

Cited by 136 publications

References 11 publications

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Partial Purification of Alkaline Protease as Thrombolytic Agent from Mutant Strain Bacillus licheniformis EMS250-O-1

Relationship Between Acrylamide Concentration and Enzymatic Activity in An Improved Single Fibrin Zymogram Gel System

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