Journal of Microbiology

Research Support, Non-U.S. Gov'ts

NOTE] Detection of a Unique Fibrinolytic Enzyme in Aeromonas sp. JH1: Han-Young Cho , Min Jeong Seo , Jeong Uck Park , Byoung Won Kang , Gi-Young Kim , Woo Hong Joo , Young-Choon Lee , Yong Kee Jeong; J. Microbiol. 2011;49(6):1018-1021. Published online December 28, 2011; DOI: https://doi.org/10.1007/s12275-011-1376-7

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Abstract: A fibrinolytic enzyme was found in a Gram-negative bacterium, Aeromonas sp. JH1. SDS-PAGE and fibrinzymography showed that it was a 36 kDa, monomeric protein. Of note, the enzyme was highly specific for fibrinogen molecules and the hydrolysis rate of fibrinogen subunits was highest for α, β, and γ chains in that order. The first 15 amino acids of N-terminal sequence were X-D-A-T-G-P-G-G-N-V-X-T-G-K-Y, which was distinguishable from other fibrinolytic enzymes. The optimum pH and temperature of the enzyme were approximately 8.0 and 40°C, respectively. Therefore, these results provide a fibrinolytic enzyme with potent thrombolytic activity from the Aeromonas genus.

Biochemical Analysis of a Fibrinolytic Enzyme Purified from Bacillus subtilis Strain A1: Won Sik Yeo , Min Jeong Seo , Min Jeong Kim , Hye Hyeon Lee , Byoung Won Kang , Jeong Uck Park , Yung Hyun Choi , Yong Kee Jeong; J. Microbiol. 2011;49(3):376-380. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1165-3

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Abstract: A fibrinolytic enzyme from Bacillus subtilis strain A1 was purified by chromatographic methods, including DEAE Sephadex A-50 column chromatography and Sephadex G-50 column gel filtration. The purified enzyme consisted of a monomeric subunit and was estimated to be approximately 28 kDa in size by SDS-PAGE. The specific activity of the fibrinolytic enzyme was 1632-fold higher than that of the crude enzyme extract. The fibrinolytic activity of the purified enzyme was approximately 0.62 and 1.33 U/ml in plasminogen-free and plasminogen-rich fibrin plates, respectively. Protease inhibitors PMSF, DIFP, chymostatin, and TPCK reduced the fibrinolytic activity of the enzyme to 13.7, 35.7, 15.7, and 23.3%, respectively. This result suggests that the enzyme purified from B. subtilis strain A1 was a chymotrypsin-like serine protease. In addition, the optimum temperature and pH range of the fibrinolytic enzyme were 50°C and 6.0-10.0, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as Q-T-G-G-S-I-I-D-P-I-N-G-Y-N, which was highly distinguished from other known fibrinolytic enzymes. Thus, these results suggest a fibrinolytic enzyme as a novel thrombolytic agent from B. subtilis strain A1.

Purification and Biochemical Characterization of a 17 kDa Fibrinolytic Enzyme from Schizophyllum commune: In Suk Park , Jeong Uck Park , Min Jeong Seo , Min Jeong Kim , Hye Hyeon Lee , Sung Ryeal Kim , Byoung Won Kang , Yung Hyun Choi , Woo Hong Joo , Yong Kee Jeong; J. Microbiol. 2010;48(6):836-841. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0384-3

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Abstract: A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.

A Fibrinolytic Enzyme from the Medicinal Mushroom Cordyceps militaris: Jae-Sung Kim , Kumar Sapkota , Se-Eun Park , Bong-Suk Choi , Seung Kim , Nguyen Thi Hiep , Chun-Sung Kim , Han-Seok Choi , Myung-Kon Kim , Hong-Sung Chun , Yeal Park , Sung-Jun Kim; J. Microbiol. 2006;44(6):622-631.; DOI: https://doi.org/2465 [pii]

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Abstract: In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9%. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and 37°C, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin α-chain followed by the γ-γ chains. It also hydrolyzed the β-chain, but more slowly. The Aα, Bβ, and γ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by Cu2+ and Co2+, but enhanced by the additions of Ca2+ and Mg2+ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it’s a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

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