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- Functional Analysis of a Subtilisin-like Serine Protease Gene from Biocontrol Fungus Trichoderma harzianum
- Haijuan Fan , Zhihua Liu , Rongshu Zhang , Na Wang , Kai Dou , Gulijimila Mijiti , Guiping Diao , Zhiying Wang
- J. Microbiol. 2014;52(2):129-138. Published online February 1, 2014
- DOI: https://doi.org/10.1007/s12275-014-3308-9
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- 20 Crossref
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Abstract
- The subtilisin-like serine protease gene ThSS45 has been cloned from Trichoderma harzianum ACCC30371. Its coding region is 1302 bp in length, encoding 433 amino acids, with a predicted protein molecular weight of 44.9 kDa and pI of 5.91. ThSS45 was shown by RT-qPCR analysis to be differentially transcribed in response to eight different treatments. The transcription of ThSS45 was up-regulated when grown in mineral medium, under carbon starvation, and nitrogen starvation, and in the presence of 1% root powder, 1% stem powder, and 1% leaf powder derived from Populus davidiana × P. bolleana (Shanxin poplar) aseptic seedlings. The highest increase in transcription approached 3.5-fold that of the control at 6 h under induction with 1% poplar root powder. The transcription of ThSS45 was also slightly up-regulated by 1% Alternaria alternata cell wall and 5% A. alternata fermentation liquid. Moreover, the analyses of coding and promoter regions of ThSS45 homologs indicated that serine protease may be involved in both mycoparasitism and antibiotic secretion. ThSS45 was cloned into the pGEX-4T-2 vector and then expressed in Escherichia coli BL21. The recombinant protein, with an expected molecular weight of approximately 69 kDa, was then purified. When transformant BL21-ss was induced with 1 mM IPTG for 6 h, the purified protease activity reached a peak of 18.25 U/ml at pH 7.0 and 40°C. In antifungal assays the purified protease obviously inhibited the growth of A. alternata mycelia.
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Citations
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- 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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