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Production, Purification and Characterisation of a Potential Fibrinolytic Protease from Endophytic Xylaria curta by Solid Substrate Fermentation

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Abstract

The present investigation highlights the optimal conditions for production of a non-toxic, bi-functional fibrinolytic enzyme xylarinase produced by endophytic fungus Xylaria curta by solid substrate fermentation using rice chaff medium. The purified enzyme is a monomeric protein with a molecular mass of ∼33 kDa. The enzyme exhibits cleavage of Aα and Bβ chains of fibrin(ogen) and has no effect on γ chain. The optimal fibrinolytic activity of the enzyme was observed at 35 °C and pH 8. The fibrinolytic activity was enhanced in the presence of Ca2+, whereas it was completely inhibited in the presence of Fe2+ and Zn2+ ions and inhibitors like EDTA and EGTA suggesting it to be a metalloprotease. The K m and V max of the enzyme for azocasein were 326 μM and 0.13 μM min−1. The N-terminal sequence of the enzyme (SNGPLPGGVVWAG) was same when compared to xylarinase isolated from culture broth of X. curta. Thus, xylarinase could be exploited as a potent clot busting enzyme which could be produced on large scale using solid substrate fermentation.

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Acknowledgments

Vineet Meshram is thankful to University Grants Commission, Govt. of India, New Delhi, for providing financial support via Rajiv Gandhi National Fellowship (F.16-1886(SC)/2010(SA-III). The authors are thankful to Department of Biotechnology (DBT), BT/PR/10083/NDB/52/95/2007, under which the endophytic culture was isolated. We express our gratitude to Dr. Girish Sahani, Director, IMTECH (Now DG CSIR), Mrs. Paramjit Kaur, Senior Technical Officer, Ms. Neha Rana and Ms. Navneet Kaur, Research Scholar, IMTECH, Chandigarh, India, for their kind cooperation in N-terminal sequencing.

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  1. Department of Biotechnology, Thapar University, Patiala, Punjab, 147004, India

    Vineet Meshram, Sanjai Saxena, Mahiti Gupta & Neha Kapoor

  2. Department of Biochemistry, DAV University, Jalandhar, Punjab, India

    Karan Paul

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  1. Vineet Meshram
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Correspondence to Sanjai Saxena.

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Meshram, V., Saxena, S., Paul, K. et al. Production, Purification and Characterisation of a Potential Fibrinolytic Protease from Endophytic Xylaria curta by Solid Substrate Fermentation. Appl Biochem Biotechnol 181, 1496–1512 (2017). https://doi.org/10.1007/s12010-016-2298-y

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