Biotechnology Letters

, Volume 39, Issue 12, pp 1911–1916 | Cite as

Isolation, purification and characterization of a pH tolerant and temperature stable proteinaceous protease inhibitor from marine Pseudomonas mendocina

  • K. Sapna
  • P. P. Manzur Ali
  • K. R. Rekha Mol
  • Sarita G. Bhat
  • M. Chandrasekaran
  • K. K. Elyas
Original Research Paper



An extracellular protease inhibitor (BTPI-301) of trypsin was purified and characterized from an isolate of Pseudomonas mendocina.


BTPI-301was purified to homogeneity by (NH4)2SO4, precipitation, DEAE Sepharose and CNBr-activated Sepharose chromatography. Homogeneity was proved by native PAGE and SDS-PAGE. The intact molecular mass was 11567 Da by MALDI-TOF analysis. BTPI-301was a competitive inhibitor with a Ki of 3.5 × 10−10 M. It was stable and active at pH 4–12 and also at 4–90 °C for 1 h. Peptide mass fingerprinting by MALDI revealed that the BTPI-301 is a new inhibitor not reported so far with protease inhibitory activity. The pI of the inhibitor was 3.8. The stoichiometry of trypsin-BTPI-301 interaction is 1:1. The inhibitor was specific towards trypsin.


A pH tolerant and thermostable protease inhibitor BTPI-301 active against trypsin was purified and characterized from P. mendocina that could be developed and used as biopreservative as well as biocontrol agent.


Inhibitory activity Protease inhibitor Pseudomonas mendocina Trypsin 



The authors gratefully acknowledge the financial support from Department of Biotechnology, Government of India (Sanction Order No.: BT/PR7906/AAQ/03/281/2006 dated 07.03.2007).


  1. Ali PP, Sapna K, Mol KR, Bhat SG, Chandrasekaran M, Elyas KK (2014) Trypsin inhibitor from edible mushroom Pleurotus floridanus active against proteases of microbial origin. Appl Biochem Biotechnol 173:167–178CrossRefPubMedGoogle Scholar
  2. Angelova L, Dalgalarrondo M, Minkov I, Danova S, Kirilov N, Serkedjieva J, Chobert JM, Haertlé T, Ivanova I (2006) Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity. Biochim Biphys Acta 1760:1210–1216CrossRefGoogle Scholar
  3. Avanzo P, Sabotič J, Anžlovar S, Popovič T, Leonardi A, Pain RH, Kos J, Brzin J (2009) Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions. Microbiology 155:3971–3981CrossRefPubMedGoogle Scholar
  4. Brzin J, Rogelj B, Popovič T, Štrukelj B, Ritonja A (2000) Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom Clitocybe nebularis. J Biol Chem 275:20104–20109CrossRefPubMedGoogle Scholar
  5. Chandrasekaran M (1985) Studies on Microbial spoilage of Penaeus indicus. Ph.D thesis Cochin University of Science and Technology School of Marine Sciences, Cochin, pp. 1–258Google Scholar
  6. Imada C, Simidu U, Taga N (1985) Isolation and characterization of marine bacteria producing alkaline protease inhibitor. Bull Jpn Soc Sci Fish 51:799–803CrossRefGoogle Scholar
  7. Imada C, Hara S, Maeda M, Simidu U (1986) Amino acid sequences of marinostatins C-1 and C-2 from marine Alteromonas sp. Bull Jpn Soc Sci Fish 52:1455–1459CrossRefGoogle Scholar
  8. Jiang CJ, Hao ZY, Zeng R, Shen PH, Li JF, Wu B (2011) Characterization of a novel serine protease inhibitor gene from a marine metagenome. Mar Drugs 9:1487–1501CrossRefPubMedPubMedCentralGoogle Scholar
  9. Kumar CG, Takagi H (1999) Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv 17:561–594CrossRefPubMedGoogle Scholar
  10. Lopez-Otin C, Bond JS (2008) Proteases: multifunctional enzymes in life and disease. J Biol Chem 283:30433–30437CrossRefPubMedPubMedCentralGoogle Scholar
  11. Maurizi MR (1992) Proteases and protein degradation in Escherichia coli. Experientia 48:178–201CrossRefPubMedGoogle Scholar
  12. Pandhare J, Zog K, Deshpande VV (2002) Differential stabilities of alkaline protease inhibitors from actinomycetes: effect of various additives on thermostability. Biores Technol 84:165–169CrossRefGoogle Scholar
  13. Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 28:425–449CrossRefGoogle Scholar
  14. Sabotič J, Kos J (2012) Microbial and fungal protease inhibitors—current and potential applications. Appl Microbiol Biotechnol 93:1351–1375CrossRefPubMedGoogle Scholar
  15. Schwarz WH, Zverlov VV (2006) Protease inhibitors in bacteria: an emerging concept for the regulation of bacterial protein complexes? Mol Microbiol 60:1323–1326CrossRefPubMedGoogle Scholar
  16. Shiga Y, Hasegawa K, Tsuboi A, Yamagata H, Udaka S (1992) Characterization of an extracellular protease Inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene. Appl Environ Microbiol 58:525–531PubMedPubMedCentralGoogle Scholar
  17. Shiga Y, Yamagata H, Tsukagoshi N, Udaka S (1995) BbrPI, an extracellular proteinase inhibitor of Bacillus brevis, protects cells from the attack of exogenous proteinase. Biosci Biotechnol Biochem 59:2348–2350CrossRefPubMedGoogle Scholar
  18. Wee KE, Yonan CR, Chang FN (2000) A new broad-spectrum protease inhibitor from the entomopathogenic bacterium Photorhabdus luminescens. Microbiology 146:3141–3147CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • K. Sapna
    • 1
  • P. P. Manzur Ali
    • 2
  • K. R. Rekha Mol
    • 1
  • Sarita G. Bhat
    • 1
  • M. Chandrasekaran
    • 1
  • K. K. Elyas
    • 3
  1. 1.Department of BiotechnologyCochin University of Science and TechnologyCochinIndia
  2. 2.Department of BiotechnologyMES CollegeAluvaIndia
  3. 3.Department of BiotechnologyCalicut UniversityMalappuramIndia

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