African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12209

Full Length Research Paper

Isolation, partial purification and characterization of antifungal trypsin inhibitor protease from the seed of Blighia sapida K.D. Koenig (Ackee)

Nwozo S. O.*
  • Nwozo S. O.*
  • Nutritional / Industrial Biochemistry unit, Department of Biochemistry, FBMS, College of Medicine, University of Ibadan, Nigeria; SensorLab, Department of Chemistry, University of Western Cape, Bellville, Cape Town 7535, South Africa.
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Iwuoha E. I.
  • Iwuoha E. I.
  • Nutritional / Industrial Biochemistry unit, Department of Biochemistry, FBMS, College of Medicine, University of Ibadan, Nigeria; SensorLab, Department of Chemistry, University of Western Cape, Bellville, Cape Town 7535, South Africa.
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Waryo T.
  • Waryo T.
  • SensorLab, Department of Chemistry, University of Western Cape, Bellville, Cape Town 7535, South Africa.
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Kgarebe B.
  • Kgarebe B.
  • SensorLab, Department of Chemistry, University of Western Cape, Bellville, Cape Town 7535, South Africa.
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  •  Received: 25 July 2013
  •  Accepted: 08 May 2014
  •  Published: 16 July 2014

Abstract

Seed proteins have been evaluated for their nutritive value and biological activity. Proteases and proteinase inhibitors have been of immense benefit to both agriculture and therapeutic purposes. The seed proteins of Blighia sapida was evaluated for both structure and biological activity in this study. The matured dried seeds were pulverized and sequentially extracted using 10 mM Tris/HCl pH 7.4, 10 mM ammonium acetate and 10 mM sulphuric acid. Crude protein extracts were concentrated and the protein concentrations were estimated. Proteins were purified by 70% ammonium sulphate precipitation, Sephadex G50 reversed phase chromatography and finally by HPLC on a C18 column. Two bands were obtained from SDS-PAGE electrophoresis and they were identified by ESI/MS using in gel tryptic digestion. The seed protein from B. Sapida consists of two single polypeptide chains each with mass of about 24 to 27 KDa as established by a combination of SDS-PAGE and ESI/MS. Proteins exhibited protease activity, which was confirmed by zymography. Protease activity was characterized for effect of temperature, pH, divalent metal ions and chelating agents. The crude protein from the seed of B. sapida showed antimicrobial activity and the antifungal activity was comparable with the reference drug, Ticonazole.

 

Key words: Blighia sapida, chromatography, protease activity, in-gel trypsin digestion/mass spectrophotometry, antimicrobial activity.