Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

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• 作者：Ali Bougatef ; Nabil Souissi ; Nahed Fakhfakh ; Yosra Ellouz-Triki ; Moncef Nasri
• 关键词：Trypsin ; Purification ; Biochemical characterization ; Viscera ; Sardina pilchardus
• 刊名：Food Chemistry
• 出版年：2007
• 出版时间：2007
• 年：2007
• 卷：102
• 期：1
• 页码：343-350
• 全文大小：251 K

文摘

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9 % recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5 % and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.