Titre du document / Document title

New alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus) with high activity at low temperature: Purification and characterisation

Auteur(s) / Author(s)

JELLOULI Kemel ⁽¹⁾ ; BOUGATEF Ali ⁽¹⁾ ; DAASSI Dalel ⁽¹⁾ ; BALTI Rafik ⁽¹⁾ ; BARKIA Ahmed ⁽¹⁾ ; NASRI Moncef ⁽¹⁾ ;

Affiliation(s) du ou des auteurs / Author(s) Affiliation(s)

⁽¹⁾ Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d'Ingénieurs de Sfax, B.P. "W' 3038, Sfax, TUNISIE

Résumé / Abstract

A highly alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus), with high activity at low temperature, was purified and characterised. The enzyme was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography, with a 13.9-fold increase in specific activity and 41.3% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 23.2 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. Purified trypsin appeared as a single band on native-PAGE. Interestingly, the enzyme was highly active over a wide range of pH, from 9.0 to 11.5, with an optimum at pH 10.5, using Nα-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 9.0, 11.5 and 12.0 were 86.5%, 92.6% and 52.4%, respectively. The enzyme was extremely stable in the pH range 7.0―12.0. In addition, the enzyme had high activity at low and moderate temperatures with an optimum at around 40 °C and had more than 80% of its maximum activity at 20 °C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. The enzyme showed extreme stability towards oxidising agents, retaining about 87% and 80% of its initial activity after 1 h incubation at 40 °C in the presence of 1% sodium perborate and 1% H₂O₂, respectively. In addition, the enzyme showed excellent stability and compatibility with some commercial solid detergents. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVG-GYECFPNST. B. capriscus trypsin, which showed high homology with trypsins from marine vertebrates, had a basic residue, Asn, at position 10, where His and Tyr are common in all marine vertebrates trypsins. The trypsin kinetic constants, K_m and k_cat for BAPNA, were 0.068 mM and 2.76 s^―1, respectively, while the catalytic efficiency, k_cat/K_m, was 40.6 s^―1 mM^―1.

Revue / Journal Title

Food chemistry   ISSN 0308-8146   CODEN FOCHDJ 

Source / Source

2009, vol. 116, n^o3, pp. 644-650 [7 page(s) (article)] (3/4 p.)

Langue / Language

Anglais

Editeur / Publisher

Elsevier, Oxford, ROYAUME-UNI  (1976) (Revue)

Mots-clés anglais / English Keywords

Vertebrata ; Pisces ; Enzyme ; Hydrolases ; Peptidases ; Serine endopeptidases ; Purification ; Low temperature ; Balistes capriscus ; Trypsin ;

Mots-clés français / French Keywords

Vertebrata ; Pisces ; Enzyme ; Hydrolases ; Peptidases ; Serine endopeptidases ; Purification ; Basse température ; Balistes capriscus ; Trypsin ;

Mots-clés espagnols / Spanish Keywords

Vertebrata ; Pisces ; Enzima ; Hydrolases ; Peptidases ; Serine endopeptidases ; Purificación ; Baja temperatura ; Balistes capriscus ; Trypsin ;

Mots-clés d'auteur / Author Keywords

Alkaline trypsin ; Purification ; Biochemical characterisation ; Grey triggerfish ; Balistes capriscus ; Intestine ;

Localisation / Location

INIST-CNRS, Cote INIST : 17810, 35400018827791.0080