Journal of Comparative Physiology B

, Volume 177, Issue 6, pp 655–666

Two trypsin isoforms from the intestine of the grass carp (Ctenopharyngodon idellus)

Original Paper

DOI: 10.1007/s00360-007-0163-6

Cite this article as:
Liu, ZY., Wang, Z., Xu, SY. et al. J Comp Physiol B (2007) 177: 655. doi:10.1007/s00360-007-0163-6
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Abstract

Two trypsin isoforms (GT-A and GT-B) from the grass carp (Ctenopharyngodon idellus) intestine were isolated and purified. SDS-PAGE electrophoresis showed that GT-A and GT-B had relative molecular masses of 30,740 and 26,400, respectively. Enzyme activity was inhibited by three organic trypsin inhibitors but not by EDTA. They had optimal pH of 8.0 and 8.5, and optimal temperatures of 38.5 and 44.0°C, respectively, when hydrolyzing N–benzoyl-l-arginine ethyl ester·HCl (BAEE). They lost 95.8 and 93.7% of their activities, respectively, after heating for 20 min at 65°C. Their thermal denaturation temperatures, respectively, were 66.3 and 67.3°C. GT-A has a Km value of 21.2 μM and a Vmax of 2.0 × 103 min−1, and GT-B has a Km value of 31.7 μM and a Vmax of 3.3 × 103 min−1. Their physiological efficiencies were 94.3 and 105.3 μM−1 min−1, respectively. The Arrhenius activation energies of GT-A and GT-B were 4.16 and 4.38 kcal/mol, respectively. The activities of GT-A and GT-B were not activated by Ca2+, but their thermostability was improved in the presence of Ca2+. Enzyme activity was reduced in presence of Zn2+, Cu2+, Hg2+ and Al3+. Thermal stabilities of GT-A and GT-B were intermediate between Arctic and tropical fish species, and consistent with the wide range of water temperatures to which grass carp are exposed in most provinces of China.

Keywords

Grass carp Intestine Trypsin Purification SDS-PAGE Kinetic Property 

Abbreviations

EDTA

Ethylenediaminetetraacetate

GT-A

Grass carp trypsin isoform A

GT-B

Grass carp trypsin isoform B

BAEE

N-benzoyl-l-arginine ethyl ester HCl

PMSF

Phenylmethylsulfonylfluoride

SBTI

Soy bean trypsin inhibitor

TLCK

Tosyl-lysine chloromethyl kotone

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Zhong-Yi Liu
    • 1
  • Zhang Wang
    • 1
  • Shi-Ying Xu
    • 1
  • Lin-Na Xu
    • 1
  1. 1.Food Science and Safety Focal Laboratory of Ministry of Education of ChinaFood Science Institute of Southern Yangtze UniversityWuxiChina

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