Purification and biochemical characterization of a trypsin-like enzyme present in the sperm of the rock shrimp, Rhynchocinetes typus

Summary

The aim of the present work was to isolate, purify and characterize a trypsin-like enzyme from the sperm of the rock shrimp, Rhynchocinetes typus. Sperm proteins were extracted with 1 mM HCl in 10% glycerol at pH 3.0. Purification of the trypsin-like substance was effected by affinity chromatography using SBTI-agarose, yielding a specific activity on BAEE as substrate of 787 U/mg, with a recovery rate of 34%. Enzymatic activity was maximal at 27°C, pH of 8.0, 50 mM Ca²⁺ and 30 mM Mg²⁺. One hundred percent inhibition of enzymatic activity was obtained at 0.05 mM Zn²⁺. Kinetic analysis showed that the K_M on BAEE as substrate at pH 8.0 was 2.5 x 10⁻⁵ M and the V _MAX, reached was 198 U. It was also found that the enzyme had a substrate inhibition at concentrations higher than 0.06 mM of BAEE. These findings suggest that this enzyme has similar characteristics to other trypsin-like enzymes including acrosin.

Key words:

• Sperm
• trypsin-like
• enzyme
• shrimp