Abstract:

In this study, the optimum isolation condition of crude cysteine proteinase inhibitors (CPIs) from silver
carp eggs that provided increased specific activity as measured by Azocasein assay was determined as homogenization
using 20 mmol/L phosphate buffer containing 0.1 mmol/L phenylmethanesulfonyl fluoride (PMSF) at pH 6.0, and pH
readjustment to 8.0 after acid (pH 3.0) treatment of the homogenate at 30 ℃ for 10 min. Under the optimized conditions, the
purity of CPIs was enhanced 16.54 folds. SP Sepharose fast flow chromatography analysis demonstrated effective removal
of acid, alkali and protein impurities with poor thermal stability using acid treatment at pH 3.0 than at pH 4.0, giving rise to
a purification factor of 48.22. After further chromatography on a Sephacryl S-200 column, partially purified low-molecularweight
fractions named as Ⅱ-b and Ⅱ-c were obtained with a specific activity of 1 098.59 and 1 769.23 U/mg, respectively,
and their purification folds were 312.10 and 502.62 times, respectively. The electrophoresis analysis showed that Ⅱ-b and
Ⅱ-c could not be identified by gelatin substrate-SDS-reverse zymography. However, after reaction with papain under the
appropriate condition, both fractions were found to contain at least two low-molecule-weight CPIs (7 and 10 kD) as
indicated by SDS-PAGE analysis. When added to silver carp surimi at a level of 5 U/g, each fraction significantly improved
the gel strength significantly by 48.77% and 55.55%, respectively, and inhibited softening.

Key words: silver carp eggs, cysteine protease inhibitors, purification, characterization, surimi gel strength