Studies on cathepsin L in the surimi of southern blue whiting

Abstract

According to the conventional method,actomyosin was extracted from the surimi of southern blue whiting (Micromesistius australias). After Sepharose-6B gel filtration of the actomyosin obtained,cathepsin L activity was monitored in each of the fractions collected. The results showed that the activity values in the actomyosin samples were much higher than that in the washing water,suggesting that cathepsin L could not be removed completely during conventional leaching and still remained in actomyosin sample. Further purification of actomyosin by dilution-precipitation could not remove cathepsin L effectively,indicating that cathepsin L might be dissolved together with actomyosin in the presence of high ion-strength solutions. The profile of the gel filtration showed hat the main peak of cathepsin L was obviously separated from that of actomyosin,suggesting that cathepsin L was non-binding with actomyosin. Fractions that showing the main peak of cathepsin L were pooled to be called L_ mix ,partially purified cathepsin L. L_ mix could strongly hydrolyze Z-Phe-Arg-MCA,a specific substrate for cathepsin L,rather than Z-Arg-Arg-MCA,spcefic substrate for cathepsin B,nor Arg-MCA,substrate for cathepsin H,as well as Boc-Gln-Ala-Arg-MCA,substrate for trypsin and trypsin-like proteases. The above results indicate that L_ mix was a crude cathepsin L. Cathepsin L specific inhibitors,such as E-64,specific inhibitor for cathepsins,and leupeptin,inhibitor for both cathepsins and trypsin-like proteases,could suppress the activity of L_ mix completely. Activators for cathepsin L,such as DTT,EDTA+DTT,could effectively improve the activity of L_ mix . As a result,the effect of activators and inhibitors confirmed the partially purified crude enzyme,L_ mix ,to be a thiol-type cysteine protease. The temperature dependence and pH dependence of the activity of L_ mix were also investigated. L_ mix had optimum temperature of 45 ℃ which was right in the temperature range of modori phenomena. L_ mix had optimum pH of 5.5,but kept high residue activity near neutral pH,and the pH range of surimi processing,suggesting its high potential ability in modori phenomena.