Abstract
An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.
Highlights
Lysosomal cysteine proteases, cathepsins B, H, L, and so on, play an important role in intracellular protein turnover
We decided that pool B was the cathepsin B fraction, and it was used for further purification
This paper was the first report that cathepsin B was purified and characterized from horse mackerel muscle
Summary
Cathepsins B, H, L, and so on, play an important role in intracellular protein turnover. It is generally considered that a major cause of post-mortem muscle softening in fish is proteolysis of muscle structural proteins by endogenous proteases such as lysosomal cysteine proteases. It is a general concept that endogenous proteases cause thermal gel-weakening, which is called the modori phenomenon. It is one of the urgent problems in the fisheries industry. Cathepsin B was the most active cysteine protease in horse makerel muscle at 50 °C, suggesting that the enzyme was likely to participate in thermal gel disintegration. The purpose in this study is to contribute to the quality improvement of surimi-based products using natural protease inhibitors and the possible application of endogenous proteases to seafood processing
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