Study On Collagenolytic Proteinases In The Softening Of Common Carp (Cyprinus Carpio) Muscle

Fresh water fish is abundant in our country and fish muscle is regarded as high qualityprotein resource for consumption and as crude material for aquatic products processing.Post-mortem tenderization of fish muscle is an unfavorable change to affect the quality of muscletexture occurred during chilled storage. Disintegration of myofibrillar proteins and collagenscaused by different types of endogenous proteinases are proposed to be the main responsibilityfor the post-mortem changes. In contrast to Cathepsin, Calpain and myofibril-bound serineproteinase （MBSP）, which were proved to take part in the degradation of myofibrillar proteins offish muscle, few reports are available concering collagenolytic proteinases in fish muscle. In thepresent study, using common carp as material, we tried to elucidate the effect of collagenolyticproteinases on the softening of muscle.The present study investigated the activities of serine proteinase and metalloproteinaseduring the cold storage of muscle and found that the activity of metalloproteinase increasedsignificantly. At the same time, the texture index of hardness, cohesiveness, gumminess,springness, chewiness of fish muscle decreased accompanied with the metalloproteinase activityascending. Thus, the change of fish texture was proposed to be tightly associated with theactivity of metalloproteinase in the sarcoplasmic fraction.In order to take a comprehensive analysis of the functional of collagenolytic proteinases incarp muscle, serine proteinase and metalloproteinases were purified to homogeneity byammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Phenyl-Sepharoseand Gelatin-Sepharose. The purified serine proteinase showed a molecular weight of70kDa, andwas named collagenolytic proteinase-1（CP-1）. The purified metalloproteinases revealed twobands with molecular masses of66and65kDa as estimated by SDS–PAGE, which were namedcollagenolytic proteinase-2A（CP-2A）and collagenolytic proteinase-2B（CP-2B）. These twokinds of enzymes showed activities under alkaline conditions, and the optimum temperature forthe enzymes were30℃and40℃, respectively. CP-1was specifically inhibited by serineproteinase inhibitors while CP-2was specifically inhibited by metalloproteinase inhibitors. Metalions of Ca²⁺and Ba²⁺activated CP-2to some degree. By matrix-assisted laserdesorption/ionization-tandem time-of-flight mass spectrometry （MALDI-TOF/TOF-MS）analysis,6peptide fragments with107amino acid residues were obtained from CP-2A while5 peptide fragments with69amino acid residues were obtained from CP-2B. These peptidefragments were identical to MMP-2from grass carp muscle, confirming the purified CP-2isMMP-2.In fish intramuscular connective tissue, type I and V collagen are major and typical fibrillarcollagen molecules. Thus, these two kinds of collagens were isolated from carp muscle forinvestigation of the hydrolyzing ability and degradation effect of purified collagenolyticproteinases. At optimum temparature, both CP-1and CP-2significantly degraded type I and typeV collagen. Moreover, the two collagenolytic proteinases persisted their activities even at4℃.This result suggested that the two enzymes are able to degrade collagens in fish muscle duringchilled storage.In summary, our present study investigated different kinds of collagenolytic proteinase inthe sarcoplasmic proteins of common carp muscle. The relationship between collagenolyticactivities and texture profile parameters of fish muscle during chilled storage was revealed. Thismanuscript also constructed a series of process to purify different kinds of collagenolyticproteinase. The impact of temperature, pH, proteinase inhibitors and metal ions on the activitiesof enzymes was also discussed. Particularly, using peptide mass fingerprinting （PMF） technology,the purified metalloproteinases were proved to be matrix metalloproteinase-2（MMP-2）, givingan adequate evidence to prove the involvement of MMP-2in the softening of fish muscle.