Study On Proteinases Related To Texture From The Muscle Of Abalone(Haliotis Discus Hannai)

Abalone is regarded as one of the finest type of seafood,valued for its unique texture,flavor and its background of Chinese traditional culture.With the development of economy and the improvement of people's living standard,abalone consumption is getting more and more popular.Adductor muscle of the abalone,mainly consists of myofibril and collagen,the inseparable combination of the two proteins make its unique texture.Postmortem tenderization of abalone muscle is a complex biochemical process and is also one of the most important indicators of texture change.Moreover,endogenous proteinases and inhibitors existing in the body wall play important roles in the softening process by affecting the major structural protein component.To elucidate the texture change mechanism,we set up a study to purify and characterize two kinds of protein including paramyosin and collagen,and a proteinase named cathepsin L as well as a serine protease inhibitor,what's more,and to study the most important characterization of them.This aim of reserach is to understand the intrinsic nature of the texture change of abalone and to link textural attributes to proteins and proteinases and endogenous inhibitors of the muscle tissue.A paramyosin?PM?was purified from the myofibril of abalone.The purification procedure consisted of ammonium sulfate fractionation and hydroxyapatide chromatography.The molecular weight of PM as estimated by SDS-PAGE was 97.0 kDa.Peptide mass fingerprinting of PM obtained 36 peptide fragments with a total of 323 amino acid residues,which were 99.7% identical to paramyosin from Haliotis discus discus.Circular dichroism?CD?spectrum analysis demonstrated that PM have typical spectra characteristics of ?-helix structure;Meanwhile,the denaturation temperature?Td?of PM measured by circular dichroism spectrum was about 58.1 ?.Purification and characterization of pepsin-soluble collagen from adductor?PSC1?of abalone was conducted.The results of SDS-PAGE suggested that the structure of PSC1 was??1?3 and the molecular weight of ?1 chain was approximately 140 kDa,which was the characteristic of type?collagen from aquatic invertebrates.Peptide mass fingerprinting?PMF?of PSC1 analysis obtained 6 peptide fragments including 75 amino acid residues,which were identical to collagen from H.discus with 100% identity.FTIR spectra further confirmed that PSC1 have complete triple helical structure.Meanwhile,the denaturation temperature?Td?of PM measured by circular dichroism spectrum was about 27.3 ?.What's more,a polyclonal antibody was prepared against pepsin-soluble collagen from abalone adductor?PSC1?.According to Western blot analysis,the polyclonal antibody only positively reacted with ?,? and ? chains of PSC1 from abalone,indicating high specificity of the polyclonal antibody.A cathepsin L was isolated from the muscle of abalone by processes including ammonium sulfate fractionation,ion exchange chromatography,gel filtration chromatography and hydrophobic chromatography.The enzyme was in homogeneity with a molecular weight of approximately 30 kDa on SDS-PAGE.Using Z-Phe-Arg-MCA as substrate,the purified proteinase revealed optimal activity at temperature 35 ? and pH 5.5,respectively.The enzymatic activity of CL was completely suppressed by cystatin proteinases inhibitor E-64,while other proteinase inhibitors did not show any inhibitory effect.In the present study,we found that there existed a relationship between the increasing activity of cathepsin L and the decreasing textural attributes during the cold storage of abalone muscle,and what,s more,the enzyme hydrolyzed abalone native myofibrillar proteins at 37 ?,suggesting its possible involvement in the degradation of myofibrils from abalone during storaging and processing.A trypsin inhibitor was isolated from the muscle of abalone by heat treatment,acid-alkali treatment,ammonium sulfate fractionation and chromatographies including SP-Sepharose and Phenyl-Sepharose.The trypsin inhibitor,named as HhTI?H.discus hannai trypsin inhibitor?,was in homogeneity with a molecular weight of approximately 7 kDa on SDS-PAGE.HhTI maintained high inhibitory activity at pH 2.0¹0.0 and 0⁸0 ?.HhTI was specifically active against trypsin,while showed no inhibitory activity to pepsin,papain and chymotrypsin.Moreover,HhTI completely inhibited trypsin at a molar ratio of 2:1?HhTI : trypsin?analyzed by inhibition activity assay.In addition,we found that there existed myofibril-bound proteinase?MBP?,and the seasonal change had a significant impact on the activity of MBP.According to SDS-PAGE analysis,MBP showed high enzymatic activity in degradating MHC and PM at the temperature range from 50 to 70 ?.The enzymatic activity of MBP was completely suppressed by metalloproteinase inhibitors?EDTA,EGTA and 1,10-Phenanthroline?,and partially inhibited by serine proteinase inhibitor?Benzamidine?.The protease was scarcely inhibited by Ca²⁺ and Mg²⁺,moderately actived by Cu²⁺,and suppressed by Zn²⁺ and Mn²⁺ to some degrees.Hence,this research supplies a new way about MHC and PM degradation and is helpful for studying myofibril protein metabolism.Proteinases and inhibitors play important roles in protein physiological metabolism and protein degredation during postmortem.In the present study,two structural proteins,a cathepsin L as well as a serine proteinase inhibitor were purified,and their characteristics were investigated.This study is beneficial for clarifying the mechanism of abalone texture change during storaging and processing,and providing some significant reference value for ablone processing and preservation.