【作者】 吴国平；

【导师】 苏文金； 曹敏杰；

【作者基本信息】 厦门大学 ， 微生物学， 2009， 博士

【摘要】 蛋白酶及其内源性抑制剂在细胞和生物体的生命活动过程中起着十分重要的作用，在生产实践中也具有广泛的用途。鱼类作为动物中最丰富的物种之一，为人类提供优质蛋白质资源。鱼类肌肉蛋白代谢和死后储藏过程中的降解等生化变化与其内源性蛋白酶和抑制剂密切相关，而这些变化又直接影响鱼肉的品质。因此，对鱼类蛋白及其内源性抑制剂的研究具有重要的理论意义和潜在的应用价值。本文以海水鱼真鲷为研究对象，对其肌肉中三种蛋白酶和一种内源性蛋白酶抑制剂进行了研究，为阐明鱼类肌肉蛋白新陈代谢机制、鱼死后肌肉软化机理等提供实验依据和理论参考。通过硫酸铵沉淀、DEAE-Sephacel离子交换层析、Sephacryl S-200凝胶过滤、羟基磷灰石层析和Phenyl-Sepharose疏水层析等5步纯化，首次从海水名贵鱼真鲷肌肉中纯化到一种亮氨酸氨肽酶（LAP）。LAP是一种结合Zn²⁺和（或）Mn²⁺的金属蛋白酶，分子量为96 kDa，酶的最适温度和最适pH分别为40℃和7．5。37℃下对荧光底物Leu-MCA分解的米氏常数K_m和k_cat值分别为1．55μmol／L和26．4s^-1，水解反应的活化能（E_a）为59．6 kJ／mol。蛋白酶抑制剂Bestatin对LAP表现为竞争性抑制作用，抑制常数（K_I）为1．44μmol／L。Zn²⁺和Cd²⁺对真鲷LAP的抑制作用类型均为混合型抑制，抑制常数（K_I）分别为3．54 mmol／L和0．92mmol／L，对酶-底物络合物的抑制常数(K_IS)则分别为4．49 mmol／L和5．9 mmol／L。亮氨酸、色氨酸和苯丙氨酸对LAP的抑制均为竞争性抑制作用，其抑制常数（K_I）分别为0．54、0．21和1．19 mmol／L；半胱氨酸对LAP为混合型抑制效应，抑制常数K_I为0．26 mmol／L，对酶-底物络合物的抑制常数K_IS为9．43 mmol／L。利用制备的兔抗真鲷LAP多克隆抗血清进行免疫组化分析表明，LAP在真鲷肌肉细胞的胞质内主要呈点状小颗粒样分布，免疫学检测表明在真鲷不同组织器官中广泛分布有LAP。此外，LAP在鲤鱼、鲫鱼、草鱼、鲢鱼、胡子鲶和蓝园鲹等鱼类肌肉中都有存在。通过硫酸铵沉淀、DEAE-Sephacel离子交换、Phenyl-Sepharose疏水层析和羟基磷灰石层析，从真鲷肌肉中纯化到一种具有分解明胶能力的丝氨酸蛋白酶（SP）。SP的分子量为85 kDa，酶的最适温度和最适pH分别为40℃和8．0。丝氨酸蛋白酶抑制剂可明显抑制SP活性，其他类型蛋白酶抑制剂对其活性没有明显影响。该蛋白酶对荧光合成底物Boc-Leu-Lys-Arg-MCA的米氏常数K_m和k_cat值分别为3．58μmol／L和0．13s^-1。实验表明，SP与其它已报道的肌肉肌浆可溶性丝氨酸蛋白酶（MSSP）和肌原纤维结合型丝氨酸蛋白酶（MBSP）的酶学特性均有较明显差异，可能是一种具有独特酶学性质的新型丝氨酸蛋白酶。SP对明胶和真鲷Ⅰ型胶原蛋白具有明显分解能力，对真鲷肌球蛋白重链也有明显分解作用，推测其可能是参与鱼肉保鲜过程肌肉软化的一种重要蛋白酶。通过硫酸铵沉淀、DEAE-Sephacel离子交换层析、Phenyl-Sepharose疏水层析和Gelatin-Sepharose亲和层析，从真鲷肌肉中纯化到一种依赖钙离子的金属蛋白酶（MP）。MP的分子量为52 kDa，酶的最适温度和最适pH分别为40℃和8．0，金属蛋白酶抑制剂可明显抑制其活性，其它蛋白酶抑制剂对MP活性则没有抑制作用。MP活性完全依赖Ca²⁺的存在，性质分析显示MP可能是一种类哺乳动物基质金属蛋白酶。MP对明胶和真鲷Ⅰ型胶原蛋白具有较明显的分解作用，对真鲷肌原纤维蛋白如肌球蛋白重链、肌动蛋白、α-辅肌动蛋白和伴肌动蛋白等具有明显分解作用，推测其很可能是引起鱼肉保鲜过程中肌肉软化的主要蛋白酶之一。利用分子克隆技术，从真鲷肌肉组织中克隆了类哺乳动物金属蛋白酶组织抑制剂2（TIMP-2）编码成熟蛋白区域的基因序列，该基因序列为585bp，推导编码194个氨基酸残基，与来自真鲷囊胚细胞、牙鲆、斑马鱼、大西洋鲑鱼和人TIMP-2氨基酸序列同源性分别为100％、91％、80％、72％和70％，与人TIMP1，3和4的同源性均约为40％。通过基因工程技术，构建了重组酵母系统分泌性表达TIMP-2。在摇瓶培养状态下，rTIMP-2表达水平可达10 mg／L，用Ni-NTA亲和层析可一步高度纯化rTIM P-2。rTIMP-2对真鲷和鲤鱼肌肉肌浆金属蛋白酶有抑制作用，对明胶酶A样的基质金属蛋白酶抑制作用明显。鱼类肌肉中蛋白酶及其内源性抑制剂种类多样，功能各异，它们共同参与肌肉蛋白新陈代谢和鱼死后冷藏过程中的肌肉软化。本文以真鲷为研究对象，首次建立了三种不同类型蛋白酶（LAP、SP和MP）的纯化方法，对三种蛋白酶的生物化学特性、酶学性质等进行了研究；克隆并首次在酵母系统中表达了TIMP-2，对表达的重组蛋白性质进行了分析。本文研究结果为解析鱼类肌肉蛋白新陈代谢和死后肌肉软化机制提供了理论依据，对鱼类保鲜、加工也有潜在的应用价值。更多还原

【Abstract】 Protei nases and their endogenous i nhi bi tors are extremely important proteins thatcomprise life and they are also widely applied in practice for different purposes.Fishare abundant species which supply high quality proteins for human beings.Musclemetabolism and postmortem changes of animals are relative to proteinases andinhibitors.Fish muscle postmortem tenderization is a combination of physical,chemical,biochemical and microbial processes and is also one of the most importantindicators of quality change.The initial changes occurring during postmortem of fishmuscle are due to endogenous enzymes promoting proteolysis of musde proteins andconnective tissues as well as fat hydrolysis.Thus,a detailed study on protei nases andendogenous inhibitors from fish muscle is essential both for theoretical purpose andapplication.In the present paper,three proteinases （leucine aminopeptidase,serineproteinase and metalloproteinase） and a tissue inhibitor of metalloproteinase 2（TIMP-2） from the skeletal muscle of red sea bream were studied.A leucine aminopeptidase （LAP） was purified from red sea bream （pagrus major）skeletal musdeto homogeneity with 4,850-fold and ayield of 7.4 %.The purificationprocedure consisted of ammonium sulfate fractionation and chromatographiesi ncl udi ng D EA E-Sephacel,Sephacryl S-200,Hydroxyapati te,and Phenyl-Sepharose.The molecular mass of the enzyme was approximately 96 kDa as estimated bySDS-PAGE and gel-filtration and it preferentially hydrolyzed substrate Leu-MCA.The enzymatic activity was optimal at 45℃and pH 7.5.The K_m and k_cat values ofLAP for Leu-MCA were 1.55 pmol/L and 26.4 s^-1 at 37℃,respectively.Activationenergy （E_α） of the enzyme was 59.6 kJ/mol.LAP was specifically inhibited bymetal-chelating agents,Zn²⁺ and （or） Mn²⁺ are quite possibly its metal cofactor（s）.Inaddition,bestatin strongly inhibited its activity,and K_I was 1.44μmol/L.Zn²⁺ andCd²⁺ both were mixed type inhibitors to LAP.their inhibition constants （K_I） weredetermined to be 3.54 mmol/L and 0.92 mmol/L,while enzyme-substrate complexes (K_IS) were 4.49 mmol/L and 5.9 mmol/L,respectively.L-Leu,L-Trp,L-Phe werecompetitive inhibitiors,and their inhibition constants were 0.54,0.21 and 1.19mmol/L.L-Cys was a mixed type inhibitor to LAP.the inhibition constant （K_I） andenzyme-substrate complexes (K_IS) were 0.26 mmol/L and 9.43 mmol/L,respectively.Using a highly specific polydonal antibody,the location of LAP was detectedintracellularly and distributed in different tissues of red sea bream.The existence ofLAP was also detected in the skeletal muscle of different fish species,includingcommon carp,crucian carp,grass carp,silver carp,striped catfish,and round scad.A serine proteinase （SP） was purified from red sea bream skeletal muscle tohomogeneity by ammonium sulfate fractionation and chromatographies includingDEAE-Sephacel,PhenyI-Sepharose and Hydroxyapatite.The molecular mass of SPwas approximately 85 kDa as estimated by SDS-PAGE and gel-filtration and itpreferentially hydrolyzed substrate Boc-Leu-Lys-Arg-MCA.The enzymatic activitywas optimal at 40℃and pH 8.0.The enzyme was specifically inhibited by serineproteinase inhibitors,while inhibitors to other type proteinases did not show muchinhibitory effects.The K_m and k_cat values of SPfor Boc-Lea-Lys-Arg-M CA were 3.58μmol/L and 0.13 s^-1 at 37℃,respectively.Furthermore,SP effectively hydrolyzedgelatin,native typeⅠcollagen and myofibrillar proteins such as myosin heavy chain（MHC）.These data suggest that SP is different from muscle sarcoplasmic serineproteinase （MSSP） and myofibril-bound serine proteinase （MBSP）.SP is quitepossibly a novel serine proteinase which play important role during postmortemtenderization of fish muscle.A metalloproteinase （MP） was also purified from red sea bream skeletal muscleto homogeneity by ammonium sul fate fracti onati on and chromatographi es includingDEAE-Sephacel,Phenyl-Sepharose and Gelatin-Sepharose.The molecular mass ofMP was approximately 52 kDa as estimated by SDS-PAGE and gel-filtration.Theenzymatic activity was optimal at 40℃and pH 8.0.The enzyme was almostcompletely inhibited by metalloproteinase inhibitors such as EDTA,EGTA and1,10-phenanthroline,while other proteinase inhibitors did not show any inhibitory effect.Divalent metal ion Ca²⁺ is essential for the gelatinolytic activity.Furthermore,MP effectively hydrolyzed gelatin and native typeⅠcollagen,and also digestedmyofibrillar proteins such as myosin heavy chain （MHC）,actin,α-actinin and nebulin,strongly suggesting its involvement in the texture softening of fish muscle during thepostmortem stage.A TIMP-2 encoding 195 amino acid residues was doned from red sea breamskeletal muscle.The identity of the amino acid sequence of TIMP-2 is 100%,91%,80%,72% and 70% to embryo cell of red sea bream,flounder,zebra fish,salmon andhuman,while only about 40% to other three types of TIM P from human.Furthermore,a recombinant TIM P-2 yeast expression system was constructed for the first ti me and10 mg/L reombinant TIMP-2 could be obtained under flask culture.ReombinantTIMP-2 was purified by Ni-NTA affinity column and inhibited metalloproteinasesfrom red sea bream and a gelatinase A like metalloproteinase from common carpmuscle to different degrees.Proteinases and endogenous inhibitors participate in muscle metabolism andprotein degradati on during postmortem.In the present study,purification methods ofthree different types of protei nase were established and thei r enzymatic characteristicswere investigated.A TIMP-2 gene was also cloned from red sea bream skeletalmuscle and successfully expressed in yeast GS115.The biological activity of therecombinant TIMP-2 to metalloprotei nase and gelatinase A like metalloprotei nase wasdetected.This study is beneficial for understanding the biochemical role ofproteinases and endogenous inhibitors in muscle metabolism and especially related topostmortem tenderization of fish muscle.It also provides some new information forenzymology study and potential applications of these proteinases and inhibitor foraquatic food processing.更多还原