【作者】 杨锋；

【导师】 苏文金； 曹敏杰；

【作者基本信息】 集美大学 ， 食品科学， 2009， 硕士

【摘要】 胰蛋白酶(Trypsin,EC 3.4.21.4)和胰凝乳蛋白酶(Chymotrypsin,EC 3.4.21.1)是丝氨酸蛋白酶家族中的主要水解酶,也是肽链内切酶。胰蛋白酶专一裂解碱性氨基酸残基(Lys,Arg)羧基侧链的肽键;而胰凝乳蛋白酶则专一水解Phe、Trp和Tyr等疏水氨基酸残基羧基侧链的肽键。胰蛋白酶和胰凝乳蛋白酶不但在鱼体内起着重要的生理功能,而且在低温下具有较高的酶活性,是具有潜在重要应用价值的工具酶。目前,国内外关于鱼类胰蛋白酶的研究较多,但对胰凝乳蛋白酶的研究却很少。本课题首次以淡水鲫鱼为研究对象,对其肝胰脏中胰凝乳蛋白酶和胰蛋白酶进行了分离纯化,研究其酶学性质,并制备了抗胰凝乳蛋白酶和胰蛋白酶多克隆抗体,为今后研究淡水鱼胰凝乳蛋白酶和胰蛋白酶的生理功能以及这类酶在生物、医药以及食品加工中的应用提供了理论基础。本课题分离纯化胰凝乳蛋白酶和胰蛋白酶的方法包括硫酸铵分级盐析、DEAE-Sepharose阴离子交换层析、Sephacryl S-200凝胶过滤层析、SP-Sepharose阳离子交换层析和Phenyl-Sepharose疏水层析等。本研究得到了两种类型的胰凝乳蛋白酶(阴离子型胰凝乳蛋白酶Chymotrypsin A和阳离子型胰凝乳蛋白酶Chymotrypsin B)和一种阴离子型胰蛋白酶。SDS-PAGE和Native-PAGE结果表明Chymotrypsin A、Chymotrypsin B和Trypsin都得到了高度纯化。SDS-PAGE显示Chymotrypsin A、Chymotrypsin B和Trypsin的分子量分别为28 kDa、27 kDa和21 kDa。以Suc-Leu-Leu-Val-Tyr-MCA为底物,Chymotrypsin A、Chymotrypsin B的最适温度分别为40°C和50°C,最适pH值分别为pH 7.5和pH 8.5;当水解底物Boc-Phe-Ser-Arg-MCA时,Trypsin的最适温度和最适pH值分别是35°C和pH 8.5。Chymotrypsin A、Chymotrypsin B和Trypsin在40°C以下稳定性较好, pH值的耐受范围为5.5至11.0。丝氨酸类蛋白酶抑制剂对Chymotrypsin A、Chymotrypsin B和Trypsin产生强烈抑制,并且两种酶的抑制效果相似。Trypsin底物特异性实验表明,该酶对荧光底物Boc-Gln-Arg-Arg-MCA的分解能力最强。Chymotrypsin A、Chymotrypsin B和Trypsin可被Ca2+和Mg2+离子激活,而被Fe2+、Zn2+、Mn2+、Cu2+、Ba2+和Cd2+离子在一定程度上抑制。动力学实验表明,以Suc-Leu-Leu-Val-Tyr-MCA为底物,Chymotrypsin A、Chymotrypsin B的Km值分别为1.4和0.5μmol/L,所对应的Kcat值分别为2.7和3.4 s-1;而Trypsin的Km值和Kcat值分别为0.8μmol/L和203.5 s-1。免疫印迹实验表明,Chymotrypsin A和Chymotrypsin B可以与抗鲫鱼Chymotrypsin B抗体呈阳性反应;Trypsin可与抗鲤鱼trypsin抗体呈阳性反应。更多还原

【Abstract】 Trypsin (EC 3.4.21.4) and Chymotrypsin (EC 3.4.21.1) are endopeptidases and are two members of the large family of serine proteinases. Trypsin specifically hydrolyzes proteins and peptides at the carboxyl side of arginine and lysine residues. Chymotrypsin cleaves on the carboxyl side of phenylalanine, tyrosine and tryptophan residues. Trypsin and Chymotrypsin are not only play important physiological and digestive functions in fish body, but also play a critical role in protein digestion for food processing. As fish enzymes that have high acitivy at low temperatures, trypsin and chymotrypsin have also been used in food processing industry. However, till now,information about fish chymotrypsins and trypsins from fresh water fish are less available. In order to study the physiological and digestive functions of trypsins and chymotrypsins from fresh water fish and provide the basic information for application in food processing, we studied the purification, characterization and preparation of polyclonal antibodies against chymotrypsin B and trypsin from the hpatopancreas of crucian carp, an important fresh water fish in China.An anionic form (Chymotrypsin A) and a cationic form (Chymotrypsin B) of chymotrypsins and an anionic form trypsin from the hepatopancreas of crucian carp (Carassius auratus) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation, column chromatographies on DEAE-Sepharose, Sephacryl S-200 HR, Phenyl-Sepharose or SP-Sepharose.Purified chymotrypsins and trypsin revealed single bands on native-PAGE and SDS-PAGE. The molecular weights of chymotrypsin A, chymotrypsin B and trypsin were 28 kDa, 27 kDa and 21 kDa, respectively on SDS-PAGE both under reducing and non-reducing conditions. Chymotrypsin A and chymotrypsin B exhibited maximal activity at 40°C and 50°C, respectively, and optimal pHs were 7.5 and 8.0 using Suc-Leu-Leu-Val-Tyr-MCA as substrate. However, trypsin respectively revealed maximal activity at 35°C and pH 8.5, using Boc-Phe-Ser-Arg- MCA as substrate. The two chymotrypsins and trypsin were stable up to 40°C and in the pH range from 5.5 to 11.0. Serine proteinases inhibitors are effective to chymotrypsins and trypsin and their susceptibilities were similar. Chymotrypsin A, chymotrypsin B and trypsins were activated by metal ions such as Ca2+ and Mg2+ but inactivated by Fe2+, Zn2+, Mn2+, Cu2+ and Cd2+ to different degrees. Apparent Kms of chymotrypsin A and B were 1.4 and 0.5μmol/L and Kcats of the two enzymes were 2.7 and 3.4 S-1 using Suc-Leu-Leu-Val-Tyr-MCA as substrate, respectively. Apparent Km and Kcat of trypsin were 0.8μmol/L and 203.5 s-1, respectively using Boc-Phe-Ser-Arg-MCA as substrate. Immunoblotting analysis using anti-chymotrypsin B weakly cross reacted with chymotrypsin A, suggesting their homogeneity. Immunoblotting analysis using anti-common carp trypsin A cross reacted with the purified trypsin, suggesting common carp trypsin A is homogenous to crucian carp trypsin.更多还原