【作者】 翁凌；

【导师】 曹敏杰； 张农；

【作者基本信息】 集美大学 ， 食品加工与安全（专业学位）， 2011， 硕士

【摘要】 本研究以绿豆为原料，通过硫酸抽提、加热处理、超滤浓缩，DEAE-Sepharose阴离子交换柱、Superdex75凝胶过滤等方法，纯化得到绿豆胰蛋白酶抑制剂（Mung bean trypsininhibitor, MBTI）。SDS-PAGE显示其分子量约为9ku, MALTI-QIT-TOF MS质谱分析得到其分子量为8887.25u。性质分析表明MBTI对鲢肌原纤维结合型丝氨酸蛋白酶（Myofibril-bound Serine Proteinase, MBSP）和猪胰蛋白酶有明显的抑制作用，其活力为3812U/mL。MBTI对MBSP的抑制机理为竞争性抑制，MBTI对由MBSP所引起的鱼类肌原纤维蛋白质分解具有显著的抑制作用，推测其对鱼糜制品凝胶形成能力、弹性及品质也有改善效果。针对我国南美白对虾产量大、虾死后肌肉易降解等品质保藏实际问题，通过硫酸铵盐析、DEAE-Sepharose，Phenyl-Sepharose，Hydroxyapatite，Superdex-75等方法从南美白对虾消化腺中分离得到一种丝氨酸蛋白酶。SDS-PAGE结果显示其分子量约为28ku左右，最适pH与最适温度分别为9.0和40℃，该酶在35℃以下以及pH7.0-10.0之间有较高的稳定性。利用Edman降解法测定了该酶的N末端氨基酸序列为IVGGTDAKPGELPYQLSFQDI，与报道的丝氨酸蛋白酶有高度同源性。底物特异性实验与抑制剂实验结果进一步表明该酶属于类胰蛋白酶的丝氨酸蛋白酶。动力学实验显示，以Boc-Phe-Ser-Arg-MCA为底物时，Km=0.69nmol/L，kcat=0.33S-1，kcat/Km=5×10（8mol/L）-1S-1。本研究发现，MBTI对南美白对虾肌肉的自身降解有明显的抑制效果，可作为对虾保鲜剂的重要成分。更多还原

【Abstract】 Mung bean trypsin inhibitor (MBTI) was purified to homogeneity. The purificationprocedures consisted of soaking and extracting with0.1mol/L sulfuric acid, heat treatment,ultrafiltration, DEAE-Sepharose ion exchange column chromatography and Superdex75gel-filtration. The molecular mass of MBTI was approximately9ku, as determined bySDS-PAGE and8887.25u by MALDI-QIT-TOF MS. MBTI effectively inhibited the enzymaticactivity of silver carp myofibril-bound serine protease (MBSP) and porcine trypsin. Theinhibition type of MBTI to MBSP is reversible inhibition. MBTI is an effective inhibitor inpreventing myofibrillar protein (such as myosin heavy chain, MHC, actin and tropomyosin)degradation caused by MBSP and was thus supposed to be effective in preventing surimi geldegradation.On the other hand, with the increasing production of Pacific white shrimp in our country, thequality protection, especially muscle autolysis during storage should be notified. In the presentstudy, a serine proteinase from the hepatopancreas of Pacific white shrimp was purified by aseries of procedures, including ammonium sulfate precipitation, column chromatographies onDEAE-Sepharose, Phenyl-Sepharose, Hydroxyapatite and Superdex75. Purified serineproteinase revealed a single band on SDS-PAGE. The molecular weight was approximately28ku, The optimum pH and temperature of the enzyme were9.0and40℃, respectivety. Theenzyme was stable up to35C in the pH range from7.0to10.0. The N-terminal amino acidsequence of the enzyme as determined by Edman degradation method wasIVGGTDAKPGELPYQLSFQDI which revealed higher idetities to other serine proteinasesreported. Substrate specificity and inhibitor sensitivity experiments suggested that the enzyme isa trypsin-type serine proteinase. Kinetic constants of Kmwas0.69nmol/L, kcatwas0.33S-1andkcat/Kmwas5×10（8mol/L）-1S-1using Boc-Phe-Ser-Arg-MCA as substrate. The suppressing effectof MBTI on Pacific white shrimp muscular protein degradation strongly suggested that MBTIcan be used as an important ingredient as shrimp preservative.更多还原