【作者】 胡重江；

【导师】 李英文；

【作者基本信息】 西南农业大学 ， 水产养殖， 2005， 硕士

【摘要】 动物体内有多种蛋白消化酶。蛋白消化酶类协同作用,分解蛋白质,利于肠道对氨基酸的吸收。胰蛋白酶、糜蛋白酶和弹性蛋白酶存在于几乎所有脊椎动物和无脊椎动物的消化道内,它们属于肽链内切酶。胰蛋白酶是其中一种重要的动物蛋白水解酶,专一性强,专一水解精氨酸、赖氨酸（三种碱性氨基酸之两种）羧基参与形成的肽键、酰胺键和酯键,其高度专一性表现为对碱性氨基酸一端的选择,属于族专一性（基团专一性）。本研究目的是草鱼肝胰脏中纯化胰蛋白酶并对其性质进行分析研究。 草鱼胰蛋白酶（GT）的纯化: 本实验采用硫酸铵分级沉淀和鸡卵类粘蛋白偶联的琼脂糖凝胶亲和层析,得到两种酶,即胰蛋白酶A（GTA）和胰蛋白酶B（GTB）。 胰蛋白酶用0.5 M NaCl,0.05 M CACl₂,0.05 M Tris-HCI（pH8.0）粗酶抽提缓冲液抽提出来,得到粗酶抽提液。将粗酶抽提液加入固体硫酸铵至30%饱和度,4℃静置3h后,离心（8000 rpm,0℃,20 min）,将沉淀弃置,上清液中继续加入固体硫酸铵至70%饱和浓度,4℃静置过夜,离心（8000rpm,0℃,15 min）,收集沉淀,将其用适量的亲和柱平衡液Tris-HCI缓冲液（pH8.0,50 mM）溶解。得粗酶液.装柱:取一支层析柱,先装入1/4体积的亲和柱平衡液（0.1 M pH=8.0,含0.05 MCaCl₂的Tris-HCI溶液）。然后将亲和吸附剂轻轻搅匀,缓缓加入柱内,待其自然沉降,调好流速3 mL/10 min左右,用亲和柱平衡液平衡,检测流出液A₂₈₀值小于0.02。上样:将上一步得到的硫酸铵分级沉淀粗酶液用5 M NaOH调至pH=8.0,（若有沉淀,过滤去除之）。沉淀用0.5 M NaCl,0.05 M CaCl₂,0.05 MTris-HCI（pH7.0）缓冲液溶解。然后,取一定体积上述澄清溶液上柱吸附。亲和层析柱用同一缓冲液洗去杂蛋白,再用0.5 M glycine-HCI（pH3.5）缓冲液洗脱,收集一个有胰蛋白酶活性的蛋白峰,是为GTA。然后再用0.5 M glycine-HCI（pH3.0）缓冲液洗脱,收集另一个有胰蛋白酶活性的蛋白峰,即GTB。立即加入pH调节液,将pH调到9.0左右。结果GTA纯化了243倍,比活力61388 TAME单位/mg,产率30.3%。GTB纯化了5倍,比活力1447 TAME单位/mg,产率0.2%。SDS-PAGE后银染,结果显示两种酶均为一条带,两种酶分子量约为27 kDa。更多还原

【Abstract】 Trypsin, chymotrypsin, and elastase occur in the alimentary canal of virtually all invertebrate and vertebrate organisms (Solomon et al., 1996) , they are all endopeptidases, which catalyze the hydrolysis of peptide bonds in the interior of a polypeptide chain or protein molecule .Trypsin is one of several animal digestive proteases whose activities collectively breakdown ingested proteins to facilitate gut ammo acid absorption. Trypsin is an enzyme specific for peptides and esters of the amino acids lysine and arginine. The goals of this research were to purify and characterize trypsin from hepatopancreas of grass carp (Ctenopharyngodon idellus).Purification of grass carp trypsin (GT)Enzyme was purified by using ammonium sulfate fractionation, CHOM sepharose 4B affinity column, two kinds of trypsin were isolated, called GTA and GTB .Trypsin was extracted from the hepatopancreas with 0.5 M NaCl-0.05 M CaC12-0.05 M, Tris-HCl extraction buffer (pH8.0), some guts were added in an effort to active trypsinogen. After actived, the crude enzyme extract was brought to 30% saturation with ammonium sulphate and subsequently centrifuged at 8, 000 rpm for 20 minutes at 0 ℃ , The 30% supernatant solution was brought to 70% saturation with ammonium sulphate, so that the 30% to 70% precipitate were collected. The precipitate was resolved with 0.5 MNaCl, 0.05 M CaCI2, 0.05 M, Tris-HCl buffer (pH7.0) . Afterwards, the crude enzyme solution was loaded on CHOM sepharose4B affinity column chromatograph, which was pre-equilibrated by 0.5 M NaCl-0.05 M CaC12-0.05 M, Tris-HCl buffer (pH7.0) fully. The column was washed with the same buffer to wash off all of the impurity proteins, then the column was eluted with 0.5 M glycine-HCl buffer (pH3.5) . The unique peak of trypsin activity was pooled , named GTA. Then the column was eluted with 0.5 M glycine-HCl buffer(pH3.0) and another enzyme with trypsin activity was obtained ,named GTB. As a result. GTA was 243-fold purified , with the specific activity of 61388 TAME units/mg protein and recovery of 30.3%, while GTB 5-fold purified, with the specific activity of 1447 TAME units/mg protein andrecovery of 0.2%. SDS-PAGE electrophoresis was performed as per the modified Laemmli method, using a discontinuous buffer system, 5 % stacking gel was made with a 1 MTris-HCI (pH6.8) buffer, while 15 % resolving ge11.5 M Tris-HCI (pH8.8) buffer. It is notable that lanes which containing proteins representing the highest level of trypsins-GTA and GTB purification obtained in this study, revealed a single band using silver stain. The purified trypsins themselves indicates an approximate molecular weight of 27 kDa.Some properties of two enzymes were studied.The pH optima for the grass carp trypsinsActivity of GT was measured in buffers of different pH value. It indicated that:The activities were influenced significantly by pH value. They were very low at pH below 5.5and increased rapidly in the range of pHS.S-8.0. But they also decreased rapidly when pH exceeded8.0. Their optimal activities were observed at approximately pH8.0.pH stability of GTStability of GT was measured in buffers of different pH value. It indicated that:Stability of GTA increased continuously in the range of pH5.5-9.0. It decreased gradually whenpH exceeded 9.0. GTA was most stable at pH9.0.Stability of GTB increased continuously in the range of pH5.5-9.5. It decreased gradually whenpH exceeded 9.0. GTA was most stable at pH9.5.Temperature optima for the grass carp trypsinsActivity of GT was measured at different temperature. It indicated that:The activity of GTA increased when temperature increased. The temperature optimum for GTAwas 60℃. But the activity decreased gradually when temperature exceeded 60℃. It decreasedrapidly when temperature exceed 70℃.The activity of GTB increased when temperature increased. The temperature optimum for GTAwas 60℃. But the activity decreased rapidly when temperature exceeded 60℃.Thermostabilities of GTGT was stable at temperature under 40℃. When maintained at 40℃ for 30 minutes, theremaining activity of GTA was 91.7 percent of the highest activity of the enzyme, while that of GTB80.6 percent. GT lost activity rapidly when temperature exceeded 40℃ .When it reached 60℃, theremaining activity of GTA was 2.6 percent of the highest activity of the enzyme, while that of GTB2.0percent. When it reached 80℃, the remaining activity of GTA was 1.0 percent of the highestactivity of the enzyme, while that of GTB 0.5 percent.Summary: The Michaelis constant of GTA is 0.37 mmolBAPA/ mL. Both GTA and GTBexhibited optimal activity at a pH of 8.0 and at a temperature of 60℃, using TAME as substrate.更多还原