大菱鲆主要消化酶—蛋白酶、脂肪酶、淀粉酶的研究
摘要
本论文从消化酶的角度，研究了大菱鲆体内主要消化酶如蛋白酶、脂肪酶、淀粉酶的分布和活性，鱼龄、温度、盐度等因素对大菱鲆消化酶活性的影响，并对其主要蛋白酶进行了纯化和理化性质的研究，分析了大菱鲆粗酶液对饲料中常用蛋白质饲料原料的体外消化率，对各种蛋白质饲料原料进行营养评价，最后探讨了植物蛋白替代鱼粉的可行性及外源酶制剂应用的情况。主要研究结论如下：
(1)大菱鲆对蛋白质的消化主要在胃和前肠中进行，对淀粉的消化则主要依靠前肠和幽门盲囊，而脂肪消化主要集中在肠中。
不同消化部位如胃、幽门盲囊、前肠、中肠、后肠的蛋白酶最适作用温度和pH依次为：40℃、pH2.0，40℃、pH8.0，60℃、pH8.0，40℃、pH8.5，60℃、pH8.0；脂肪酶最适温度和pH依次为：40℃、pH6.5，40℃、pH8.0，40℃、pH7.5，50℃、pH7.0，40℃、pH8.0；淀粉酶最适温度和pH依次为：40℃、pH7.5，40℃、pH6.5，40℃、pH7.0，40℃、pH7.5，40℃、pH7.5。另外，各酶均有低温下最适pH升高，及作用时间延长时最适温度降低的特性，适应生理条件下的消化。
(2)大菱鲆生长发育过程中，消化生理和各消化酶分泌机能逐步完善。蛋白酶和脂肪酶活力在整个发育阶段持续增高，淀粉酶活力在幼鱼期呈增大趋势，180天鱼龄后开始下降。整个生长过程中蛋白酶活力处于绝对优势。180天鱼龄是大菱鲆生长发育的一个转折点，可作为大菱鲆配合饲料分阶段研制的一个参考分界点。
(3)环境水温对大菱鲆消化生理有明显的影响作用：随水体温度的提高，大菱鲆消化器官比重呈明显的下降趋势；大菱鲆各消化器官中分泌的酶蛋白量随水体温度的提高持续下降，肠中酶蛋白分泌量降幅最大；各消化器官中总酶活力随水温的升高呈先上升后下降的趋势，其中蛋白酶受影响最大。
(4)水体盐度对大菱鲆消化酶活性影响显著。在0M-0.5M的盐度变化范围内，幽门盲囊蛋白酶、肠蛋白酶随盐度增高被激活，胃蛋白酶、脂肪酶和淀粉酶均先被激活后被抑制。脂肪酶受盐度影响最大，蛋白酶最小；消化器官中幽门盲囊受盐度影响最显著，胃和肠次之。在0.2M-0.3M的盐度范围内，各消化部位的
重要消化酶均处于比较活跃的状态。
(5)对大菱坪主要蛋白酶进行了分离纯化，并研究了其理化性质:
采用硫酸钱分级沉淀、DEAE一S即har0Se F.F.阴离子交换层析、Sephadex
G一100凝胶过滤层析、Sephacry1S一200凝胶过滤层析等纯化技术得到三种蛋白
酶电泳纯样品，分别为胃蛋白酶、幽门盲囊蛋白酶、肠蛋白酶I。经电泳检测分
子量依次为39900Dal、66009Dal、86776Dal。纯酶最适反应pH依次为2.0、9.0、
8.5，最适反应温度依次为40℃、30一40℃、50℃;PH稳定范围依次为pHI.0一9.0、
pH6.0一10.0、pH7.0一10.5，40oC以下酶活性较稳定;Mn，‘和Cu，‘激活大菱虾胃
蛋白酶，Fe3+、丝氨酸蛋白酶抑制剂、糜蛋白酶抑制剂、胃酶抑制剂、琉基蛋白
酶抑制剂显著抑制大菱鲜胃蛋白酶活性;Mn2+、M扩激活幽门盲囊蛋白酶，Cuz+、
Zn2+、琉基蛋白酶抑制剂和胰蛋白酶抑制剂抑制其活性;Mnz+激活肠蛋白酶I，Cuz+、
Zn2+、Ag‘、Fe3+、琉基蛋白酶抑制剂、胰蛋白酶抑制剂抑制大菱娜肠蛋白酶I活
性。双倒数作图法得大菱坪胃蛋白酶Iha为2.559/L、肠蛋白酶I的Km为2.929/L。
(6)大菱坪不同消化器官粗酶液对饲料原料干物质和蛋白质的体外消化能
力均表明胃、前肠、幽门盲囊为营养物质主要消化部位。十种饲料原料的干物质
体外消化率依次为进口秘鲁鱼粉>国产鱼粉>虾粉>肉粉>饲料酵母>豆粕>
花生粕>棉粕>麦鼓>次粉，蛋白质体外消化率依次为进口秘鲁鱼粉>虾粉>国
产鱼粉>肉粉>饲料酵母>豆粕>花生粕>次粉>棉粕>麦数，动物性饲料原料
的干物质、蛋白质体外消化率明显高于植物性饲料的相应体外消化率。氨基酸指
数EAAI表明，除饲料酵母为良好蛋白源外，其余均为优质蛋白源。
综合分析表明十种饲料原料中动物性饲料以虾粉、肉粉较好，植物性饲料中
则以豆粕最佳。
(7)通过不同豆粕含量及外源酶制剂的六个实验组在18℃下50天的饲喂
实验，结果表明:无外源消化酶饲料配方下的大菱虾对蛋白质和碳水化合物的表
观消化率随饲料中豆粕含量的增加呈下降的趋势。20%豆粕替代组和全鱼粉组实
验鱼的生长状态明显好于40%豆粕替代组及全豆粕组。各实验组大菱虾消化器官
比重随豆粕添加量的增多而增大，以比肝重变化最为显著。不同饲料配方组大菱
醉蛋白酶、淀粉酶活力随豆粕含量的增多而升高，脂肪酶无明显变化。添加外源
酶制剂组相比于未添加组生长状态及消化生理有明显改善。
实验结果表明，饲料中以豆粕替代鱼粉量功%对大菱虾生长无负面影响，在
添加外源酶制剂的情况下豆粕替代量可增加到100k仍能保证大菱坪的正常生长。
Turbot Scophthalmus maximus L. is a culture species naturally living in Europe. It is newly imported to China by Yellow Sea Fisheries Research Institute in 1992. Turbot is adapted to living in low temperature, enduring low oxygen and can breed in high density. Now the turbot culture becomes a new industry in our sea economy. In order to promote its culture in our country, it is very necessary to do some work on its basic research. In this study, the main digestive enzymes in turbot were fully discussed. Main proteases were purified and their characters were studied. The vitro coefficient of feed material by turbot alimentaiy tract homogenates was studied. Finally, partial or total replacement offish meal by soybean meal in diet for turbot and the dietary enzyme were researched.
(1) The protein is digested mainly by stomach and foregut, and starch mainly by foregut and pylorus cecum, and fat mainly by gui. The optimum temperature and pH of protease in each digestive organ such as stomach, pylorus cecum, foregut, midgut, hindgut are 40癈 pH2.0, 40癈 pHS.O, 60癈 pH8.0,40癈 pH8.5, 60癈 pH8.0, And for the amylase are 40 pH6.5, 40 pHS.O 40 pH7.5, 50 pH7.0, 40 pH8.0, for the lipase are 40 pH7.5, 40 pH6.5,40 pH7.0,40 pH 7.5, 40 pH 7.5. Otherwise, the optimum pH of all digestive enzymes increases in low temperature. And the optimum of temperature decrease when the reaction time is longer.
(2)The protease activity of turbot is increased continually in all growth phases, and the maximum activity increase occurs between 30-day old and 90-day old. The lipase activity is also increased along with the gro \vth and the difference between each phase is not very great. The amylase activity s increased in the parr phase and decreased after 180-day. The dominant enzyme i> protease in all growth phases, and 180-day phase is the turning point during the growth of turbot.
(3)Within a water temperature range of 10 ~ 25 , the weight of digestive organs in percentage of body weight tends to decrease with the increase of temperature. And
the quantity of enzyme protein secreted by each digestive organ also tends to decrease, and the enzyme protein in intestine decreased farthest. The total enzyme activity is increased firstly and then decrease with the increase of the water temperature. Among the digestive enzymes, the activity of protease is affected most significantly.
(4)At salt concentration range of OM-0.5M, the activity of proteases in pylorus cecum and gut of turbot are activated when the salt concentration is increased. And the activity of stomach protease is activated in low salt concentration but inhibited in high salt concentration. The activity of lipase and amylase is also activated firstly and inhibited in high salt concentration. The activity of lipase is affected strongly and protease is least affected by salt. Most digestive enzymes of each digestive organ are activated in the salt concentration between 0.2M-0.3M.
(5)The stomach protease^ pylorus cecum protease and gut protease I are purified by using ammonium sulfate precipitation, DEAE-Sepharose F.F. , Sephadex G-100 column and Sephacryl S -200 column chromatography. The purified enzymes exhibit a single band on SDS-polyacrolymide gel electrophoresis (SDS-PAGE). Their molecular weights are determined to be about 39900Dal, 66009Dal and 86776Dal respectively. The stomach protease is stable at pH ranging from 1.0-9.0 and below 40 癈. The optimum temperature and pH of enzyme are 40 癈 and pH2.0 for stomach protease, 30-40癈 and pH9.0 for pylorus cecum protease , 50 癈 and pH8.5 for gut protease I . The stomach protease is activated by Mn2+, Cu2+, and inactivated by Fe3+, PMSF, TPCK, Pepstatin, PCMB. The pylorus cecum protease is activated by Mn2+, Mg2+ and inhibited by Cu2+, Zn2+, PCMB, TLCK. The gut protease I is activated by Mn2+ and inhibited by Cu2+, Zn2+, Ag+, Fe3+, PCMB and TLCK. The Km value of stomach protease and gut protease I are determined to be 2.55g/L and 2.92g/L respectively.
(6) The vitro dry matter digestive ability of each digestive organ is in the seq
(1)大菱鲆对蛋白质的消化主要在胃和前肠中进行，对淀粉的消化则主要依靠前肠和幽门盲囊，而脂肪消化主要集中在肠中。
不同消化部位如胃、幽门盲囊、前肠、中肠、后肠的蛋白酶最适作用温度和pH依次为：40℃、pH2.0，40℃、pH8.0，60℃、pH8.0，40℃、pH8.5，60℃、pH8.0；脂肪酶最适温度和pH依次为：40℃、pH6.5，40℃、pH8.0，40℃、pH7.5，50℃、pH7.0，40℃、pH8.0；淀粉酶最适温度和pH依次为：40℃、pH7.5，40℃、pH6.5，40℃、pH7.0，40℃、pH7.5，40℃、pH7.5。另外，各酶均有低温下最适pH升高，及作用时间延长时最适温度降低的特性，适应生理条件下的消化。
(2)大菱鲆生长发育过程中，消化生理和各消化酶分泌机能逐步完善。蛋白酶和脂肪酶活力在整个发育阶段持续增高，淀粉酶活力在幼鱼期呈增大趋势，180天鱼龄后开始下降。整个生长过程中蛋白酶活力处于绝对优势。180天鱼龄是大菱鲆生长发育的一个转折点，可作为大菱鲆配合饲料分阶段研制的一个参考分界点。
(3)环境水温对大菱鲆消化生理有明显的影响作用：随水体温度的提高，大菱鲆消化器官比重呈明显的下降趋势；大菱鲆各消化器官中分泌的酶蛋白量随水体温度的提高持续下降，肠中酶蛋白分泌量降幅最大；各消化器官中总酶活力随水温的升高呈先上升后下降的趋势，其中蛋白酶受影响最大。
(4)水体盐度对大菱鲆消化酶活性影响显著。在0M-0.5M的盐度变化范围内，幽门盲囊蛋白酶、肠蛋白酶随盐度增高被激活，胃蛋白酶、脂肪酶和淀粉酶均先被激活后被抑制。脂肪酶受盐度影响最大，蛋白酶最小；消化器官中幽门盲囊受盐度影响最显著，胃和肠次之。在0.2M-0.3M的盐度范围内，各消化部位的
重要消化酶均处于比较活跃的状态。
(5)对大菱坪主要蛋白酶进行了分离纯化，并研究了其理化性质:
采用硫酸钱分级沉淀、DEAE一S即har0Se F.F.阴离子交换层析、Sephadex
G一100凝胶过滤层析、Sephacry1S一200凝胶过滤层析等纯化技术得到三种蛋白
酶电泳纯样品，分别为胃蛋白酶、幽门盲囊蛋白酶、肠蛋白酶I。经电泳检测分
子量依次为39900Dal、66009Dal、86776Dal。纯酶最适反应pH依次为2.0、9.0、
8.5，最适反应温度依次为40℃、30一40℃、50℃;PH稳定范围依次为pHI.0一9.0、
pH6.0一10.0、pH7.0一10.5，40oC以下酶活性较稳定;Mn，‘和Cu，‘激活大菱虾胃
蛋白酶，Fe3+、丝氨酸蛋白酶抑制剂、糜蛋白酶抑制剂、胃酶抑制剂、琉基蛋白
酶抑制剂显著抑制大菱鲜胃蛋白酶活性;Mn2+、M扩激活幽门盲囊蛋白酶，Cuz+、
Zn2+、琉基蛋白酶抑制剂和胰蛋白酶抑制剂抑制其活性;Mnz+激活肠蛋白酶I，Cuz+、
Zn2+、Ag‘、Fe3+、琉基蛋白酶抑制剂、胰蛋白酶抑制剂抑制大菱娜肠蛋白酶I活
性。双倒数作图法得大菱坪胃蛋白酶Iha为2.559/L、肠蛋白酶I的Km为2.929/L。
(6)大菱坪不同消化器官粗酶液对饲料原料干物质和蛋白质的体外消化能
力均表明胃、前肠、幽门盲囊为营养物质主要消化部位。十种饲料原料的干物质
体外消化率依次为进口秘鲁鱼粉>国产鱼粉>虾粉>肉粉>饲料酵母>豆粕>
花生粕>棉粕>麦鼓>次粉，蛋白质体外消化率依次为进口秘鲁鱼粉>虾粉>国
产鱼粉>肉粉>饲料酵母>豆粕>花生粕>次粉>棉粕>麦数，动物性饲料原料
的干物质、蛋白质体外消化率明显高于植物性饲料的相应体外消化率。氨基酸指
数EAAI表明，除饲料酵母为良好蛋白源外，其余均为优质蛋白源。
综合分析表明十种饲料原料中动物性饲料以虾粉、肉粉较好，植物性饲料中
则以豆粕最佳。
(7)通过不同豆粕含量及外源酶制剂的六个实验组在18℃下50天的饲喂
实验，结果表明:无外源消化酶饲料配方下的大菱虾对蛋白质和碳水化合物的表
观消化率随饲料中豆粕含量的增加呈下降的趋势。20%豆粕替代组和全鱼粉组实
验鱼的生长状态明显好于40%豆粕替代组及全豆粕组。各实验组大菱虾消化器官
比重随豆粕添加量的增多而增大，以比肝重变化最为显著。不同饲料配方组大菱
醉蛋白酶、淀粉酶活力随豆粕含量的增多而升高，脂肪酶无明显变化。添加外源
酶制剂组相比于未添加组生长状态及消化生理有明显改善。
实验结果表明，饲料中以豆粕替代鱼粉量功%对大菱虾生长无负面影响，在
添加外源酶制剂的情况下豆粕替代量可增加到100k仍能保证大菱坪的正常生长。
Turbot Scophthalmus maximus L. is a culture species naturally living in Europe. It is newly imported to China by Yellow Sea Fisheries Research Institute in 1992. Turbot is adapted to living in low temperature, enduring low oxygen and can breed in high density. Now the turbot culture becomes a new industry in our sea economy. In order to promote its culture in our country, it is very necessary to do some work on its basic research. In this study, the main digestive enzymes in turbot were fully discussed. Main proteases were purified and their characters were studied. The vitro coefficient of feed material by turbot alimentaiy tract homogenates was studied. Finally, partial or total replacement offish meal by soybean meal in diet for turbot and the dietary enzyme were researched.
(1) The protein is digested mainly by stomach and foregut, and starch mainly by foregut and pylorus cecum, and fat mainly by gui. The optimum temperature and pH of protease in each digestive organ such as stomach, pylorus cecum, foregut, midgut, hindgut are 40癈 pH2.0, 40癈 pHS.O, 60癈 pH8.0,40癈 pH8.5, 60癈 pH8.0, And for the amylase are 40 pH6.5, 40 pHS.O 40 pH7.5, 50 pH7.0, 40 pH8.0, for the lipase are 40 pH7.5, 40 pH6.5,40 pH7.0,40 pH 7.5, 40 pH 7.5. Otherwise, the optimum pH of all digestive enzymes increases in low temperature. And the optimum of temperature decrease when the reaction time is longer.
(2)The protease activity of turbot is increased continually in all growth phases, and the maximum activity increase occurs between 30-day old and 90-day old. The lipase activity is also increased along with the gro \vth and the difference between each phase is not very great. The amylase activity s increased in the parr phase and decreased after 180-day. The dominant enzyme i> protease in all growth phases, and 180-day phase is the turning point during the growth of turbot.
(3)Within a water temperature range of 10 ~ 25 , the weight of digestive organs in percentage of body weight tends to decrease with the increase of temperature. And
the quantity of enzyme protein secreted by each digestive organ also tends to decrease, and the enzyme protein in intestine decreased farthest. The total enzyme activity is increased firstly and then decrease with the increase of the water temperature. Among the digestive enzymes, the activity of protease is affected most significantly.
(4)At salt concentration range of OM-0.5M, the activity of proteases in pylorus cecum and gut of turbot are activated when the salt concentration is increased. And the activity of stomach protease is activated in low salt concentration but inhibited in high salt concentration. The activity of lipase and amylase is also activated firstly and inhibited in high salt concentration. The activity of lipase is affected strongly and protease is least affected by salt. Most digestive enzymes of each digestive organ are activated in the salt concentration between 0.2M-0.3M.
(5)The stomach protease^ pylorus cecum protease and gut protease I are purified by using ammonium sulfate precipitation, DEAE-Sepharose F.F. , Sephadex G-100 column and Sephacryl S -200 column chromatography. The purified enzymes exhibit a single band on SDS-polyacrolymide gel electrophoresis (SDS-PAGE). Their molecular weights are determined to be about 39900Dal, 66009Dal and 86776Dal respectively. The stomach protease is stable at pH ranging from 1.0-9.0 and below 40 癈. The optimum temperature and pH of enzyme are 40 癈 and pH2.0 for stomach protease, 30-40癈 and pH9.0 for pylorus cecum protease , 50 癈 and pH8.5 for gut protease I . The stomach protease is activated by Mn2+, Cu2+, and inactivated by Fe3+, PMSF, TPCK, Pepstatin, PCMB. The pylorus cecum protease is activated by Mn2+, Mg2+ and inhibited by Cu2+, Zn2+, PCMB, TLCK. The gut protease I is activated by Mn2+ and inhibited by Cu2+, Zn2+, Ag+, Fe3+, PCMB and TLCK. The Km value of stomach protease and gut protease I are determined to be 2.55g/L and 2.92g/L respectively.
(6) The vitro dry matter digestive ability of each digestive organ is in the seq
引文
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