【作者】 刘忠义；

【导师】 王璋；

【作者基本信息】 江南大学 ， 食品科学， 2007， 博士

【摘要】 酶、不饱和脂肪酸以及抗冷冻蛋白是水生生物体内适应变温环境的几种重要的功能物质。酶是鱼以及水生生物体内非常重要的功能物质,具有比较典型的生长环境温度适应性。来源于低温寒冷地区的鱼及其他水生生物体内的酶具有冷酶的某些性质,在低温下有比较高的催化活性,能有效催化生物化学反应;对热不稳定,在中等以上温度即迅速失活;有比较低的Arrhenius活化能。而在热带地区的鱼体内发现有耐热的消化蛋白酶。已经研究的大多数来自鱼虾等水生生物体内的消化蛋白酶表现出一定的不是很严格的冷酶的特性。本论文选择我国主要淡水鱼草鱼作为研究对象,研究其消化道蛋白酶的主要生物学特性。主要目的是探讨草鱼消化器官中蛋白酶的可开发利用前景并深入研究探讨草鱼消化生理和营养。研究了草鱼和青鱼主要消化酶的活性分布。在草鱼和青鱼各消化器官中均检出酸性蛋白酶、碱性蛋白酶、淀粉酶和三酰基甘油脂酶的活性。上述主要消化酶在2种鱼的消化器官中有各自不同的分布。这些酶在2种鱼的肝和胰腺中活性比肠道高得多。这些消化酶大多沿青鱼和草鱼的整个肠道分布。除青鱼肠道中的淀粉酶外,所有其他酶在2种鱼的肠中、前段的活性均高于肠道尾部的活性（酸性蛋白酶在青鱼尾肠段中的活性尤其低）,但是2种鱼尾肠段的酶活性也相当高,尤其是草鱼。青鱼肠道中消化酶活性的分布更复杂些,每种酶都有不同的分布规律。和青鱼相比,这些酶在草鱼肠道中的分布更均匀一些。碱性蛋白酶在草鱼肠道中前部的活性只略高于尾段。整个肠道表现出的高碱性蛋白酶活性可能是草鱼快速生长的原因之一。研究了草鱼消化道中粗酸性和碱性蛋白酶的提取及部分性质。粗酶的热稳定性研究表明粗草鱼酸性蛋白酶在50℃保温30 min后损失其80%的酶活力,而使粗草鱼碱性蛋白酶损失约80%的酶活力需要60℃保温30 min。粗草鱼碱性蛋白酶的热稳定性表现出某种生长环境适应性的趋势。采用SDS-底物-PAGE电泳方法并经适当改进,分析鉴定了草鱼肠道粗碱性蛋白酶的种类。草鱼肠道中只有胰蛋白酶和金属蛋白酶,没有胰凝乳蛋白酶,这种简单的酶谱可能更加有效,尤其是在很强的环境适应能力和独特的食性方面。通过凝胶过滤色谱和离子交换色谱纯化了2种草鱼肠道胰蛋白酶（胰蛋白酶同工酶GT-A和GT-B）。纯化的酶在SDS-聚丙烯酰胺凝胶电泳胶片上以单带迁移,其相对分子质量分别为26,400和30,750。高效液相表明2种酶是纯纯酶。GT-A和GT-B均能水解酪蛋白和BAEE（N-苯酰-1-精氨酸乙酯）,被SBTI（大豆胰蛋白酶抑制因子）、PMSF（甲苯磺酰氟）和TLCK（甲苯磺酰赖氨酸氯甲酮）等强烈抑制,不被EDTA（乙二胺四乙酸二钠）抑制,依据其分子质量、水解特性和抑制特性,判断这是2种胰蛋白酶。研究了2种纯化的胰蛋白酶的各种性质。GT-A的最适作用pH为8.0,GT-B为8.5。GT-A的最适作用温度为40℃,GT-B为45℃。GT-B的热稳定性略高于GT-A,在中性磷酸盐缓冲溶液中,2种酶都在加热温度达到65℃时迅速失去活性,其热稳定性高于极地或者长期生长在寒冷环境里的鱼胰蛋白酶而低于热带鱼胰蛋白酶,这是草鱼生长环境温度适应性的表现之一。草鱼胰蛋白酶有高的生理转化效率V_max/K_m,有低的Arrhenius活化能值（E_a）。氯化钙的存在基本上不影响酶的活性,但是提高酶的热稳定性。DSC（差示量热扫描分析）表明酶的热变性温度分别为66.3℃（GT-A）和67.3℃（GT-B）。钙离子的存在分别提高2种酶的变性温度6.7℃（GT-A）和7.1℃（GT-B）。草鱼胰蛋白酶的热稳定性介于北极鱼和热带鱼胰蛋白酶之间,这和中国大部分省内草鱼生长的广泛的水域环境温度相适应。研究了2种草鱼胰蛋白酶同工酶的氨基酸组成以及圆二色性。2种酶都只含有比较低的芳香族氨基酸、半胱氨酸以及蛋氨酸,丝氨酸和甘氨酸的含量很高,尤其酸性氨基酸含量极高,而碱性氨基酸含量相对比较低。酶溶液在280 nm处只有微弱的吸光度,而在210 nm附近有很高的吸光度。在变性剂存在下,GT-B的紫外吸收大幅度增加,依据“双状态模型”计算得到其变性自由能为39.825KJ/mol。CD测定表明2种酶蛋白分子均具有较少的规则二级构象单元（包括α-螺旋、β-折叠以及β-转角）和较多的无规卷曲构象。这些信息结合起来可以较好地解释2种酶的理化和动力学等性质。此外,2种酶的活性中心肯定含有丝氨酸和组氨酸残基。此外,结合底物特异性以及底物-PAGE分析了草鱼肠道的酸性蛋白酶的活性组分。草鱼肠道酸性蛋白酶中有1种组织蛋白酶B、L或者H,另外有1种酶能水解牛血红蛋白而不能水解BAA（N-苯酰-1-精氨酰苯胺）。部分纯化了从草鱼肠道中提取的粗酸性蛋白酶,其主要活性组分是一种能水解牛血红蛋白而不能水解BAA的酶。部分纯化的酶最适作用温度为37℃,最适作用pH约为2.3,酶不耐热,60℃加热30min即丧失几乎全部活力。酶被胃抑酶素A强烈抑制,被EDTA抑制,但是不被PMSF和SBTI抑制。更多还原

【Abstract】 Enzymes, unsaturated fat acids, and antifreeze protein （AFP） are important functional compositions for the aquatic organisms adapting to cold environment. Enzymes, which are typical environment temperature dependent, play an important role in fish and other aquatic organisms. The digestive enzymes from fish or other aquatic organisms residing in the cold temperature environment have the properties of cold-adapted enzyme: performing high catalysis activity at low temperature; thermal unstable, denaturing fast at medium temperature; low Arrhenius active energy. In contrast, the digestive enzymes from tropic fish are thermal stable. Most of the investigated proteinases from fish and shrimp exhibit some properties of cold-adapted enzyme, but their adaptations to low temperature are discrete comparing to those occurring "real" cold enzymes.This thesis investigated the biological properties of proteinases from the intestines of grass carp, which is one of major fresh water fishes in China. The objectives of this project were evaluating the utilization potentiality of proteinases from intestines of grass carp, and intensively studying the digest physiology and nutrition of grass carp.Activity distributions of the main digestive enzymes of grass carp and black carp were studied. In the all digestive organs of grass carp and black carp, the acidic and basic protease, amylase and lipase were detected and were diversely distributed in the digestive organs of the two species. These enzymes are far more active in fish liver and pancreas than in intestine, and most of them distribute along the whole intestine of the two species. All the enzymes except amylase are more active in the forepart and middle part than the end of the intestines of the fishes （especially for the acidic protease of the black carp）, but all the digestive enzymes are rather active in the distal part, especially for grass carp. The enzymes’ activity distribution in intestines of the black carp are more sophisticated, and each has its own distribution patterns, and the enzymes distribute more uniformly in intestine of grass carp than that of black carp. The alkaline proteases activity is slightly higher in the forepart and middle parts than in the end of the Grass carp intestines. High protease activity in the whole intestines of grass carp may be one of the reasons why they can grow fast.The crude acidic and alkaline proteinases from the digestive tracts of grass carp were extracted, and some their properties have been studied. The thermostability assay results of the enzymes showed that the acidic proteinase lost 80% of its activity after heating at 50℃for 30 min, and the alkaline proteinase lost 80% of its activity after heating at 60℃for 30 min. The thermostability of alkaline proteinase showed the trend of environmental temperature adaptability.SDS-substrate-PAGE method was slightly modified and used to characterize classes and types of the grass carp alkaline proteinase. There were trypsins and metalloprotease （or elastase） in the grass carp intestine but not chymotrypsin. The simple zymogram might be more efficient especially to strong adaptability to the environments in that it resided and to its special food habit.Two types of trypsin isozymes were purified by gel filtration chromatography and ion-exchange chromatography. Their relative molecular masses were determined to be 26,400 and 30,750, respectively, using SDS-PAGE electrophoresis. The HPLC results showed that the enzymes were pure. All two isozymes could hydrolyze casein and BAEE, and be strongly inhibited by SBTI, PMSF and TLCK, but not by EDTA. They could be defined as trypsin according to their relative molecular mass, hydrolyzing characteristic and inhibition properties.The properties of the both purified trypsin isozymes （GT-A and GT-B） were determined. The optimum pH and temperature for GT-A are 8.0 and 40℃, and the optimum pH and temperature for GT-B are 8.5 and 45℃. The thermostability of GT-B is slightly higher than that of GT-A. Both trypsins in neutral phosphate buffer solution quickly lose their activities as temperature exceeds 65℃. The higher thermostability of both trypsins than that of fish trypsins from freezing conditions is an indication of adaptability to living environments’ temperature for grass carp. Trypsins from Grass carp have high physiological efficiency of V_max/K_m and low Arrhenius activation energy （E_a）. In the presence of CaCl₂, the activities of enzyme might not be influenced, however, their thermostability can be improved. Differencial scan calorimetry （DSC） analysis results showed that their heat-induced denature temperatures were 66.3℃（GT-A） and 67.3℃（GT-B）, respectively. In the presence of Ca²⁺, the thermo denatured temperature is elevated by 6.7℃for GT-A and 7.1℃for GT-B. Thermal stabilities of both GT-A and GT-B were intermediate between Arctic and tropical fish species, and consistent with the wide range of water temperatures to which grass carp are exposed in most provinces of China.The amino acid composition and circular dichroism （CD） of two fish trypsin isozymes were determined. They contained low content of aromatic amino acids, cysteine and methionine, high content of serine and glycine. The content of acid amino acids was very high while that of alkaline amino acids was relatively low. Weak absorbance was read at wavelength of 280 nm for enzyme solution, but very high absorbance was read near 210 nm. And in the present of denaturant, the absorption increased significantly. According to "di-state model", the denaturation free energy （ΔG） was calculated to be 39.825 kJ/mol for GT-B. The results of CD indicated that both GT-A and GT-B contained small ratio ofα-helix,β-sheet, andβ-return but mostly random. The information of compositions and structures from both GT-A and GT-B can explain their physic-chemic and dynamical properties. Furthermore, it is verified that the active central of both GT-A and GT-B contains serine and histidine resides.The active components of acid proteases from Grass carp intestines were analyzed according to the substrate specificity of enzymes using substrate-PAGE electrophoresis. The acidic proteases from Grass carp intestines might be composed Cathepsin B, L or H and an enzyme which can hydrolyze bovine hemoglobin but can’t hydrolyze N-bezoyl-1-argininamide （BAA）. The crude acidic proteases extracted from Grass carp intestine were partially purified, the main active component of the partially purified acidic protease was an enzyme which can hydrolyze bovine hemoglobin while can’t hydrolyze BAA. The optimal temperature and pH of the partially purified protease were 37℃and pH 2.3, respectively. The protease had poor thermostability, almost completely losing its activity when it was heated at 60℃for 30 min. It was strongly inhibited by pepstatin A and inhibited by ethylenediaminetetraacetate （EDTA）, but it was not inhibited by PMST and SBTI.更多还原