【作者】 李洪军；

【导师】 陈宗道；

【作者基本信息】 西南农业大学 ， 农产品加工及贮藏工程， 2003， 博士

【摘要】 弹性蛋白酶是一种以分解不溶性弹性蛋白为特征的广谱水解内肽酶，也属一种丝氨酸蛋白酶。弹性蛋白酶可影响人的脂质代谢、降低胆固醇、抑制动脉硬化发展；抑制实验性肝纤维化、脂肪肝及增强肝中脂肪分解；具有降血压耐缺氧和保护心肌作用；此外还可抑制糖尿病等。同时，弹性蛋白酶可作为产品的原料，用以制造抑制衰老的高级化妆品。在食品工业中，弹性蛋白酶具有良好的肉类嫩化作用，可研制出新的肉类嫩化酶制剂，通过处理难以食用的韧带、筋键等蛋白原料，开发出营养食品和功能食品，丰富人类食物资源，大力提高农副产品的附加值。研究表明，弹性蛋白酶在医药、食品、日用化工等方面具有较高的应用价值。因此，弹性蛋白酶是一种应用广泛的功能性蛋白酶，其市场前景十分广阔。目前，国内外大多利用胰脏来提取生产弹性蛋白酶，生产成本较高，产量十分有限，大大限制了该酶的应用范围。由于受胰脏资源的限制，产品价格十分昂贵，因此我国弹性蛋白酶大多只能用于医药上，在食品工业中的应用尚处于研究阶段。本研究从弹性蛋白酶产生菌种的选育入手，经过大量的菌种筛选，从动物副产物牛肚中筛选出了46种菌种，并从其中分离选育了1株弹性蛋白酶产生菌，系统地研究了菌种形态学特征和生理生化性能，同时，研究了菌株的产酶特性，优化了培养条件和发酵工艺，确定了变温发酵参数与条件。应用柱层析和凝胶电泳分离纯化了微生物弹性蛋白酶，研究了弹性蛋白酶的酶学性质与酶促反应动力学，确定了酶的催化动力学模型，并探讨了弹性蛋白酶对各种肉类的降解作用及嫩化效果，比较了弹性蛋白酶与现有蛋白酶的作用特点，并探讨了酶解动力学特点。 1．应用微生物菌种分离筛选与复合诱变技术，在25种动物副产品牛肚(牛瓣胃)中分离筛选得到了生长速度快，产弹性蛋白酶稳定，且酶活力较高的菌株。系统地研究了菌株的形态学特征、生长特性及其生理生化性质，研究结果表明，该菌为红色的革兰 中 文 摘 要氏阴性杆菌，在显微镜下观察，该菌体中等大小，菌体长为2．53urn，宽为0石4urn，两端钝园，无芽抱，单个排列无规律；在蛋白陈培养基上生长良好，菌落黄色，圆形，凸起；在弹性蛋白平板上生长形成明显的蛋白水解透明圈。在不同温度条件下，其生长情况显著不同，该菌在37OC－40C生长较好，培养时间相对较短，一般为18－24h。应用活菌暗视野观察，发现该菌有微弱的运动性，可见直线运动，但距离不远。弹性蛋白酶产生菌严格好氧，化能异养，过氧化氢酶和氧化酶均为阳性，在37oC培养能产生色素，色素在中性和碱性培养基上呈墨绿色，在酸性培养基上呈红色；不水解淀粉；能液化明胶；不产生脓青素。 经鉴定该菌株为假单胞杆菌（PseudomonasspSlfYI人通过利用紫外线（UV＊2537X 10””m，30Cm处，辐照 605X氯化理和 EMS复合诱变，获得了一株突变菌株（Pseudomonas Sp S…人其产酶水平较野生出发菌株提高了 35．49％。研究表明有机氮源有利于弹性蛋白酶的产生，其作用比无机氮源好干酪素、酵母浸膏、胰蛋自晚是较理想的氮源，其中，干酪素不仅能促进产酶提高，而且对发酵液的pH影响较小，因此，选择干酪素作为最佳氮源。同时，利用蔗糖、葡萄糖、甘油、可溶性淀粉、木糖等复合碳源对产酶具有明显的促进作用，其中，葡萄糖能够促进菌体生物量的积累，但量大对弹性蛋白酶的产生存在一定的阻遏作用。在发酵基础培养基中以各种不同无机盐试验，摇瓶培养，结果表明，磷酸氢二盐对产酶有促进作用，同时也利于菌体的生长，尤以磷酸氢一销的效果最好。而Znh、Ca》在发酵过程中对酶的产量没有促进作用。在250mL的三角烧瓶中，摇瓶发酵培养，通过调节回旋式摇床的转速，结果表明，增大溶氧量能促进菌体生长及酶的产生。 L采用h ＊5）正交试验优化假单胞杆菌（Pseudomonas Sp SWUM）生长培养基，经方差分析表明，影响弹性蛋白酶产生的显著因素由强到弱的排列顺序为葡萄糖。Na。HPO。、蔗糖、干酪素、pH，发酵培养基组成最佳配方为：葡萄糖0．5％，蔗糖3％，干酪素3％，NazHPO。0．4％，酵母膏1％，0刀1％MgSO广7H。O。应用优化的培养基进行发酵动力学研究，结果表明，选择起始 pH为 7，最适发酵温度为 37oC，250ml三角烧 一2一 弹性蛋白酶忧良菌种选育及酸功能特性研究瓶装液量为20ml，接种量3％、种龄为12 ’J’时的种子培养液，变温发酵24h，弹性蛋白酶的产量达到最高，其酶活性为32．3U／mL。在发酵过程中补加碳源、氮源、分段变温培养有利于弹性蛋白酶的产生。在发酵液中按 0刀 1％（W／叫的量添加氨基酸和维生素等生长回于进行产酶发酵试验，结果发现，氨基酸和维生素对Pseudomonas Sp SWUM菌种的产酶有重要作用，其中精氨酸、酪氨酸和维生素C作用明显。同时发现，诱导物能激活菌体的产酶机制，弹性蛋白能够诱导弹性蛋白酶的产生，当其诱导浓度在0％－0石％时呈现线性关系。菌体细胞?更多还原

【Abstract】 Elastase is a kind of broad-spectrum endopeptidase that can degrade specially undissolvable elastin, which also belongs to serine protease family. Elastase benefits the bodies of people in many aspects, such as effecting on lipid metabolism, reducing cholesterin, inhibiting the development of arteriosclerosis, promoting the lipid hydrolyzation in liver as to preventing experimental liver fibrosis and fat liver, reducing blood pressure, helping to resist the lack of oxygen, protecting cardiac muscle and preventing diabetes. Furthermore, as the resource, elastase can be made into high-class cosmetics that postpone the aging of people. In food industry, elastase can be made into the new type of enzyme preparation because of its excellent tenderizing effect. Nutrient and functional foods can be researched and produced when many originally inedible protein resources, such as ligaments and tendons, are processed by elastase, so food resources will be widened and the additional values of animal by-product will be improved. In many previous studies, it was indicated that elastase has broad applications in medical therapy, food industry, consumer goods industry, chemistry industry and so on. Thus, elastase is a kind of widely-used functional protease, and it has a wide market prospect. Nowadays elastase has high production costs and low yields, which is almost isolated from animal pancreas home and abroad, limit its application fields. Short supply of animal pancreas causes the high price of commercial elastase. So in China, elastase is only used in medicament, and its application in food industry still rests on the experimental stage. In this study, elastase-producing strain was screened, isolated and breed, and we got 1 strain from 46 possible strains which came from cattle stomach. Elastase morphologic character and biochemical characteristic was systematically studied. And investigated its capability of producing elastase, optimized its culture conditions and fermentation technology,and subsequently established the temperature-changed constant and dynamic model of fermentation. We purified the elastase with the aid of SephadexG-75 column chromatograph and SDS-PAGE, and studied its enzymology property as well as its kinetics of enzyme-catalyzed reactions. Later elastase hydrolyzation and tenderizing effect on meat was studie, contrasted the operating characteristic of elastase to that of other existing protease, finally set up the model of its kinetics of enzyme-catalyzed reactions.l.Obtainning elastase-producing strain that has high growth speed and steady capability of producing elastase with high enzyme activity from the animal by-product (cattle stomach) by screening and mutagenetic technique.The strain that had higher growth speed and stable capability producing elastase with high activity had was obtained from 25 kinds of animal by-products (cattle stomach) by microbiology screening and compound mutagenetic techniques. It was studied systematically on morphology feature, growth characteristics, physical and biochemical property. Under the microscope, research results sljow that the strain was a kind of Gram-negative bacillus and they had middle-sized cells (length: 2.53um, width: 0.64um) that were singly arranged in disorder without spores, but both ends of the cell were a little arc. It could grow well on the peptone medium, and form a protruding light yellow round bacterial colony, and there was apparently clear zone around the bacteria colony when cultured in elastin medium. The growth state varied with the cultural temperature, and the cultural time of the strain was the shortest at the temperature from 37 to 40 , usually 18-24h. By observing the bacteria under dark-field microscope, it was found that could move slightly along a straight way. The bacterium, capable of producing elastase, was a kind of chemoheterotrophic microbe, and its tests of oxidase and peroxidase both produced positive result. Cultured at 37 , the bacteria could produce pigment which was deep green on neutral and alkalin更多还原