海洋低温碱性蛋白酶的化学特性研究
摘要
本论文对碱性蛋白酶在国内外的研究与进展及目前海洋低温酶的研究状况进行了综述，阐明了海洋低温碱性蛋白酶的研究对国民经济的发展所起的重大作用，并指出了现有研究工作的不足之处和今后的发展方向。
论文首次对一种应用我国海洋嗜极微生物代谢产物并通过现代生物工程手段开发出的一类新型酶——海洋低温碱性蛋白酶(YS-80-122)的化学特性展开研究，通过对其分离纯化与制备工艺的探讨，理化性质和催化动力学的研究，及利用计算机模建方式对该酶的结构进行预测，获得了该碱性蛋白酶的各项性能参数和许多有关于结构与活性中心的有价值信息，为今后进一步开展该酶的研究与改造、生产与应用奠定理论基础。本文主要开展了以下几个方面的工作：
海洋低温碱性蛋白酶的分离纯化与制备工艺研究：①将该酶的发酵液分别经过高速冷冻离心机离心(6000转／分，4℃)去除菌体和其它有机粘性物质，错流超滤系统(截留分子量10，000Dal)去除小分子量杂质，并经冷冻干燥获得可供工业使用的粗酶；②将上一步经超滤获得的澄清发酵液分别经过盐析(30％-65％)，等电聚焦制备电泳(12W)，SephacryI S-200凝胶过滤，最后冷冻干燥，获得试剂酶，经反相C8 HPLC和SDS-PAGE聚丙烯酰胺凝胶电泳图谱，该酶纯度已达色谱纯及电泳纯。
海洋低温碱性蛋白酶的化学性质研究：①该酶经SDS-PAGE聚丙烯酰胺凝胶电泳和HPLC凝胶过滤测得其分子量为49300±1000Dal，经薄层聚丙烯酰胺凝胶等电聚焦测定酶的等电点为pH8.5，与已知碱性蛋白酶不同，由于其来自于海洋，属于一种新型碱性蛋白酶。②讨论了pH和温度对酶活性和稳定性的影响。通过采取两维实验数据结果和三维数据模拟相结合的手段，计算出酶活方程，r~2＞0.9，求得其反应最适pH为9.5，最适温度为30℃。在对该酶的pH和温度稳定性研究中发现，该酶在低温条件下(t≤30℃)具有较好的稳定性，保持相对低pH(pH≤8)有利于保持酶的稳定，且酶活性随pH和温度的升高而下降。⑧研究了常见金属离子，增稠剂、表面活性剂和氧化剂H_2O_2对酶活的影响。研究发现多数的金属离子对该酶不具有抑制作用，Pb~(2+)、Ag~+、Cu~(2+)对该酶有较强的抑制作用。该酶与常见
海洋低沮碱性蛋白醉的化学特性研究
的增稠剂、表面活性剂、氧化剂H八有良好的配伍性，其性质十分有利于今后该酶
在洗涤行业的进一步开发。但实验也发现，金属鳌合剂如EDTA能严重引起该酶失
活，这一性质与以往发现的碱性蛋白酶有很大的不同，这与其独特的活性中心有
关。
海洋低温碱性蛋白酶的催化动力学研究:①在以酪蛋白为底物的催化反应体
系中研究了PH和温度对酶催化反应动力学参数的影响。求得其反应活化能
Ea=33.ZkJ/mol;反应的温度系数Q:0=10，，‘33f“‘’，、习;pKa=9.3、p介10.7、最适p除10.0，
并根据所测的PK值可以推断出该低温碱性蛋白酶的活性部位可能会存在酪氨酸
酚轻基或疏基等结构，而不包含a一梭基、a一氨基、门冬氨酸p一梭基、以及
谷氨酸丫一竣基等常见氨基酸侧链基团。②EDTA对酶的抑制作用研究，通过动力
学参数凡的变化，经过计算得到EDTA种酶主要产生“竞争性”抑制，所以该酶应
该具有某些金属蛋白酶的性质，其活性中心应该包含M扩+，znz十，c尹，Fe2+，cu2+
等金属元素，EDTA的加入将金属原子从酶蛋白剥离而引起失活，在实验中发现，
加入乙萨+后，失活的酶活性又逐渐恢复，因此根据动力学数据推断，ZnZ+是该酶活
性中心所包含的金属元素。
海洋低温碱性蛋白酶的同源模建与结构预测:①用DNASTAR分析此酶得出其
分子量为49903.92Dal，等电点PI为8.52，与前面实验基本吻合。此酶含有459
个氨基酸。疏水性氨基酸148个，极性氨基酸153个。BLAST同源性分析其与碱性
蛋白酶的相似性很高，同源性可达76%，应该具有的碱性蛋白酶性质。②进行结构
模建获得结构比较合理的模建结果，显示了锌离子的三个组氨酸残基结合位点，
也是其催化位点。与前述实验推断其活性中心应有金属离子ZnZ+存在的结果吻合，
表明该模建具有较高的理论价值和实际意义。
Alkaline proteases constitute one of the most important groups of industrial enzymes, and have established roles in the detergent, food, pharmaceutical, leather and silk industries. And the low-temperature alkaline proteases producing by some kinds of marine bacterium are attracting more and more concerns all over the world for their special characters nowadays. We summarize the studies about the respects of the marine low-temperature alkaline protease all over the world and point out the developing direction for later research.
Marine Low-temperature Alkaline Protease(MLAP) is a kind of extracellular enzyme from a kind of marine bacterium of Flavobacterium YS-80-122.The study mainly concerned about the study of chemical characters of MLAP. During our experiments, we studied the purification conditions of enzyme, the characters of purified enzyme and enzyme kinetic parameters, and structural model by computer analysis. Through these methods, we got the data of the enzyme's characters and many useful structural information about the area of protein-ligand interactions. These results can provide theoretical foundation for further industrial use of this kind of enzyme.
The main results and conclusions are as follows:
Isolation and purification of MLAP: (1)A preliminary study on industrial abstraction technology is carried out by centrifuging the fermentation medium with high speed (6000r7m) at low temperature(4 ) and ultra filtrating (capturing molecular weight 10000Dal). After cooling dryness we get the raw samples. (2)Enzyme were precipitated by, ammonium sulphate (30%-65%). The precipitate was dialyzed against 20mM Na2HPO4-KH2PO4 (pH=6.5 ) buffer. The dialyzate was applied to IEF producing system(12W) and column Sephacry I S-200. At last we get the electrophoreticly pure sample after cooling dryness and the purity is checked by SDS- PAGE and C8 HPLC.
Chemical characterization of MLAP: (1)We get its molecular weight--49,300+
1000Dal by SDS PAGE gel and HPLC gel, and its pI-8.5 by IEF analysis system. For its native character is different from all the alkaline protease people knewed, we can classify it as a new kind of protease. (2)We have studied the effects of temperature and pH on enzyme activity and stability. With the experiments results, we have made a model of enzyme activity- temperature-pH (r2>0.9). And through this model we knew its pH optima is at 9.5, temperature optima is at 30 . The enzyme was stable at low temperature (t 30 ) and keeping relatively low pH C pH 8 ) is helpful for its stability. (3)In addition we studied the effects of various metal ions and some other ingredients which often occurr in detergents such as some kinds of surfactants and oxidants on enzyme activity. In our experiments we knew that the MLAP is compatible with most of the metal ions and surfactants. Few metal ions such as Pb2+, Ag+ , Cu2+act as strong inhibitors of enzyme activity. Also strong inhibition was observed with EDTA, and the finding was different from former alkaline protease.
Study of kinetics parameters of MLAP: (1)We studied the effects of pH and temperature on the kinetic parameters of the enzyme hydrolyzing casein reaction. We got the data of activation energy(Ea=33.2kJ/mol) and reaction temperature modulus( Q10=1017433[l/ (T1-T2) ]). Also we get the values of pKa=9.3 and pKb=10.7, and which offers many useful information concerning the groups involvedin enzyme funcion. (2)By studying the kinetics parameters changing by adding its inhibitor such as EDTA, we deduce that the inhibitor EDTA is a kind of competitive inhibitor to this enzyme. So we knew there must be some metal elements in the protein-ligand interactions area.
Homoibgy modeling and structural prediction of MLAP: (1)By analyzing the sequencing result with DNASTAR software, we get the result as follows: The molecule weight of the enzyme is 49903.92Dal; the pi is 8.52; there are 459 amide acids and 148 ones of which are hydrophobic and 153 ones are polar. It has a high homology to pseudomonas(nearly 76%), so it should has the similar
论文首次对一种应用我国海洋嗜极微生物代谢产物并通过现代生物工程手段开发出的一类新型酶——海洋低温碱性蛋白酶(YS-80-122)的化学特性展开研究，通过对其分离纯化与制备工艺的探讨，理化性质和催化动力学的研究，及利用计算机模建方式对该酶的结构进行预测，获得了该碱性蛋白酶的各项性能参数和许多有关于结构与活性中心的有价值信息，为今后进一步开展该酶的研究与改造、生产与应用奠定理论基础。本文主要开展了以下几个方面的工作：
海洋低温碱性蛋白酶的分离纯化与制备工艺研究：①将该酶的发酵液分别经过高速冷冻离心机离心(6000转／分，4℃)去除菌体和其它有机粘性物质，错流超滤系统(截留分子量10，000Dal)去除小分子量杂质，并经冷冻干燥获得可供工业使用的粗酶；②将上一步经超滤获得的澄清发酵液分别经过盐析(30％-65％)，等电聚焦制备电泳(12W)，SephacryI S-200凝胶过滤，最后冷冻干燥，获得试剂酶，经反相C8 HPLC和SDS-PAGE聚丙烯酰胺凝胶电泳图谱，该酶纯度已达色谱纯及电泳纯。
海洋低温碱性蛋白酶的化学性质研究：①该酶经SDS-PAGE聚丙烯酰胺凝胶电泳和HPLC凝胶过滤测得其分子量为49300±1000Dal，经薄层聚丙烯酰胺凝胶等电聚焦测定酶的等电点为pH8.5，与已知碱性蛋白酶不同，由于其来自于海洋，属于一种新型碱性蛋白酶。②讨论了pH和温度对酶活性和稳定性的影响。通过采取两维实验数据结果和三维数据模拟相结合的手段，计算出酶活方程，r~2＞0.9，求得其反应最适pH为9.5，最适温度为30℃。在对该酶的pH和温度稳定性研究中发现，该酶在低温条件下(t≤30℃)具有较好的稳定性，保持相对低pH(pH≤8)有利于保持酶的稳定，且酶活性随pH和温度的升高而下降。⑧研究了常见金属离子，增稠剂、表面活性剂和氧化剂H_2O_2对酶活的影响。研究发现多数的金属离子对该酶不具有抑制作用，Pb~(2+)、Ag~+、Cu~(2+)对该酶有较强的抑制作用。该酶与常见
海洋低沮碱性蛋白醉的化学特性研究
的增稠剂、表面活性剂、氧化剂H八有良好的配伍性，其性质十分有利于今后该酶
在洗涤行业的进一步开发。但实验也发现，金属鳌合剂如EDTA能严重引起该酶失
活，这一性质与以往发现的碱性蛋白酶有很大的不同，这与其独特的活性中心有
关。
海洋低温碱性蛋白酶的催化动力学研究:①在以酪蛋白为底物的催化反应体
系中研究了PH和温度对酶催化反应动力学参数的影响。求得其反应活化能
Ea=33.ZkJ/mol;反应的温度系数Q:0=10，，‘33f“‘’，、习;pKa=9.3、p介10.7、最适p除10.0，
并根据所测的PK值可以推断出该低温碱性蛋白酶的活性部位可能会存在酪氨酸
酚轻基或疏基等结构，而不包含a一梭基、a一氨基、门冬氨酸p一梭基、以及
谷氨酸丫一竣基等常见氨基酸侧链基团。②EDTA对酶的抑制作用研究，通过动力
学参数凡的变化，经过计算得到EDTA种酶主要产生“竞争性”抑制，所以该酶应
该具有某些金属蛋白酶的性质，其活性中心应该包含M扩+，znz十，c尹，Fe2+，cu2+
等金属元素，EDTA的加入将金属原子从酶蛋白剥离而引起失活，在实验中发现，
加入乙萨+后，失活的酶活性又逐渐恢复，因此根据动力学数据推断，ZnZ+是该酶活
性中心所包含的金属元素。
海洋低温碱性蛋白酶的同源模建与结构预测:①用DNASTAR分析此酶得出其
分子量为49903.92Dal，等电点PI为8.52，与前面实验基本吻合。此酶含有459
个氨基酸。疏水性氨基酸148个，极性氨基酸153个。BLAST同源性分析其与碱性
蛋白酶的相似性很高，同源性可达76%，应该具有的碱性蛋白酶性质。②进行结构
模建获得结构比较合理的模建结果，显示了锌离子的三个组氨酸残基结合位点，
也是其催化位点。与前述实验推断其活性中心应有金属离子ZnZ+存在的结果吻合，
表明该模建具有较高的理论价值和实际意义。
Alkaline proteases constitute one of the most important groups of industrial enzymes, and have established roles in the detergent, food, pharmaceutical, leather and silk industries. And the low-temperature alkaline proteases producing by some kinds of marine bacterium are attracting more and more concerns all over the world for their special characters nowadays. We summarize the studies about the respects of the marine low-temperature alkaline protease all over the world and point out the developing direction for later research.
Marine Low-temperature Alkaline Protease(MLAP) is a kind of extracellular enzyme from a kind of marine bacterium of Flavobacterium YS-80-122.The study mainly concerned about the study of chemical characters of MLAP. During our experiments, we studied the purification conditions of enzyme, the characters of purified enzyme and enzyme kinetic parameters, and structural model by computer analysis. Through these methods, we got the data of the enzyme's characters and many useful structural information about the area of protein-ligand interactions. These results can provide theoretical foundation for further industrial use of this kind of enzyme.
The main results and conclusions are as follows:
Isolation and purification of MLAP: (1)A preliminary study on industrial abstraction technology is carried out by centrifuging the fermentation medium with high speed (6000r7m) at low temperature(4 ) and ultra filtrating (capturing molecular weight 10000Dal). After cooling dryness we get the raw samples. (2)Enzyme were precipitated by, ammonium sulphate (30%-65%). The precipitate was dialyzed against 20mM Na2HPO4-KH2PO4 (pH=6.5 ) buffer. The dialyzate was applied to IEF producing system(12W) and column Sephacry I S-200. At last we get the electrophoreticly pure sample after cooling dryness and the purity is checked by SDS- PAGE and C8 HPLC.
Chemical characterization of MLAP: (1)We get its molecular weight--49,300+
1000Dal by SDS PAGE gel and HPLC gel, and its pI-8.5 by IEF analysis system. For its native character is different from all the alkaline protease people knewed, we can classify it as a new kind of protease. (2)We have studied the effects of temperature and pH on enzyme activity and stability. With the experiments results, we have made a model of enzyme activity- temperature-pH (r2>0.9). And through this model we knew its pH optima is at 9.5, temperature optima is at 30 . The enzyme was stable at low temperature (t 30 ) and keeping relatively low pH C pH 8 ) is helpful for its stability. (3)In addition we studied the effects of various metal ions and some other ingredients which often occurr in detergents such as some kinds of surfactants and oxidants on enzyme activity. In our experiments we knew that the MLAP is compatible with most of the metal ions and surfactants. Few metal ions such as Pb2+, Ag+ , Cu2+act as strong inhibitors of enzyme activity. Also strong inhibition was observed with EDTA, and the finding was different from former alkaline protease.
Study of kinetics parameters of MLAP: (1)We studied the effects of pH and temperature on the kinetic parameters of the enzyme hydrolyzing casein reaction. We got the data of activation energy(Ea=33.2kJ/mol) and reaction temperature modulus( Q10=1017433[l/ (T1-T2) ]). Also we get the values of pKa=9.3 and pKb=10.7, and which offers many useful information concerning the groups involvedin enzyme funcion. (2)By studying the kinetics parameters changing by adding its inhibitor such as EDTA, we deduce that the inhibitor EDTA is a kind of competitive inhibitor to this enzyme. So we knew there must be some metal elements in the protein-ligand interactions area.
Homoibgy modeling and structural prediction of MLAP: (1)By analyzing the sequencing result with DNASTAR software, we get the result as follows: The molecule weight of the enzyme is 49903.92Dal; the pi is 8.52; there are 459 amide acids and 148 ones of which are hydrophobic and 153 ones are polar. It has a high homology to pseudomonas(nearly 76%), so it should has the similar
引文
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