Structure, Function, and Application of a Halophilic Enzyme, Thermolysin

嗜盐酶嗜热菌蛋白酶的结构、功能和应用

基本信息

批准号：
07660109
负责人：
INOUYE Kuniyo
金额：
$ 1.28万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1.Substrate specificity in the salt-activation of thermolysin : There is 10,000 times or more difference in the activity between good and poor substrates, although the degree of activation (DOA) by salt was not dependent on the substrates. The enzyme activity increases with increase in hydrophobicity of the amino acid at the cleavable bond. The difference in the hydrophobicity has no effects on the salt-activation.2.Effects of ions : The orders of the effectiveness of cations and anions were Na^+>K^+>Li^+ and Cl^->Br^-, respectively. No relationship was observed between the size of ions and DOA,and specific ion-enzyme interaction was suggested.3.The solubility and molecular weight of thermolysin : The solubility is 1 mg/ml in the ordinary buffers, and increased greatly by addition of salts. It is suggested that thermolysin is highly hydrophobic. The enzyme exists as a monomer.4.Electrostatic interaction : The pH-dependence of DOA was bell-shaped. DOA was 16 at pH 7.0, and 2 at pH 6 and 9. When temperature was raised from 5 to 35ﾟC,DOA decreased from 20 to 4. DOA decreased with the addition of alcohols. The activation is controlled by the electrostatic interaction between thermolysin and ions of salts.5.Chemical modification of tyrosines : Thermolysin contains 28 tyrosyl residues. Their ionization were changed by nitration of the tyrosyl residues and amination ofthe nitrated tyrosines. DOA was lowered by introducing negative charges onto the enzyme surface by the nitration, and was recovered to the level observed for native enzyme after amination of the nitrated tyrosines. The salt-activation is cancelled by the increase in negative charges on the enzyme surface.

1.热蛋白的盐激活中的底物特异性：良好和贫穷底物之间的活性差异10,000倍或更高，尽管盐的激活程度（DOA）并不依赖于底物。酶活性随着可裂解键处氨基酸的疏水性增加而增加。疏水性的差异对盐激活没有影响。2。离子的效果：阳离子和阴离子的有效性的顺序分别为Na^+> k^+> li^+和cl^ - > br^ - 。离子和DOA之间没有观察到关系，并提出了特定的离子 - 酶相互作用。3。热蛋白的溶解度和分子量：在普通缓冲液中的溶解度为1 mg/ml，并且通过添加盐而大大增加。建议热素蛋白高度疏水。酶作为单体存在。4。电压相互作用：DOA的pH依赖性是钟形的。 DOA在pH 7.0时为16，在pH 6和9处为2。当温度从5到35 c时，DOA从20降低到4。DOA随着醇的添加而降低。激活受热蛋白和盐离子之间的静电相互作用控制。5。酪氨酸的化学修饰：热溶素含有28个酪糖基保留。通过硝化酪氨酸的酪氨酸保留和胺化来改变它们的电离。通过将负电荷通过硝化引入酶表面，并通过硝化酪氨酸胺化后发现天然酶的水平来降低DOA。盐激活因酶表面负电荷的增加而取消。