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Preparation of Chimeric Enzymes and Their Properties

嵌合酶的制备及其性质

基本信息

批准号：
07558224
负责人：
KURAMITSU Seiki
金额：
$ 0.96万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

The "homologous recombination" method is an in vivo ligation method that is independent of restriction-endonuclease site. The limit of usefulness of this method was checked. First, a 40-50-mer double stranded oligonucleotide is synthesized. This oligonucleotide includes the sequence around the junction in the final expected product. This synthetic oligonucleotide is then inserted up or down stream from the original DNA fragment. The plasmid is cleaved by a restriction endonuclease within two homologous regions. When E.Coli JC8679 (recBC,sbcA) is transformed with this linearized plasmid, the recombination occurs between the two homologous regions in the cell. We have developed the "homologous ligation" method for overproducing chimeric enzymes and an extremely thermophilic enzyme of aminotransferases.Aminotransferases catalyze the reversible transamination reaction via the ping-pong bi-bi mechanism. Escherichia coli aspartate aminotransferase (AspAT) and aromatic amino acid aminotransfe … More rase (AroAT) have high specificity for both acidic and hydrophobic substrates. This interesting phenomenon was analyzed by site-directed mutagenesis, chimera studies, X-ray crystallography and steady-state and presteady-state kinetic studies.Some chimeric enzymes constructed by homologous recombination in E.coli cells lost their activity for either the acidic or hydrophobic substrate, but retained their activity for the other. These results suggest that aminotransferases have two substrate-binding sites for acidic and hydrophobic substrates and that construction of two substrate-binding pockets might be a general strategy employed by transferases.We investigated the activity of aminotransferases using a series of aliphatic substrates. Enzyme activity was found to increase with an increase in substrate hydrophobicity. For large hydrophobic substrates, the enzyme did not distinguish their shapes. These results indicated that the substrate-binding pocket had a uniform hydrophobic environment and that steric hindrance for the substrate was very weak. Less

“同源重组”方法是一种不依赖于限制性内切酶位点的体内连接方法。检验了这种方法的实用限度。首先，合成40-50聚双链寡核苷酸。该寡核苷酸包括最终预期产物中连接周围的序列。然后将合成的寡核苷酸插入原始DNA片段的上游或下游。质粒在两个同源区域内被一个限制性内切酶切割。用该线性化质粒转化大肠杆菌JC8679 （recBC,sbcA）时，细胞内的两个同源区域之间发生重组。我们开发了“同源连接”方法来过量生产嵌合酶和氨基转移酶的一种极端嗜热酶。转氨酶通过乒乓- bi-bi机制催化可逆转氨化反应。大肠杆菌天冬氨酸转氨酶（AspAT）和芳香氨基酸转氨酶（AroAT）对酸性和疏水性底物都有很高的特异性。这一有趣的现象通过定点诱变、嵌合体研究、x射线晶体学、稳态和准稳态动力学研究进行了分析。在大肠杆菌细胞中，通过同源重组构建的一些嵌合酶对酸性或疏水性底物失去活性，但对另一种底物保持活性。这些结果表明，氨基转移酶对酸性和疏水性底物具有两个底物结合位点，并且两个底物结合口袋的构建可能是转移酶采用的一般策略。我们利用一系列脂肪底物研究了转氨酶的活性。酶活性随着底物疏水性的增加而增加。对于大的疏水底物，酶不能区分它们的形状。这些结果表明，底物结合袋具有均匀的疏水环境，底物的位阻非常弱。少