Design and Synthesis of Novel Artificial Enzymes by Using Recombinant DNA Technique

利用重组DNA技术设计与合成新型人工酶

• 批准号: 07405060
• 负责人: KIMURA Shunsaku
• 金额: $ 4.61万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07405060/
• 关键词: Mutants; Recombinant DNA Technique; Artifical Enzymes; Esterase; Subtilisin; Enzymatic Activity; Protein Structure; タンパク質工学; 遺伝子工学; リゾチーム; tRNA; ポリプロリン; 非天然アミノ酸

Hydrophobilized mutant enzymes of esterase from Pseudomonas sp.KWI-56 by connection of 10 or 20 tyrosine residues at the C terminus were synthesized by using the recombinant DNA technique. The mutant protein with a longer poly (tyrosine) chain became more hydrophobic than that with a shorter chain. The structure of the mutant proteins was analyzed by CD spectroscopy, which revealed the decrease of alpha-helicalconformation and the increase of beta-sheet conformation. The enzymatic activity of the mutant esterase was abolished due to the structure change induced by connection of a poly (tyrosine) chain, which should interact with hydrophobic region of the protein. On the other hand, a hydrophobilized mutant enzyme of subtilisin Carsberg by connection of poly (oxyethylene) was synthesized and the structure-activity relationship was investigated in organic solvents. The mutant enzyme showed 150-fold higher activity than the wild type in the ester-exchange reaction in benzen solution. The high activity of the mutant enzyme was also observed in dichloromethane solution, but the activity was diminished in water-miscible organic solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran. In these solvents, the molecular structure of the mutant protein was change significantly probably due to depletion of the essential water from the enzyme.

利用重组DNA技术合成了假单胞菌KWI-56酯酶C端连接10或20个酪氨酸残基的疏水突变体。具有较长聚酪氨酸链的突变蛋白比具有较短聚酪氨酸链的突变蛋白具有更强的疏水性。用圆二色谱分析了突变蛋白的结构，发现突变蛋白的α-螺旋构象减少，β-折叠构象增加。突变体酯酶的酶活性被取消，由于结构的变化引起的连接的聚（酪氨酸）链，这应该与蛋白质的疏水区域相互作用。另一方面，通过聚氧乙烯醚的连接，合成了枯草杆菌蛋白酶Carsberg的疏水化突变体，并在有机溶剂中对其构效关系进行了研究。突变酶在苯溶液中的酯交换反应中表现出比野生型高150倍的活性。在二氯甲烷溶液中也观察到突变酶的高活性，但在水溶性有机溶剂如二甲亚砜、乙腈和四氢呋喃中活性降低。在这些溶剂中，突变蛋白的分子结构发生了显著的变化，可能是由于酶中必需的水被耗尽。

项目成果

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Y.Ito：“通过在羧基末端与聚脯氨酸进行遗传组合来实现溶菌酶的Hydropuobilization” Chem.Lett.65-66 (1997)

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