Functional modification of amino-acid dehydrogenases by the combination of site-directed mutagenesis and evolutionary molecular engineering

定点突变与进化分子工程相结合对氨基酸脱氢酶进行功能修饰

• 批准号: 15580079
• 负责人: SAWA Yoshihiro
• 金额: $ 2.3万
• 依托单位: Shimane University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580079/
• 关键词: Amino acid dehydrogenase; Alanine dehydrogenase; Glutamate dehydrogenae; Aspartate dehydrogenase; L-aspartic acid; ランダムミューテーション; 耐熱化; 基質特異性改変

One of the major goals of our studies has been to understand the structural basis of amino acid substrate specificity in amino acid dehydrogenase, and to apply such knowledge to the engineering of novel substrate or cofactor specificities. The three dimensional structure of aspartate aminotransferase from Phormidium lapideum has been determined for design of aspartate binding site by X-ray crystallography. The alanine dehydrogenase gene and glutamate dehydrogenase gene were chosen as templates for the mutagenesis. The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in E.coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined as a hexameric structure (M_r 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate requiring NADH and NAD^+ as cofactors, respectively. The enzyme showed low thermostability with T_m=41℃ due to low structural integrity of hexamer. To improve thermal stability of this enzyme, we performed error-prone PCR introducing random mutagenesis on cloned GluDH. Two single mutant enzymes Q144R and E27F were isolated from mutant library whose T_m values were 61℃ and 49℃, respectively. Furthermore, Q144R has a remarkably high k_<cat> value (435 s^<-1>) for amination reaction at 37℃ which is 1.3 times higher than that of wild type. Among the residues predicted to be important involving substrate recognition, Lys80,Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild type enzyme is specific for 2-oxoglutarate with k_<cat> value 344 s^<-1>, whereas G82K and M101S are specific for oxaloacetate with k_<cat> values 3.45 and 5.68 s^<-1>, which are 265 and 473 folds higher than those for 2-oxoglutarate, respectively. We are in progress to enhance the k_<cat> values of those mutant enzymes by using molecular evolutional engineering.

一个一个一个一个一个一个一个一个somem的新型底物或辅因子特异性的工程。诱变。在E.Coli中，枯草芽孢杆菌（BS-Gludh）的ROCG基因脱氢酶（BS-Gludh）在大量中。 c在枯草芽孢杆菌中的37°C野生型中的c反应被静音为一系列野生型酶。比265倍和473倍的折叠473倍倍于2-氧化的折叠，我们正在进行使用分子进化工程，以增强这些突变酶的K_ <CAT>值。

项目成果

酵素の分子設計はどこまで可能か

酶分子的设计可以达到什么程度？

• 发表时间: 2003
• 期刊: 化学 58
• 作者: 内海俊彦;Qianghua Xu;澤 嘉弘
• 通讯作者: 澤 嘉弘

Cloning, Structural Analysis and Expression of the Gene Encoding Aspartate Aminotransferase from the Thermophilic cyanobacterium

嗜热蓝藻天冬氨酸转氨酶基因的克隆、结构分析及表达

• 发表时间: 2003
• 期刊: Journal of Bioscience and Bioengineering 95
• 作者: H.Kim;M.Nakaoka;M.Yagi;H.Ashida;K.Hamada;H.Shibata;Y.Sawa
• 通讯作者: Y.Sawa

アスパラギン酸脱水素酵素、アラニン脱水素酵素、L-アスパラギン酸製造方法および、D-リンゴ酸製造方法

天冬氨酸脱氢酶、丙氨酸脱氢酶、L-天冬氨酸生产方法和D-苹果酸生产方法

• 发表时间: 2005

Conversion of cofactor specificities of alanine dehydroge nases by site-directed mutagenesis

通过定点诱变转换丙氨酸脱氢酶的辅因子特异性

• 发表时间: 2004
• 期刊: Journal of Molecular Catalysis B : Enzymatic 30
• 作者: H.Ashida;A.Galkin;L.Kulakova;Y.Sawa.N.Nakajima;N.Esaki
• 通讯作者: N.Esaki

Conversion of cofactor specificities of alanine dehydrogenases by site-directed mutagenesis

通过定点诱变转换丙氨酸脱氢酶的辅因子特异性

• 发表时间: 2004
• 期刊: Journal of Molecular Catalysis B : Enzymatic 30
• 作者: H.Ashida;A.Galkin;L.Kulakova;Y.Sawa;N.Nakajima;N.Esaki
• 通讯作者: N.Esaki