| 嗜热共生杆菌内消旋-2,6-二氨基庚二酸脱氢酶中Y76对辅酶偏好性影响 |
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| 引用本文： | 赵雷明,刘卫东,陈曦,王敏,冯进辉,吴洽庆,朱敦明.嗜热共生杆菌内消旋-2,6-二氨基庚二酸脱氢酶中Y76对辅酶偏好性影响[J].生物工程学报,2015,31(7):1108-1118. |
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| 作者姓名： | 赵雷明 刘卫东 陈曦 王敏 冯进辉 吴洽庆 朱敦明 |
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| 作者单位： | 1 天津科技大学生物工程学院 工业发酵微生物教育部重点实验室，天津 300457；2 中国科学院天津工业生物技术研究所 工业酶国家工程实验室 天津市生物催化技术工程中心，天津 300308,2 中国科学院天津工业生物技术研究所 工业酶国家工程实验室 天津市生物催化技术工程中心，天津 300308,2 中国科学院天津工业生物技术研究所 工业酶国家工程实验室 天津市生物催化技术工程中心，天津 300308,1 天津科技大学生物工程学院 工业发酵微生物教育部重点实验室，天津 300457,2 中国科学院天津工业生物技术研究所 工业酶国家工程实验室 天津市生物催化技术工程中心，天津 300308,2 中国科学院天津工业生物技术研究所 工业酶国家工程实验室 天津市生物催化技术工程中心，天津 300308,2 中国科学院天津工业生物技术研究所 工业酶国家工程实验室 天津市生物催化技术工程中心，天津 300308 |
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| 基金项目： | 中国科学院重点部署项目 (KSZD-EW-Z-016-3)，国家自然科学基金 (Nos. 21072151, 21102100)，国家重点基础研究发展计划 (973计划) (No.2011CB710801) 资助。 |
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| 摘 要： | 辅酶NAD(H)相比NADP(H)有稳定性好、价格低廉及更广的辅酶循环方法等优势,因此在实际应用中常需将NADP(H)依赖型的脱氢酶改造成为NAD(H)依赖型的。来源于嗜热共生杆菌Symbiobacterium thermophilum的NADP(H)依赖型内消旋-2,6-二氨基庚二酸脱氢酶(meso-2,6-diaminopimelate dehydrogenase,St DAPDH)及其突变体酶是催化还原氨化合成D-氨基酸的优良催化剂,本研究试图改变其辅酶偏好性,增强其应用优势。对其晶体结构分析可知,氨基酸残基Y76距离腺嘌呤较近,R35及R36和辅酶上磷酸基团有直接相互作用。依氨基酸侧链基团性质对Y76进行了定点突变,发现不同突变子对两种辅酶的偏好性都发生了变化;对与磷酸基团直接作用的R35、R36进行的双突变R35S/R36V,导致酶对NADP+的催化活力降低;将R35S/R36V和部分Y76突变进行了组合,发现三突变组合以NAD+为辅酶时的活力均大于以NADP+为辅酶的活力,实现了辅酶偏好性转变。这些研究工作为进一步实现St DAPDH的辅酶偏好性完全转变提供依据。
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| 关 键 词： | 氨基酸脱氢酶，内消旋-2 6-二氨基庚二酸脱氢酶，辅酶偏好性，突变 |
| 收稿时间： | 2014/10/30 0:00:00 |
Effect of residue Y76 on co-enzyme specificity of meso-Diaminopimelate dehydrogenase from Symbiobacterium thermophilum |
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| Leiming Zhao,Weidong Liu,Xi Chen,Min Wang,Jinhui Feng,Qiaqing Wu and Dunming Zhu.Effect of residue Y76 on co-enzyme specificity of meso-Diaminopimelate dehydrogenase from Symbiobacterium thermophilum[J].Chinese Journal of Biotechnology,2015,31(7):1108-1118. |
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| Authors: | Leiming Zhao Weidong Liu Xi Chen Min Wang Jinhui Feng Qiaqing Wu and Dunming Zhu |
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| Institution: | 1 Key Laboratory of Industrial Microbiology Fermentation of Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China; 2 Tianjin Biocatalysis Technology Engineering Center, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,2 Tianjin Biocatalysis Technology Engineering Center, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,2 Tianjin Biocatalysis Technology Engineering Center, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,1 Key Laboratory of Industrial Microbiology Fermentation of Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China,2 Tianjin Biocatalysis Technology Engineering Center, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,2 Tianjin Biocatalysis Technology Engineering Center, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China and 2 Tianjin Biocatalysis Technology Engineering Center, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China |
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| Abstract: | In industrial application of NAD(P)H-dependent dehydrogenases, NAD(H) has the advantages over NADP(H) in higher stability, lower price and wider recycling system. Recently, a meso-2,6-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH) has been found to be a useful biocatalyst for the production of D-amino acids, but it requires NADP(H) as co-enzyme. To switch the co-enzyme specificity from NADP(H) to NAD(H), we studied the effect of Y76 on the co-enzyme specificity of StDAPDH, because the crystal structural analysis indicated that residue Y76 is near the adenine ring. The mutation of Y76 exerted significant effect on the co-enzyme specificity. Furthermore, the double mutant R35S/R36V significantly lowered the specific activity toward NADP+, and the combination of R35S/R36V with some of the Y76 mutants resulted in mutant enzymes favorable NAD+ over NADP+. This study should provide useful guidance for the further development of highly active NAD+-dependent StDAPDH by enzyme engineering. |
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| Keywords: | amino acid dehydrogenase meso-diaminopimelate dehydrogenase co-enzyme preference mutagenesis |
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