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Engineering Candida tenuis Xylose reductase for improved utilization of NADH: antagonistic effects of multiple side chain replacements and performance of site-directed mutants under simulated in vivo conditions.工程改造嗜松假丝酵母木糖还原酶以提高NADH的利用率：多个侧链替换的拮抗作用及定点突变体在模拟体内条件下的性能
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2
The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.通过定点诱变和X射线晶体学探究纤细假丝酵母木糖还原酶（AKR2B5）的辅酶特异性。
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3
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6
Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis.通过定点诱变探究纤细假丝酵母木糖还原酶（AKR2B5）的底物结合位点。
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Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis.对来自浅白假丝酵母的木糖还原酶催化的NADH依赖性醛还原机制的瞬态和稳态动力学研究。
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8
Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant.纤细假丝酵母木糖还原酶（AKR 2B5）的酶机制研究：H113A突变体的X射线结构和催化反应概况
Biochemistry. 2004 May 4;43(17):4944-54. doi: 10.1021/bi035833r.
9
Ethanol production from xylose by a recombinant Candida utilis strain expressing protein-engineered xylose reductase and xylitol dehydrogenase.通过表达蛋白质工程化木糖还原酶和木糖醇脱氢酶的重组产朊假丝酵母菌株由木糖生产乙醇。
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10
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3
Metabolic engineering and classical selection of the methylotrophic thermotolerant yeast Hansenula polymorpha for improvement of high-temperature xylose alcoholic fermentation.通过代谢工程和经典筛选方法对嗜甲基耐热酵母多形汉逊酵母进行改造，以提高其高温木糖酒精发酵性能。
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Recent advances in rational approaches for enzyme engineering.酶工程合理方法的最新进展。
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6
Analysis and prediction of the physiological effects of altered coenzyme specificity in xylose reductase and xylitol dehydrogenase during xylose fermentation by Saccharomyces cerevisiae.分析和预测酿酒酵母木糖发酵过程中木糖还原酶和木糖醇脱氢酶辅酶特异性改变对生理效应的影响。
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Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization.酿酒酵母工程菌株对混合葡萄糖-木糖底物的发酵：木糖还原酶辅酶特异性的作用，以及葡萄糖对木糖利用的影响。
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本文引用的文献

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In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations.酵母细胞内代谢动力学的活体分析：I. 实验观测。
Biotechnol Bioeng. 1997 Jul 20;55(2):305-16. doi: 10.1002/(SICI)1097-0290(19970720)55:2<305::AID-BIT8>3.0.CO;2-M.
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Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.用于快速厌氧木糖发酵的表达木糖异构酶的酿酒酵母菌株的代谢工程改造
FEMS Yeast Res. 2005 Feb;5(4-5):399-409. doi: 10.1016/j.femsyr.2004.09.010.
3
Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc.通过引入结构锌实现木糖醇脱氢酶辅酶特异性的完全逆转及热稳定性的提高。
J Biol Chem. 2005 Mar 18;280(11):10340-9. doi: 10.1074/jbc.M409443200. Epub 2004 Dec 28.
4
The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.通过定点诱变和X射线晶体学探究纤细假丝酵母木糖还原酶（AKR2B5）的辅酶特异性。
Biochem J. 2005 Jan 1;385(Pt 1):75-83. doi: 10.1042/BJ20040363.
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Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae.用于酿酒酵母中戊糖分解代谢的磷酸酮醇酶途径的代谢工程
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Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle.酿酒酵母的最小代谢工程用于高效厌氧木糖发酵：原理验证
FEMS Yeast Res. 2004 Mar;4(6):655-64. doi: 10.1016/j.femsyr.2004.01.003.
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Metabolic engineering for improved fermentation of pentoses by yeasts.通过酵母进行戊糖发酵改良的代谢工程。
Appl Microbiol Biotechnol. 2004 Feb;63(5):495-509. doi: 10.1007/s00253-003-1450-0. Epub 2003 Nov 1.
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High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?一种真菌木糖异构酶的高水平功能表达：酿酒酵母高效发酵木糖产乙醇的关键？
FEMS Yeast Res. 2003 Oct;4(1):69-78. doi: 10.1016/S1567-1356(03)00141-7.
9
Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae.工程化氧化还原辅因子再生以改善酿酒酵母中的戊糖发酵
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10
Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production.木糖发酵酿酒酵母中铵同化的代谢工程改善了乙醇产量。
Appl Environ Microbiol. 2003 Aug;69(8):4732-6. doi: 10.1128/AEM.69.8.4732-4736.2003.

工程改造嗜松假丝酵母木糖还原酶以提高NADH的利用率：多个侧链替换的拮抗作用及定点突变体在模拟体内条件下的性能

Engineering Candida tenuis Xylose reductase for improved utilization of NADH: antagonistic effects of multiple side chain replacements and performance of site-directed mutants under simulated in vivo conditions.

作者信息

Petschacher Barbara, Nidetzky Bernd

机构信息

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.

出版信息

Appl Environ Microbiol. 2005 Oct;71(10):6390-3. doi: 10.1128/AEM.71.10.6390-6393.2005.

DOI:10.1128/AEM.71.10.6390-6393.2005
PMID:16204564
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1265968/
Abstract

Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2'-phosphate binding pocket were tested for NADPH versus NADH selectivity (R(sel)) in the presence of physiological reactant concentrations. The experimental R(sel) values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274-->Arg and Arg-280-->His substitutions, which individually improved wild-type R(sel) 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.

摘要

对光滑假丝酵母木糖还原酶的六个单点和多点变体进行了测试，这些变体经设计在辅酶2'-磷酸结合口袋中具有侧链置换，在生理反应物浓度存在的情况下检测其对NADPH与NADH的选择性（R(sel)）。实验得到的R(sel)值与描述混合交替辅酶利用的动力学机制的预测结果非常吻合。赖氨酸-274→精氨酸和精氨酸-280→组氨酸替换分别使野生型R(sel)提高了50倍和20倍，当它们组合在一个双突变体中时具有相反的结构效应。