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首页> 外文期刊>Advanced synthesis & catalysis >Combinatorial Mutation Analysis of ω‐Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone
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Combinatorial Mutation Analysis of ω‐Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone

机译：ω-转氨酶的组合突变分析，以能够进行异丁烯烯酮不对称胺化的工程变体

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Abstract > ω‐Transaminase (ω‐TA) is an important enzyme for asymmetric synthesis of chiral amines. Rapid creation of a desirable ω‐TA variant, readily available for scalable process operation, is demanded and has attracted intense research efforts. In this study, we aimed to develop a quantitative mutational analysis (i.?e., R ‐analysis) that enables prediction of combinatorial mutation outcomes and thereby provides reliable guidance of enzyme engineering through combination of already characterized mutations. To this end, we determined three mutatable active‐site residues of ω‐TA from Ochrobactrum anthropi (i.?e., leucine 57, tryptophan 58 and valine 154) by examining activities of nine alanine‐scanning mutants for seven substrate pairs. The R ‐analysis of the mutatable residues is based on assessment of changes in relative activities for a series of structurally analogous substrates. Using three sets of substrates (five α‐keto acids, six arylalkylamines and three arylalkyl ketones), we found that combination of two point mutations display additive effects of each mutational outcome such as steric relaxation for bulky substrates or catalytic enhancement for amination of ketones. Consistent with the R ‐analysis‐based prediction, the ω‐TA variant harboring triple alanine mutations, i.?e. L57A, W58A and V154A, showed high activity improvements for bulky substrates, e.?g. a 3.2×10 4 ‐fold activity increase for 1‐phenylbutylamine. The triple mutant even enabled asymmetric amination of isobutyrophenone, carrying a branched‐chain alkyl substituent to be accepted in a small binding pocket that normally shows a steric limit up to an ethyl group, with 99% ee of a resulting ( S )‐amine. > <blockFixed type="graphic"> <mediaResourceGroup> <mediaResource alt="image" href="urn:x-wiley:16154150:media:adsc201900184:adsc201900184-toc-0001"/> </mediaResourceGroup> </blockFixed> </abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> <div class="translation abstracttxt"> <span class="zhankaihshouqi fivelineshidden" id="abstract"> <span>机译：</span><Abstract Type =“Main”XML：Lang =“en”> <标题类型=“main”>抽象</ title> > ω-转氨酶（ω-Ta）是对手性胺的不对称合成的重要酶。需要快速创建一个理想的ω-TA变体，随时可用于可扩展的过程操作，并吸引了激烈的研究工作。在这项研究中，我们旨在制定定量的突变分析（I.One。， R </ I> - 分析，即能够预测组合突变结果，从而通过已经表征突变的组合提供了酶工程的可靠指导。为此，我们确定了三个互相旋转的ω-Ta稳定的现场残留物 ochrobactrum anthropi </ i> （I.IE.IE.IONININE 57，色氨酸58和缬氨酸154）通过检查七个基板对的九个丙氨酸扫描突变体的活性。这 R </ I> - 互相分析的残留物是基于对一系列结构类似底物的相对活性的变化的评估。使用三套底物（五α-酮酸，六个芳基烷基胺和三个芳烷基酮），我们发现两点突变的组合显示每个突变结果的添加剂效应，例如批量底物的空间弛豫或酮胺化的催化增强。与之一致 R </ I> - 基于分析的预测，ω-Ta变体覆盆三重丙氨酸突变，I.?E。 L57A，W58A和V154A，对庞大的底物，E.?G显示出高活性改进。 3.2×10 4 </ sup> - 1-苯基丁胺的折叠活性增加。三重突变体甚至使得异丁酮的不对称胺化，携带支链烷基取代基以在通常显示乙基的空间限制的小粘合剂中待接受，具有＆ 99％ ee </ i> 由此产生（ s </ i> ）-胺。 </ p> > <blockfixed type =“图形”> <mediaresourcegroup> <MEDIARESOURCE ALT =“图像”HREF =“URN：X-Wiley：16154150：媒体：ADSC201900184：ADSC201900184-TOC-0001”/> </ mediaresourcegroup> </ blockfixed> </ p> </摘要> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> </div> <div class="record"> <h2 class="all_title" id="enpatent33" >著录项</h2> <ul> <li> <span class="lefttit">来源</span> <div style="width: 86%;vertical-align: text-top;display: inline-block;"> <a href='/journal-foreign-19407/'>《Advanced synthesis & catalysis》</a> <b style="margin: 0 2px;">|</b><span>2019年第11期</span><b style="margin: 0 2px;">|</b><span>共13页</span> </div> </li> <li> <div class="author"> <span class="lefttit">作者</span> <p id="fAuthorthree" class="threelineshidden zhankaihshouqi"> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Kim Hong‐Gon&option=202" target="_blank" rel="nofollow">Kim Hong‐Gon;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Han Sang‐Woo&option=202" target="_blank" rel="nofollow">Han Sang‐Woo;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Shin Jong‐Shik&option=202" target="_blank" rel="nofollow">Shin Jong‐Shik;</a> </p> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zkzz" style="display: none;">展开▼</span> </div> </li> <li> <div style="display: flex;"> <span class="lefttit">作者单位</span> <div style="position: relative;margin-left: 3px;max-width: 639px;"> <div class="threelineshidden zhankaihshouqi" id="fOrgthree"> <p>Department of BiotechnologyYonsei UniversityYonsei-Ro 50 Seodaemun-Gu Seoul 03722 South Korea;</p> <p>Department of BiotechnologyYonsei UniversityYonsei-Ro 50 Seodaemun-Gu Seoul 03722 South Korea;</p> <p>Department of BiotechnologyYonsei UniversityYonsei-Ro 50 Seodaemun-Gu Seoul 03722 South Korea;</p> </div> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zhdw" style="display: none;">展开▼</span> </div> </div> </li> <li > <span class="lefttit">收录信息</span> <span style="width: 86%;vertical-align: text-top;display: inline-block;"></span> </li> <li> <span class="lefttit">原文格式</span> <span>PDF</span> </li> <li> <span class="lefttit">正文语种</span> <span>eng</span> </li> <li> <span class="lefttit">中图分类</span> <span><a href="https://www.zhangqiaokeyan.com/clc/238.html" title="化学工业">化学工业;</a><a href="https://www.zhangqiaokeyan.com/clc/1186.html" title="物理化学（理论化学）、化学物理学">物理化学（理论化学）、化学物理学;</a><a href="https://www.zhangqiaokeyan.com/clc/7777.html" title="化学反应过程">化学反应过程;</a></span> </li> <li class="antistop"> <span class="lefttit">关键词</span> <p style="width: 86%;vertical-align: text-top;"> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=ω-transaminase&option=203" rel="nofollow">ω-transaminase;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=chiral amines&option=203" rel="nofollow">chiral amines;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=asymmetric synthesis&option=203" rel="nofollow">asymmetric synthesis;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=protein engineering&option=203" rel="nofollow">protein engineering;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=combinatorial mutation&option=203" rel="nofollow">combinatorial mutation;</a> </p> <div class="translation"> 机译：ω-转氨酶;手性胺;不对称合成;蛋白质工程;组合突变; </div> </li> </ul> </div> </div> <div class="literature cardcommon"> <div class="similarity "> <h3 class="all_title" id="enpatent66">相似文献</h3> <div class="similaritytab clearfix"> <ul> 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