Streptavidin–Enzyme Linked Aggregates for the One‐Step Assembly and Purification of Enzyme Cascades

摘要

Abstract > Herein, we report enzyme aggregates assembled around covalently cross‐linked streptavidin tetramers. The streptavidin oligomeric matrix (Sav Matrix ) is produced by using SpyTag/SpyCatch technology and binds tightly to fusion proteins bearing a streptavidin‐binding peptide (SBP). Fusing the SBPs to different enzymes leads to precipitation of the streptavidin–enzyme aggregates upon mixing the complementary components. This straightforward strategy can be applied to crude cell‐free extracts, allowing the one‐step assembly and purification of catalytically active aggregates. Enzyme cascade assemblies can be produced upon adding different SBP‐fused enzymes to the Sav Matrix . The reaction rate for lactate dehydrogenase (LDH) is improved tenfold (compared with the soluble enzyme) upon precipitation with the Sav Matrix from crude cell‐free extracts. Additionally, the kinetic parameters are improved. A cascade combining a transaminase with LDH for the synthesis of enantiopure amines from prochiral ketones displays nearly threefold rate enhancement for the synthesis of ( R )‐α‐methylbenzylamine compared with the free enzymes in solution. </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> > 在此，我们报告组装成共价交联的链霉抗生物素蛋白四聚体的酶聚集体。链霉抗生物素蛋白寡聚基矩阵（SAV 矩阵</ sup> 通过使用Spytag / Spycatch技术产生并将其紧密结合到携带链霉抗生物素蛋白结合肽（SBP）的融合蛋白。将Sbps融合到不同的酶导致链霉抗生物素蛋白酶聚集体在混合互补成分时沉淀。这种直接的策略可以应用于粗细胞的提取物，允许一步组装和催化活性聚集体的纯化。可以在向SAV添加不同的SBP融合酶时生产酶级联组件矩阵</ sup> 。乳酸脱氢酶（LDH）的反应速率在苏格兰沉淀后改善了十倍（与可溶性酶比较）矩阵</ sup> 来自粗细胞的提取物。另外，动力学参数得到改善。将具有LDH的转氨酶组合用于合成来自Prochiral酮的对映胺胺的级联酶显示出用于合成的几乎三倍的速率增强（ R </ I> ）-α-甲基苄基胺与溶液中的游离酶相比。 </ 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-16041/'>《ChemCatChem》</a> <b style="margin: 0 2px;">|</b><span>2018年第13期</span><b style="margin: 0 2px;">|</b><span>共7页</span> </div> </li> <li> <div class="author"> <span class="lefttit">作者</span> <p id="fAuthorthree" class="threelineshidden zhankaihshouqi"> </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"> </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/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></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=biocatalysis&option=203" rel="nofollow">biocatalysis;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=enzyme cascades&option=203" rel="nofollow">enzyme cascades;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=protein design&option=203" rel="nofollow">protein design;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=protein precipitation&option=203" rel="nofollow">protein precipitation;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=protein cross-link&option=203" rel="nofollow">protein cross-link;</a> </p> <div class="translation"> 机译：生物分析;酶级联;蛋白质设计;蛋白质沉淀;蛋白质交联; </div> </li> </ul> </div> </div> <div class="literature cardcommon" id="literaturereference" style="display:none"> <div class="similarity "> <h3 class="all_title" id="enpatent111">引文网络</h3> <div class="referencetab clearfix"> <ul id="referencedaohang"> <li dataid="referenceul">参考文献</li> <li dataid="citationul">引证文献</li> <li dataid="commonreferenceul">共引文献</li> <li dataid="commoncitationul">同被引文献</li> <li dataid="tworeferenceul">二级参考文献</li> <li dataid="twocitationul">二级引证文献</li> </ul> </div> <div class="reference_details" id="referenceList"> <ul id="referenceul"></ul> <ul id="citationul"></ul> <ul id="commonreferenceul"></ul> <ul id="commoncitationul"></ul> <ul id="tworeferenceul"></ul> <ul id="twocitationul"></ul> </div> </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> <li class="active" >外文文献</li> <li >中文文献</li> <li >专利</li> </ul> </div> <div class="similarity_details"> <ul > <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/journal-foreign-detail/0704022057395.html">Streptavidin–Enzyme Linked Aggregates for the One‐Step Assembly and Purification of Enzyme Cascades</a> <b>[J]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> </a> <a href="/journal-foreign-16041/" target="_blank" rel="nofollow" class="tuijian_authcolor">ChemCatChem .</a> <span>2018</span><span>,第13期</span> </span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：链霉抗生物素蛋白 - 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href="/academic-degree-domestic_mphd_thesis/020315999433.html">自组装氟化酶聚集体的表达纯化及催化活性研究</a> <b>[A] </b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=屠春浩&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 屠春浩</a> <span> . 2019</span> </span> </div> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/patent-detail/06120194598.html">纯化的毁丝霉属漆酶及编码该酶的核酸</a> <b>[P]</b> . <span> 中国专利： CN1192108C </span> <span> . 2005.03.09</span> </div> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/06120104346849.html">纯化的毁丝霉属漆酶及编码该酶的核酸</a> <b>[P]</b> . <span> 中国专利： CN1157008A </span> <span> . 1997-08-13</span> </div> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130417741847.html">Method for detecting the presence of a at least one target, at least two targets or a apluralidade targets, method for detection of a first facilitator assembly using a 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