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绿藻来源光脱羧酶McFAP的挖掘及其中短链脂肪酸选择性脱羧机制研究
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作者 马云建 仲宣儒 +4 位作者 吴斌 蓝东明 张皓 frank hollmann 王永华 《Chinese Journal of Catalysis》 SCIE EI CAS CSCD 2023年第1期160-170,共11页
利用脂肪酸脱羧制备烃类生物燃料是开发可再生能源的有效途径.相比于传统化学法,生物酶法具备高效、能耗低及环境友好等优势,更具有工业应用前景.光脱羧酶(FAP)是一类专一性强,催化效率高,催化过程无需额外添加昂贵辅因子,仅需利用蓝光... 利用脂肪酸脱羧制备烃类生物燃料是开发可再生能源的有效途径.相比于传统化学法,生物酶法具备高效、能耗低及环境友好等优势,更具有工业应用前景.光脱羧酶(FAP)是一类专一性强,催化效率高,催化过程无需额外添加昂贵辅因子,仅需利用蓝光即可将脂肪酸转化为烷(烯)烃的光驱动酶,在烃类生物燃料的高效可持续生物合成领域具有应用潜力.目前已报道的有偏好催化C12-C20链长脂肪酸的光脱羧酶,但其底物选择性机制尚未被深入探究.本文挖掘了来源于绿藻Micractinium conductrix的光脱羧酶Mc FAP,并开展了异源表达及制备、酶学性质表征、催化特性及底物选择性机制等研究.利用全基因合成技术获得了来源于绿藻Micractiniumconductrix假定的光脱羧酶基因序列mcfap,同时构建了N端缺失突变体Mc FAP-S(缺失1-550位氨基酸).在大肠杆菌中实现了Mc FAP-S的异源表达.重组的Mc FAP-S对链长为6-18的饱和直链脂肪酸均有脱羧活性,偏好中链脂肪酸,最适底物为正辛酸(C8:0)(转化率>99%).相同条件下,Mc FAP全细胞催化软脂酸(C16:0)脱羧的转化率是Cv FAP(来源于Chlorella variabilis)的1.7倍.重组的Mc FAP-S催化正辛酸脱羧的最适反应温度为40℃,孵育6 h后残余酶活力为70.2%;最适p H值为8.0,孵育5 d后残余酶活力为65.4%;对甲醇,DMSO等有机溶剂及Ni^(2+),Ca^(2+)等金属离子具有良好的耐受性;4℃避光条件下储存10 d残余酶活力为76.7%.考察了不同波长光对Mc FAP-S酶活力的影响,结果表明,红光照射3 h后Mc FAP-S残余酶活力为97.2%;在可见光照射下Mc FAP-S与正辛酸共孵育3 h后残余酶活力>99%.Mc FAP-S在30°C,p H 9.0,加酶量为60μmolL-1的条件下催化正辛酸脱羧,反应30 min后转化率为95.3%.构建了Mc FAP-S三维结构模型,通过与Cv FAP的三维结构对比分析,推测底物通道口大小对Mc FAP-S底物选择性有重要影响,根据二者对不同链长底物结合位置的区别,设计了突变体S338V,S338L,S338A,L339I,T340A,T340S,Mc FAP-S338A/L339I/T340S及?344-347.结果表明,S338L仅保留催化软脂酸脱羧活力(是野生型的3.7%);L339I对正庚酸(C7:0)脱羧活力相较于野生型降低了15%,对月桂酸(C12:0)脱羧活力增加了28%;T340S对正己酸(C6:0)脱羧活力降低了67%;S338A/L339I/T340S对正己酸脱羧活力降低了78%.以上表明,S338,L339,T340可能是参与该酶底物选择性调控的关键位点.综上所述,相较已报道的Cv FAP催化特性,Mc FAP具有偏好中链脂肪酸和脱羧活性更高等优势,提高光脱羧酶催化中链脂肪酸脱羧生成C5-C12烷烃的效率,同时本研究初步阐明了Mc FAP的底物选择性机制,可为光脱羧酶的研发及应用提供借鉴,为阐明光脱羧酶的结构功能关系研究提供一定的基础. 展开更多
关键词 光脱羧酶 McFAP 异源表达 脂肪酸 烃类生物燃料
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Breakthrough in the challenging P450-catalyzed chemoenzymatic synthesis of C14-functionalized steroids
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作者 Manfred T.Reetz frank hollmann 《Science China Chemistry》 SCIE EI CAS CSCD 2023年第9期2435-2436,共2页
Intensive past research has demonstrated that cytochrome P450 monooxygenases are involved in the biosynthesis of numerous natural products and in human metabolism of various pharmaceuticals [1]. This inspired syntheti... Intensive past research has demonstrated that cytochrome P450 monooxygenases are involved in the biosynthesis of numerous natural products and in human metabolism of various pharmaceuticals [1]. This inspired synthetic organic and pharmaceutical chemists to test them as catalysts in the oxidative hydroxylation of steroids. Unfortunately, wildtype P450s generally proved to show low levels of regio-and diastereoselectivity as well as suboptimal activity. 展开更多
关键词 STEROIDS CATALYZED synthesis
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Mirror,mirror on the wall,which is the greenest of them all?A critical comparison of chemo-and biocatalytic oxyfunctionalisation reactions
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作者 Yinqi Wu Caroline E.Paul frank hollmann 《Green Carbon》 2023年第2期227-241,共15页
This review article critically compares two widely used types of catalysis,chemo-and biocatalysis,and provides insights on their greenness according to specified parameters.A comparative analysis of the environmental ... This review article critically compares two widely used types of catalysis,chemo-and biocatalysis,and provides insights on their greenness according to specified parameters.A comparative analysis of the environmental impact of chemo-and biocatalytic oxyfunctionalisation reactions based on published experimental data reveals that both methods produce comparable amounts of waste,with the majority stemming from the solvent used.However,it is emphasised that the synthesis of the catalysts themselves,including biocatalysts,should also be considered when assessing their environmental impact.The review underscores the complexity of assessing the environmental impact of catalytic oxyfunctionalisation reactions.The article also discusses the relationship between solvent properties and the energy demands for chemical transformations and downstream processing,underlining that the choice of solvent can significantly influence the environmental impact of a catalytic process.Additionally,the review highlights the importance of considering the recyclability of reagents and the secondary CO_(2)emissions caused by the energy requirements of the reaction when evaluating the environmental impact of a catalytic process.Each chemo-and biocatalysis produce a certain environmental impact,the greenness of either method is dependent on several factors,including the type of waste generated,the recyclability of reagents,and secondary CO_(2)emissions.This review therefore recommends using consistent metrics and a comprehensive life cycle assessment approach to evaluate this environmental impact,and highlights the importance of considering the synthesis of the catalysts themselves. 展开更多
关键词 Green chemistry Catalysis BIOCATALYSIS Chemocatalysis Environmental impact Oxidative reactions
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