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

A photodecarboxylase from Micractinium conductrix active on medium and short-chain fatty acids

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.

Hydrocarbons are essential base chemicals as energy carriers and starting materials for chemical manufacture.So-called fatty acid photodecarboxylases(FAPs)represent interesting catalysts for the conversion of natural fatty acids into hydrocarbons thereby giving access to alkanes from renewable feedstock.Today,however,only few FAPs are known.In the current study we report a new FAP from the marine organism Micractinium conductrix(Mc FAP).In contrast to currently known FAPs Mc FAP exhibits high catalytic activity towards short and medium fatty acids.Recombinant expression and basic biochemical characterisation of this new member of the FAP family is reported.