CN117467631A - 一种脂肪酸脱羧酶P450BSβ突变体及其在γ-内酯制备中的应用 - Google Patents
一种脂肪酸脱羧酶P450BSβ突变体及其在γ-内酯制备中的应用 Download PDFInfo
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- CN117467631A CN117467631A CN202311303268.8A CN202311303268A CN117467631A CN 117467631 A CN117467631 A CN 117467631A CN 202311303268 A CN202311303268 A CN 202311303268A CN 117467631 A CN117467631 A CN 117467631A
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- 125000001424 substituent group Chemical group 0.000 description 1
- 150000003460 sulfonic acids Chemical class 0.000 description 1
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- 230000001131 transforming effect Effects 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 239000008371 vanilla flavor Substances 0.000 description 1
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Abstract
本发明公开了一种脂肪酸脱羧酶P450BSβ突变体及其在γ‑内酯制备中的应用,其中脂肪酸脱羧酶P450BSβ突变体是由野生型P450BSβ的氨基酸序列在第78位、85位、173位和290位突变得到的,可以利过氧化氢为直接的电子供体,脂肪酸为底物直接一步氧化内酯化为内酯。催化的底物范围扩展为链长为C6~C18链长的脂肪酸,含官能团的脂肪酸,含芳香(杂)环的脂肪酸。本发明所述的催化剂效率更高、反应更简洁、底物范围更广,在制备γ‑内酯方面具有良好的应用前景和工业价值。
Description
技术领域
本发明涉及一种脂肪酸脱羧酶P450BSβ突变体及其在γ-内酯制备中的应用,属于蛋白质领域。
背景技术
γ-内酯是γ位碳原子上的羟基与羧酸脱水缩合形成具有五元环结构的内酯,是自然界内酯中最丰富的类别之一。γ-内酯存在于多种天然产物的结构中,例如γ-癸内酯、γ-丁内酯和α-氨基-γ-内酯等,并且γ-内酯表现出优良的生物活性。例如,γ-癸内酯具有桃子香味,γ-辛内酯具有椰子香味,γ-己内酯具有香草的香味,它们是一类广泛用于饮料、香水和医药制剂中的天然桃香精。γ-内酯的世界市场量已达到每年数百万吨,价值在300-6000美元/公斤,但由于其生产技术的发展而呈下降趋势。此外,一些γ-内酯衍生物还具有抗结核菌(如Micromolide)和昆虫信息素等生物活性。因此,γ-内酯的制备受到广泛的关注。
化学方法合成简单内酯如γ-内酯的常用方法有环化、还原和氧化。由无机酸、磺酸、对甲苯磺酸和吡啶对甲苯磺酸盐等酸催化的γ-羟基羧酸及其衍生物的环化是最简单高效的γ-内酯合成方法之一,但相应的γ-羟基羧酸仍然难以合成;还原则需要底物γ位含碳基等官能团再反应生成γ-羟基羧酸,而这类底物自然界难以获得,也需要多步的化学合成;将γ位含碳碳双键等基团的底物氧化生成γ-羟基羧酸也能合成γ-内酯,但特定位点含有双键等基团的底物与γ-羟基羧酸有类似的化学构建难度与经济价值。这些方法或者需要繁多的反应步骤、产率较低,或者需要钌、铱等贵金属合成的手性助剂,以及使用化工产品对环境不友好和严苛的反应条件。近年来,生物催化剂也被用于内酯的合成,使得使用绿色、更温和的试剂和合成条件成为可能。目前已有的生物合成方法基本上都是通过构建工程菌株,然后以油酸等不饱和脂肪酸为底物,通过发酵生物合成γ-内酯。这些方法合成γ-内酯仍然需要一定程度活化的底物,这些底物具有与内酯相当的经济价值和工业适用性,并且生成的γ-内酯碳链长度不同难以分离纯化、产物立体异构选择性不理想。
对羧酸的γ位直接氧化获得γ-羟基羧酸进而环化成γ-内酯是一种理想、直接且经济的制备方法。先前研究表明,用铂和铜或过氧化二硫酸盐作为催化剂对脂肪族羧酸进行氧化内酯化的化学反应性和位点选择性较差。通过外部导向基团引导位点选择性的化学合成的效率也较低。为了在没有外部引导基团的情况下实现反应,利用底物中不同C-H键的固有偏向性开发出的化学催化剂如:通过Fe-和Mn-(PDP)催化剂可以实现在某些羧酸中富电子和空间位阻大的二、三级C(sp3)-H键的γ-内酯化,而改变这些催化剂的空间位阻性质,可以实现贫电子但空间位阻性小的初级C(sp3)-H键的内酯化。最近研究还发现一种配体控制的钯催化剂可以有效地对二羧酸进行γ-内酯化[38]。在以上这些显著进步中,底物通常需要一定的刚性、取代基模式或补充功能才能达到良好的选择性或反应性,在具有多个相同亚甲基的柔性、线性底物和对映选择性的控制方面仍然存在问题。因此,当前尚未实现柔性脂肪酸的不对称催化γ-C-H内酯化。而在已知的酶中,仅有来源于甲基杆菌(Methylobacteriumpopuli)的P450MP在能够实现对C12:0-C18:0脂肪酸β位氧化的主反应的同时,产生微量的γ-羟基脂肪酸副产物。因此,尚无专一直接将脂肪酸γ位羟基化的酶的报道。
发明内容
本发明为了克服现有的技术缺陷,解决金属催化剂催化效率不高、反应活性不强、位点和对映选择性低、底物范围窄等问题,提供了一种脂肪酸脱羧酶P450BSβ突变体及其在γ-内酯制备中的应用。本发明提供的脂肪酸脱羧酶P450BSβ突变体能够利用过氧化氢将脂肪酸转化为γ-内酯,该生物催化剂具有反应速率高、底物范围广、体系简单、合成效率高、高区域和对映体选择性及成本低廉等优势。
本发明脂肪酸脱羧酶P450BSβ突变体,其氨基酸序列如SEQ ID NO:1所示。
编码所述脂肪酸脱羧酶P450BSβ突变体的核苷酸序列如SEQ ID NO:2所示。
本发明脂肪酸脱羧酶P450BSβ突变体,是将野生型脂肪酸脱羧酶P450BSβ第290位的甘氨酸突变成异亮氨酸,将第173位的苯丙氨酸突变成丝氨酸,将第85位的谷氨酰胺突变成苯丙氨酸,将第78位的亮氨酸突变成甘氨酸后获得,具体包括如下步骤:
步骤1:构建表达脂肪酸脱羧酶P450BSβ的质粒:将上述SEQ ID NO:2的核苷酸序列经酶切重组到表达载体上;
步骤2:将步骤1构建的表达脂肪酸脱羧酶P450BSβ的质粒转化到大肠杆菌中;
步骤3:脂肪酸脱羧酶P450BSβ的表达:将步骤2得到的菌种在LB液体培养基中过夜培养,然后加入TB液体培养基中扩培,于对数期后期加入血红素前体δ-氨基乙酰丙酸和IPTG诱导表达后,离心收菌;
步骤4:含有脂肪酸脱羧酶P450BSβ的细胞裂解液的制备:用缓冲液将步骤3得到的菌体重悬,通过超声破碎裂解细胞,离心,获得上清液;
步骤5:脂肪酸脱羧酶P450BSβ的纯化:将步骤4获得的上清液加入事先预处理的镍离子亲和柱,使带有His标签的目的蛋白与镍柱结合,然后于较低咪唑浓度下除去杂蛋白,最后用含有高浓度咪唑的缓冲液将目的蛋白洗脱;
步骤6:将洗脱下来的目的蛋白透析以除去咪唑,蛋白经浓缩后冻干制成冻干粉,测定目的蛋白浓度后,-80℃保存。
本发明脂肪酸脱羧酶P450BSβ突变体的应用,是以C6~C18链长的脂肪酸为底物,以过氧化氢为氧化剂,以脂肪酸脱羧酶P450BSβ突变体为催化剂,催化脂肪酸γ位碳原子上的羟基与羧酸脱水缩合形成γ-内酯。
上述催化反应中,反应在室温下进行,反应体系为水相缓冲液体系,以Triton X-100(0.625%),5%DMSO(v/v)作为助溶剂。
所述缓冲液成分为:0.1M KPi,0.3M KCl,pH 7.0。
所述脂肪酸选自C6~C18链长的天然饱和脂肪酸或非天然羧酸。
进一步的,所述非天然羧酸的结构中含有末端或内部烯烃或炔烃、溴、酯、胺、腈、对氧敏感的羟基基序、环己基中的一种或多种;所述脂肪酸为含芳环或芳杂环的脂肪酸,其结构中含有N-、O-、S-的杂芳环、噻唑、吡啶、喹啉、苯并噻唑、呋喃、噻吩、噁唑、苯并呋喃、苯并噻吩中的一种或多种官能团。
进一步的,所述γ-内酯为S型γ-内酯。
本发明脂肪酸脱羧酶P450BSβ突变体催化脂肪酸γ位碳原子上的羟基与羧酸脱水缩合形成γ-内酯的方法,包括如下步骤:
γ-内酯化的反应体系包括bufferA(0.1M KPi,0.3M KCl,pH 7.0),含脂肪酸脱羧P450BSβ突变体蛋白(2μM),底物(1mM),过氧化氢(5mM×3,间隔时间=5min),Triton X-100(0.625%),5%DMSO(v/v)。其中,Triton X-100(0.625%),5%DMSO(v/v)作为助溶剂。该反应在室温下以100mL的最终体积反应1h;反应结束后,用HCl终止反应,取出1mL反应混合物,萃取,用TMSCHN2处理,通过GC进行定量分析产物和未反应的底物,手性GC分析对映比例。剩余的反应溶液用乙酸乙酯萃取,用无水MgSO4干燥并过滤。将滤液真空浓缩,并通过硅胶柱色谱法纯化。所有的实验平行进行三次计算产率。
本发明脂肪酸脱羧酶P450BSβ突变体催化产生γ-内酯,该生物催化剂具有反应速率高、底物范围广、体系简单、合成效率高、高区域和对映体选择性及成本低廉等优势。
本发明脂肪酸脱羧酶P450BSβ突变体是由野生型P450BSβ的氨基酸序列在第78位、85位、173位和290位突变得到的。脂肪酸脱羧酶P450BSβ突变体反应活性高,直接利用廉价的过氧化氢将链长为C6~C18链长的天然饱和脂肪酸和含有末端或内部烯烃/炔烃、溴、酯、胺、腈、对氧敏感的羟基基序、环己基、N-,O-,S-的杂芳环、噻唑、吡啶、喹啉、苯并噻唑、呋喃、噻吩、噁唑、苯并呋喃、苯并噻吩的一种或几种官能团的非天然羧酸底物,合成相应的γ-内酯。以辛酸为模式底物,反应速率(TOF)高达364/min,转换数(TON)高达3278,因反应活性高、选择性高、成本低廉,脂肪酸脱羧酶P450BSβ突变体在合成γ-内酯方面具有良好的工业生产前景。
本发明的有益效果体现在:
1、本发明构建的生物催化剂利用廉价的过氧化氢作为直接电子供体,以链长为C6~C18链长的天然饱和脂肪酸和含有末端或内部烯烃/炔烃、溴、酯、胺、腈、对氧敏感的羟基基序、环己基、N-,O-,S-的杂芳环、噻唑、吡啶、喹啉、苯并噻唑、呋喃、噻吩、噁唑、苯并呋喃、苯并噻吩的一种或几种官能团的非天然羧酸为底物,合成相应的(S)-γ-内酯,其催化底物范围更广。
2、大部分P450羟基化酶进行羟基化反应都需要昂贵的生物辅酶NAD(P)H和额外制备的氧化还原伴侣介导电子传递,因而反应体系复杂且成本高昂。该新型酶脂肪酸脱羧酶P450BSβ突变体只需过氧化氢作为直接的氧化还原供体且不需伴侣蛋白和昂贵的辅助因子(如:NADPH),直接将脂肪酸催化合成γ-内酯,反应体系简单且经济。
3、反应过程条件温和,在室温下水相溶液中进行,合成效率高,区域选择性和对映选择性高。
具体实施方式
下面对本发明作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案,而不能以此来限制本发明的保护范围。除非特定说明,以下所有的化合物和试剂都是从Sigma-Aldrich,EMD Millipore,Tokyo Chemical Industry,Macklin Biochemical,Bidepharm Biochemical和New England Biolabs购买。
实施例1:脂肪酸脱羧酶P450BSβ基因表达载体的构建
将全基因合成的野生P450BSβ片段(由苏州金唯智生物有限公司合成),经限制性内切酶NcoI和XhoI(New EnglandBiolabs公司)依据说明书进行双酶切操作,用T4连接酶(New EnglandBiolabs公司)连接到被NcoI和XhoI双酶切过的表达载体pET28a上。将连接产物转化到大肠杆菌DH5α感受态细胞(天根生化科技有限公司)。从含有50μg/ml卡那霉素的固体LB培养基平板上挑取转化成功的单克隆菌落,在含有同浓度卡那霉素的LB液体培养基中,37℃,摇床转速为220rpm/min的条件下培养过夜。从培养过夜的菌液中用质粒小抽试剂盒(天根生化科技有限公司),依据说明书提取重组质粒,并将提取的质粒送测序鉴定(苏州金唯智生物有限公司)。
实施例2:脂肪酸脱羧酶P450BSβ突变体的制备
1、P450BSβ突变体文库的构建和表达:以测序成功的含有野生型P450BSβ基因的重组质粒为模板,对野生型P450BSβ的相关氨基酸残基位点进行重叠饱和突变。在每一轮中,将获得的饱和突变库转化到大肠杆菌Rosetta(DE3)中,在37℃下于96孔深孔板中培养,每孔为含50μg/mL卡那霉素和34μg/mL氯霉素的300μLTB培养基。在摇床转速250rpm下振荡过夜后,将15μL的过夜培养物转移到新深孔板上,每孔含有1.5mL TB(50μg/mL卡那霉素和34μg/mL氯霉素)。待OD600达到0.6时,在培养液中添加0.5mM的δ-氨基乙酰丙酸和1mM的IPTG诱导。在25℃,摇床转速220rpm下培养16h后,离心转速为5000rpm离心10min收获细胞。并用0.5ml的bufferA(0.1M KPi,0.3M KCl,pH 7.0)将菌体重悬,之后利用超声进行破碎。在5000rpm/min、4℃离心30分钟后,获得上清液。反应中加入裂解液、辛酸(1mM)、H2O2(2mM)、及作为助溶剂的5%DMSO(v/v)、0.625%Triton X-100(v/v),提高底物在反应溶液中的溶解度。
2、脂肪酸脱羧酶P450BSβ突变质粒的筛选:为了进行筛选,将细胞在bufferA(0.1MKPi,0.1M KCl,pH 7.0,每孔0.5mL)中重悬,并通过超声破坏。在5000rpm/min、4℃离心30分钟后,获得上清液,反应中加入裂解液、辛酸(1mM)、H2O2(2mM)、0.625%Triton X-100(v/v)、5%DMSO(v/v)作为助溶剂,提高底物在反应溶液中的溶解度。反应在室温下密封瓶中进行1小时。采用顶空气相色谱法直接定量1-庚烯。为了量化未反应的底物、2-和3-羟基产物以及γ-和δ-内酯,用75μLHCl(5N)淬灭反应,振荡1小时(用于γ-和δ-内酯化),并用乙酸乙酯提取。有机相在无水MgSO4上干燥。羟基羧酸和底物衍生为相应的甲酯的方法是将干燥的有机相与MeOH混合,然后添加TMSCHN2。在25℃下孵育1h后,用气相色谱法测定辛酸甲酯、2-羟基和3-羟基辛酸甲酯以及γ-和δ-辛内酯的含量。辛酸、1-庚烯、2-羟基辛酸和3-羟基辛酸、γ-辛内酯和δ-辛内酯的标准品按同样的方法进行处理,并作为参考。对C-γ选择性高于亲本的变体的催化性能进行三次重复实验,取三次实验的平均值。
实施例3:P450BSβ突变体蛋白的大量表达纯化
预培养后,将含有重组质粒的细胞转移到含有50μg/mL卡那霉素和34μg/mL氯霉素的TB培养基中。在37℃,摇床转速220rpm/min下进行扩培。当OD600达到0.6时,添加0.5mM的前体ALA(δ-氨基乙酰丙酸)和1mM IPTG诱导。在25℃,摇床转速为220rpm,振荡培养16h,离心转速5000rpm离心10min,收获细胞。用bufferA将菌体重悬,并用超声破碎裂解细胞。并于4℃,离心转速10000rpm,离心45min,获得上清细胞裂解液,将其装入镍离子亲和柱上。用含35mM咪唑的bufferA洗涤柱,去除杂蛋白,用含250mM咪唑的缓冲液将P450BSβ突变体蛋白从柱上系脱下来。对bufferA进行透析,以去除咪唑,将纯化后的蛋白浓缩,-80℃保存。蛋白质浓度由Omura和Sato开发的P450-CO光谱法测定。
实施例4:脂肪酸脱酸酶P450BSβ突变体产物鉴定分析
GC:在Agilent 7890A气相色谱系统上,采用FID检测器,色谱柱:DB-WAX(30m×0.25mm,0.25μm膜),N2为载气,对诱变文库进行气相色谱筛选。注射温度:250℃,不分流模式。探测器温度:300℃。程序:40℃保持1min,10℃保持1分钟至250℃保持15min,总共40min。
GC-MS:在以链长为C6~C18链长的天然饱和脂肪酸和含有末端或内部烯烃/炔烃、溴、酯、胺、腈、对氧敏感的羟基基序、环己基、N-,O-,S-的杂芳环、噻唑、吡啶、喹啉、苯并噻唑、呋喃、噻吩、噁唑、苯并呋喃、苯并噻吩的一种或几种官能团的非天然羧酸为底物的反应结束后,用5N的HCl终止反应并用等体积的乙酸乙酯进行萃取。取样,通过气相色谱-质谱联用仪对相应的γ-内酯定量。色谱柱:TG-5MS(30m×0.25mm,0.25μm膜)。注射温度:250℃,不分流模式。程序:60℃保持1分钟,20℃min-1到320℃,共14分钟。
GC:手性气相色谱,在Agilent 7890A气相色谱系统上,使用FID检测器,色谱柱:CP-Chirasil-Dex CB(25m×0.25mm,0.25μm膜),N2为载气,注射温度:250℃,分流方式,分流比100。探测器温度:275℃。程序:100℃保温1分钟,2℃保温1分钟至180℃保温4分钟,共45分钟。
HPLC:在高效液相色谱仪(Agilent 1260Infinity)上,色谱柱为CHIRALPAK IA(Daicel,4.6×250mm,5μm色谱柱),流动相为己烷和异丙醇(异丙醇:己烷:=1:99~15:85),流速为1.0mL/min,检测波长为210nm。按照公开的程序,将酶促反应制备的纯化γ-内酯及其外消旋标准物转化为相应的苯酰胺。
NMR:在Agilent DD2400 MHz光谱仪或BrukerAvance 600MHz仪器上记录1H-和13C-核磁共振谱。
HEMS:对AB 5800MALDI TOF/TOF进行了高分辨率质谱分析(HRMS),以m/z表示。
实施例5:脂肪酸脱羧酶P450BSβ突变体催化生成γ-内酯
γ-内酯化反应体系包含bufferA(0.1M KPi,0.3M KCl,pH 7.0),纯化的P450BSβ突变体(5或7.5μM),底物(5mM),Triton X-100(0.625%v/v)和5%DMSO(v/v)为助溶剂、H2O2(5mM×3,间隔时间=5min)。该反应在室温下以100mL的最终体积反应1h。用HCl(5N)淬灭反应后,pH为1.0~2.0,再用氢氧化铵中和反应溶液至pH=7.0,取出1mL反应混合物,萃取,用TMSCHN2处理,并通过GC进行定量分析产物和未反应的底物,手性GC分析对映比例。乙酸乙酯提取剩余的反应溶液,无水MgSO4干燥,过滤。滤液真空浓缩,硅胶柱层析纯化,得到γ-内酯。所有的实验平行进行三次计算产率,结果如下表所示:
P450BSβ-L78G/Q85F/F173S/G290I催化γ-内酯实例
本发明构建的生物催化剂利用廉价的过氧化氢作为直接电子供体,以链长为C6~C18链长的天然饱和脂肪酸和含有末端或内部烯烃/炔烃、溴、酯、胺、腈、对氧敏感的羟基基序、环己基、N-,O-,S-的杂芳环、噻唑、吡啶、喹啉、苯并噻唑、呋喃、噻吩、噁唑、苯并呋喃、苯并噻吩的一种或几种官能团的非天然羧酸为底物,一步合成相应的(S)-γ-内酯。该方法具有的优点有:1)该新型脂肪酸脱酸酶P450BSβ突变体催化底物范围更广,不仅可以催化天然直链饱和脂肪酸,而且能催化非天然羧酸。2)该新型脂肪酸脱酸酶P450BSβ突变体将羧酸催化合成γ-内酯,无需NAD(P)H氧化还原伴侣蛋白和辅助因子,反应体系简单且价格低廉。3)反应过程条件温和,在室温下水相溶液中进行,反应活性高、高度区域选择性和对映体选择性、反应条件温和。在合成γ-内酯方面有良好的工业生产前景。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变形,这些改进和变形也应视为本发明的保护范围。
Claims (8)
1.一种脂肪酸脱羧酶P450BSβ突变体,其特征在于其氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述脂肪酸脱羧酶P450BSβ突变体的核苷酸序列如SEQ ID NO:2所示。
3.权利要求1所述脂肪酸脱羧酶P450BSβ突变体的应用,其特征在于:
以C6~C18链长的脂肪酸为底物,以过氧化氢为氧化剂,以脂肪酸脱羧酶P450BSβ突变体为催化剂,催化脂肪酸γ位碳原子上的羟基与羧酸脱水缩合形成γ-内酯。
4.根据权利要求3所述的应用,其特征在于:
催化反应过程中,反应在室温下进行,反应体系为水相缓冲液体系,以Triton X-100和DMSO作为助溶剂。
5.根据权利要求4所述的应用,其特征在于:
所述缓冲液成分为:0.1M KPi,0.3M KCl,pH 7.0。
6.根据权利要求3所述的应用,其特征在于:
所述脂肪酸选自C6~C18链长的天然饱和脂肪酸或非天然羧酸。
7.根据权利要求6所述的应用,其特征在于:
所述非天然羧酸的结构中含有末端或内部烯烃或炔烃、溴、酯、胺、腈、对氧敏感的羟基基序、环己基中的一种或多种;所述脂肪酸为含芳环或芳杂环的脂肪酸,其结构中含有N-、O-、S-的杂芳环、噻唑、吡啶、喹啉、苯并噻唑、呋喃、噻吩、噁唑、苯并呋喃、苯并噻吩中的一种或多种官能团。
8.根据权利要求3所述的应用,其特征在于:
所述γ-内酯为S型γ-内酯。
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