CN115247192A - 酰基/芳酰基辅酶a的酶法制备技术 - Google Patents

酰基/芳酰基辅酶a的酶法制备技术 Download PDF

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CN115247192A
CN115247192A CN202110452209.1A CN202110452209A CN115247192A CN 115247192 A CN115247192 A CN 115247192A CN 202110452209 A CN202110452209 A CN 202110452209A CN 115247192 A CN115247192 A CN 115247192A
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朱平
张波泳
王豪
巩婷
陈天娇
陈晶晶
杨金玲
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Abstract

本发明属于生物技术领域，公开了一种酶法大量制备酰基/芳酰基辅酶A的方法，即利用一种酰基转移酶，把来自廉价酰基/芳酰基供体的酰基/芳酰基转移到酰基/芳酰基受体辅酶A上，从而大量合成酰基/芳酰基辅酶A，以及该方法在制备酰基/芳酰基辅酶A中的用途。

Description

酰基/芳酰基辅酶A的酶法制备技术
技术领域
本发明属于生物技术领域，具体涉及酰基/芳酰基辅酶A的酶法合成。
背景技术
酰基/芳酰基辅酶A广泛存在于生物体内，参与生物的新陈代谢。例如：乙酰辅酶A(Acetyl-CoA)是生物体内能源物质代谢的重要中间代谢产物，作为一个枢纽性的物质，它将糖、脂肪、蛋白质三大营养物质汇聚成一条共同的代谢通路——三羧酸循环和氧化磷酸化，并彻底氧化成二氧化碳和水，释放能量用以合成ATP。酰基辅酶A是辅酶A(CoASH)的酰化形式，其携带的酰基与辅酶A的半胱氨酸残基SH-基团以高能键硫酯键的形式相连。目前，由于化学合成的成本问题，酰基/芳酰基辅酶A尚未能形成大宗商品，因而其市场价格十分昂贵。有文献报道，MilliporeSigma的酰基/芳酰基辅酶A硫酯(Acyl/Aroyl CoA thioesters)的价格在200～300美元/5mg(Sullivan SA,Nawarathne IN和WalkerKD.Arch.Biochem.Biophys.2020,683:108276-86)。
10-去乙酰巴卡亭III-10-β-O-乙酰转移酶(10-Deacetylbaccatin III-10-β-O-acetyltransferase，DBAT)来自红豆杉植物，它可以将乙酰辅酶A上的乙酰基转移到紫杉醇生物合成的前体10-去乙酰巴卡亭III(10-DAB)上，生成紫杉醇生物合成的中间体巴卡亭III(Walker KD和Croteau R.Proc.Natl.Acad.Sci.USA.2000，97:583-587)。DBAT突变体不仅可以乙酰化10-DAB为巴卡亭III，还能以10-去乙酰紫杉醇(DT)为底物乙酰化合成紫杉醇(Li BJ，等.Nat.Commun.2017，8:15544)。
发明内容
本发明涉及利用DBAT或其突变体酶法制备酰基/芳酰基辅酶A的技术：首先，筛选到一批酰基/芳酰基供体，然后，通过DBAT或其突变体将优选的酰基/芳酰基供体上的酰基/芳酰基转移到辅酶A上，生成酰基/芳酰基辅酶A。
为解决本发明的技术问题，提供了下列技术方案：
本发明的技术方案是：以表达DBAT(或其突变体)的重组菌；或纯化的DBAT(或其突变体)为催化剂，以辅酶A作为酰基/芳酰基受体，筛选如下中的一种或多种酰基/芳酰基供体：乙酰化白藜芦醇、乙酰水杨酸、乙酰水杨酸甲酯、乙酰水杨酸乙酯、乙酰水杨酸酐、乙酰水杨酰水杨酸、4-乙酰氧基乙酰苯胺、贝诺酯、赖氨匹林、卡巴匹林钙、丙酸苯酯、对苯二酚二丙酸酯、1-萘基丁酸酯、2-硝基苯基丁酸酯、2-萘基丁酸酯、丁酸2-甲基-5氧代-1-环戊烯-1-基酯、丁酸卞酯、丁酸-1-苯乙酯，酶法合成酰基/芳酰基辅酶A。最后，优选乙酰水杨酸甲酯、乙酰化白藜芦醇为乙酰基供体；优选丙酸苯酯为丙酰基供体；优选2-硝基苯基丁酸酯为丁酰基供体。
为提高酰基/芳酰基辅酶A的酶法合成效率，对反应条件做了优化，其中一定浓度钾离子的添加有利于酰基/芳酰基辅酶A的生成。
有益技术效果：
本发明的的技术优势为：利用本发明所提供的技术方法，可实现规模化制备乙酰辅酶A等酰基/芳酰基辅酶A。
附图说明
图1.反应通式.
图2.乙酰基供体的筛选.1:乙酰化白藜芦醇；2:乙酰水杨酸甲酯；3:乙酰水杨酰水杨酸；4:乙酰水杨酸乙酯；5:4-乙酰氧基乙酰苯胺；6:贝诺酯；7:乙酰水杨酸；8:赖氨匹林；9:乙酰水杨酸酐；10:卡巴匹林钙.
图3.产物乙酰辅酶AHPLC-MS图谱.
图4.DBAT催化丙酸苯酯合成丙酰辅酶A.
图5.DBAT催化2-硝基苯基丁酸酯合成丁酰辅酶A.
具体实施方式
本发明通过下列实施例予以进一步说明，这些实施例是仅用于说明性的，而不是以任何方式限制本发明权利要求的范围。
反应通式如图1所示。
实施例1：乙酰基供体的筛选
在200μL反应体系中加入1mg/mL DBAT、1mM辅酶A、含有3mM乙酰基的乙酰化白藜芦醇、乙酰水杨酸、乙酰水杨酸甲酯、乙酰水杨酸乙酯、乙酰水杨酸酐、乙酰水杨酰水杨酸、4-乙酰氧基乙酰苯胺、贝诺酯、赖氨匹林和卡巴匹林钙化合物,pH7.0,37℃反应3h，其中当采用乙酰化白藜芦醇和乙酰水杨酸甲酯为乙酰基供体时，乙酰辅酶A产量分别达到1.0mg/mL和1.15mg/mL(图2)。
实施例2:以乙酰化白藜芦醇为酰基供体酶法合成乙酰辅酶A
在200μL反应体系中加入2mg/mL DBAT、7mM辅酶A、7mM乙酰化白藜芦醇,pH5～8,37℃反应3h，乙酰辅酶A产量可高达3.4mg/mL。
实施例3:以乙酰水杨酸甲酯为酰基供体酶法合成乙酰辅酶A
在200μL反应体系中加入0.1～2mg/mL DBAT、7mM辅酶A、21mM乙酰水杨酸甲酯、1～20mM KH2PO4、1～80mM K2HPO4·3H2O,pH6～8,37℃反应0.5～3h，乙酰辅酶A产量可高达6.0mg/mL，HPLC-MS测定结果见图3。
实施例4:以丙酸苯酯为酰基供体酶法合成丙酰辅酶A
在200μL反应体系中加入1mg/mL DBAT、1mM辅酶A、3mM丙酸苯酯、1.7mM KH2PO4、7.2mM K2HPO4·3H2O,pH7,37℃分别反应0h、3h，通过质谱检测到有丙酰辅酶A生成，HPLC-MS提取的离子流图及质谱图见图4。
实施例5:以2-硝基苯基丁酸酯为酰基供体酶法合成丁酰辅酶A
在200μL反应体系中加入1mg/mL DBAT、1mM辅酶A、3mM 2-硝基苯基丁酸酯、1.7mMKH2PO4、7.2mM K2HPO4·3H2O,pH7,37℃分别反应0h、3h，通过质谱检测到有丁酰辅酶A生成，HPLC-MS提取的离子流图及质谱图见图5。

Claims (11)

1.一种制备酰基/芳酰基辅酶A的方法，其特征在于，所述的方法包括：利用酰基转移酶将酰基/芳酰基供体的酰基/芳酰基转移到辅酶A酰基/芳酰基受体上，产生酰基/芳酰基辅酶A。
2.根据权利要求1所述的方法，其特征在于，所述的酰基转移酶的来源选自植物、真菌或细菌。
3.根据权利要求2所述的方法，其中，所述的酰基转移酶为植物来源的酰基转移酶。
4.根据权利要求3所述的方法，其中，所述的酰基转移酶为红豆杉来源的酰基转移酶。
5.根据权利要求4所述的方法，其中，所述的酰基转移酶为红豆杉来源的10-去乙酰巴卡亭III-10-β-O-乙酰转移酶(DBAT)或其突变体。
6.根据权利要求1～5中任一项所述的方法，其特征在于，所述的酰基/芳酰基供体选自如下中的一种或多种：乙酰化白藜芦醇、乙酰水杨酸、乙酰水杨酸甲酯、乙酰水杨酸乙酯、乙酰水杨酸酐、乙酰水杨酰水杨酸、4-乙酰氧基乙酰苯胺、贝诺酯、赖氨匹林、卡巴匹林钙、丙酸苯酯、对苯二酚二丙酸酯、1-萘基丁酸酯、2-硝基苯基丁酸酯、2-萘基丁酸酯、丁酸2-甲基-5氧代-1-环戊烯-1-基酯、丁酸卞酯、丁酸-1-苯乙酯。
7.根据权利要求6所述的方法，其特征在于，所述的酰基/芳酰基供体为乙酰化白藜芦醇、乙酰水杨酸甲酯、乙酰水杨酰水杨酸、乙酰水杨酸乙酯、4-乙酰氧基乙酰苯胺、贝诺酯、乙酰水杨酸、丙酸苯酯、对苯二酚二丙酸酯、2-硝基苯基丁酸酯、2-萘基丁酸酯、丁酸2-甲基-5氧代-1-环戊烯-1-基酯。
8.根据权利要求7所述的方法，其中，所述的乙酰基供体优选乙酰水杨酸甲酯、乙酰化白藜芦醇，丙酰基供体优选丙酸苯酯，丁酰基供体优选2-硝基苯基丁酸酯。
9.根据权利要求1～8中任一项所述的方法，其特征在于，相对于辅酶A酰基/芳酰基受体，所述酰基转移酶的量为0.5～3mg/mL。
10.根据权利要求1～9中任一项所述的方法，其特征在于，所述酰基/芳酰基供体与所述辅酶A酰基/芳酰基受体的摩尔之比为1:1～1:4。
11.根据权利要求1～10中任一项所述的方法，其特征在于，反应体系中K+离子的存在有利于酰基/芳酰基辅酶A的生成。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107418938A (zh) * 2016-05-24 2017-12-01 中国医学科学院药物研究所 10-去乙酰巴卡亭III 10β-O-乙酰转移酶突变体及其在催化合成紫杉醇及其类似物中的应用
WO2018107880A1 (zh) * 2016-12-16 2018-06-21 中国科学院天津工业生物技术研究所 利用乙醇醛合成乙酰辅酶a及其衍生产品的新途径

Patent Citations (2)

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CN107418938A (zh) * 2016-05-24 2017-12-01 中国医学科学院药物研究所 10-去乙酰巴卡亭III 10β-O-乙酰转移酶突变体及其在催化合成紫杉醇及其类似物中的应用
WO2018107880A1 (zh) * 2016-12-16 2018-06-21 中国科学院天津工业生物技术研究所 利用乙醇醛合成乙酰辅酶a及其衍生产品的新途径

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张育楠等: "10-去乙酰巴卡亭Ⅲ-10-β-O-乙酰转移酶迭代饱和突变与活性位点分析", 中国医药生物技术, vol. 13, no. 6, pages 481 - 487 *
程抒劼等: "南方红豆杉10-去乙酰巴卡亭Ⅲ-10-乙酰转移酶基因的克隆与生物信息学分析", 生物技术通报, no. 1, pages 107 - 112 *

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