CN114507652A - 一株伯克霍尔德氏菌酯合成酶Bur01的纯化和晶体制备方法 - Google Patents
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Abstract
本发明公开了一株伯克霍尔德氏菌酯合成酶Bur01的纯化和晶体制备方法,具体包括Bur01蛋白纯化、阴离子交换层析和晶体培养过程。本发明提供了一种快速纯化Bur01蛋白用于晶体培养,从而达到对Bur01蛋白的结构进行解析的目的,为其它微生物来源短链脂肪酸乙酯合成酶的催化机理或将其工程化奠定了基础。
Description
技术领域
本发明涉及蛋白质工程技术领域,特别是涉及一种制备伯克霍尔德氏菌高纯度脂肪酸酯合成酶Bur01及其蛋白质晶体的方法。
背景技术
酯合成酶被广泛应用于改善食品的口感、风味和品质。白酒酿造中,酯合成酶对风味乙酯的合成起着至关重要的作用。酯化酶即广义的羧基酯酶,是催化合成低级脂肪酸酯的酶类的总称。白酒生产中的酯化酶主要为脂肪酶、酯酶及醇酰基转移酶等。该类酶既能催化酯的合成,也能催化酯的水解。在发酵过程中,细菌酯化酶能够将酸与醇催化合成对应的酯,包括乙酸乙酯、戊酸乙酯、丁酸乙酯、己酸乙酯、辛酸乙酯、癸酸乙酯等。
目前,酯酶催化分子机制研究仍以脂肪酶为基础。脂肪酶能够催化油-水界面(界面激活)不溶性底物的酯键水解反应和有机相(微水相)体系甘油和脂肪酸的酯化反应。与脂肪酶催化特性不同,酿造微生物来源酯酶在水相体系下催化短链脂肪酸和乙醇合成相应的酯,且具有较高的底物选择性。结构上,不同酯酶的α/β水解酶折叠(α/β hydrolasefold)结构域构象十分保守,而“帽子”(Cap)结构域的氨基酸组成和整体结构都有较大差异。Cap结构域的主要功能为识别不同碳链长度的底物,控制底物/产物的进出。同时,其表面的特征性基序能够感应环境中的溶剂类型、盐浓度、pH等变化,通过局部构象转变影响酶的活性,但是这种结构与功能之间的关系尚未完全了解。
酶的提纯技术应用广泛,以酶的结构与功能为基础,从分子水平上认识酶的催化机制和反应特性,是认识酶、改造酶的首要前提。而研究酶,首先要得到高纯度并具有生物活性的酶分子。酶具有独特的物理、化学性质,而酶的纯化也是基于这些性质进行的。常用的蛋白质分离方法有盐析法、聚乙二醇沉淀法、有机浴剂分级沉淀法、等电点法、选择性沉淀法、各种柱层析法(吸附层析、离子交换层析、凝胶过滤)、各种电泳法及亲和层析等;酶的纯化过程要尽可能地简单有效,防止酶在纯化过程中活力下降或失活。虽然蛋白质的纯化技术已经非常成熟,但是对于一些结构柔性较大、疏水性较强的酶仍然很难纯化到结晶的程度。
因此,提供一种制备细菌小分子脂肪酸乙酯合成酶Bur01蛋白晶体的方法,从而达到对Bur01蛋白的结构进行解析的目的是本领域技术人员亟需解决的问题。
发明内容
本发明的目的在于提供了一种高效制备伯克霍尔德氏菌小分子脂肪酸酯合成酶Bur01蛋白晶体的方法。
本发明是通过以下技术方案来实现:
本发明公开了一种伯克霍尔德氏菌小分子脂肪酸酯合成酶Bur01通过阴离子交换介质的特殊洗脱流程高效获得纯蛋白的方法。
本发明利用阴离子交换层析纯化后的蛋白能够在含0.1M MES pH 5.6-6.0,0.2MMg(CH3COO)2,18%-22%(w/v)PEG2000的晶体生长条件下培养获得高分辨率晶体。
本发明提供的伯克霍尔德氏菌小分子脂肪酸酯合成酶Bur01的纯化和结晶方法为小分子脂肪酸酯合成酶的三维结构和催化机制解析奠定基础。
与现有技术相比,本发明具有以下有益的技术效果:
通过减少蛋白质纯化步骤,提高单步纯化效果,制备高纯度、高均一度酶蛋白,操作过程简便,重复性强。本发明提供的伯克霍尔德氏菌小分子脂肪酸乙酯合成酶Bur01纯化和结晶方法可快速获得高衍射分辨率单晶。
附图说明
图1为伯克霍尔德氏菌脂肪酸酯合成酶Bur01阴离子交换层析纯化结果
图2为伯克霍尔德氏菌脂肪酸酯合成酶Bur01蛋白SDS-PAGE检测
图3为伯克霍尔德氏菌脂肪酸酯合成酶Bur01蛋白晶体及X-ray晶体衍射
具体实施方式
下面结合具体的附图和实施例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。下述实施例中未详细述及的操作步骤或条件,均按照本领域常规技术、条件实现。
实施例1 Bur01蛋白表达
将质粒pET28a-Bur01转入宿主细胞中,挑取单克隆进行抗性鉴定和接种,培养3-6h后,加入IPTG诱导8-12h,后离心收集菌体,并用buffer I悬浮菌体;
实施例2 Bur01蛋白亲和层析纯化
将实施例1中得到的菌株悬浮液进行破碎,上清液经Ni2+亲和层析介质结合,之后分别用Buffer II、Buffer III和Buffer IV洗脱。将Buffer IV洗脱液收集超滤浓缩,用Buffer V将蛋白溶液电导强度稀释至约30ms/cm。将降盐后的蛋白溶液注入阴离子交换介质,经如下过程洗脱(表1),收集第一个洗脱峰蛋白。
表1 洗脱液配方
实施例3 蛋白质浓缩结晶
将实施例2中得到的蛋白进行超滤浓缩,得到浓度为5mg/mL的蛋白,并利用SDS-PAGE验证蛋白的纯度和浓度,之后于0.1M MES pH 5.6-6.0,0.2M Mg(CH3COO)2,18%-22%(w/v)PEG2000进行晶体培养,培养环境为18±2℃。
缓冲液成分说明:
Buffer I:20mM Tris-HCl pH 7.5,1M NaCl;
Buffer II:20mM Tris-HCl pH 7.5,150mM NaCl,20mM咪唑;
Buffer III:20mM Tris-HCl pH 7.5,150mM NaCl,50mM咪唑;
Buffer IV:20mM Tris-HCl pH 7.5,150mM NaCl,150mM咪唑;
Buffer V:20mM Tris-HCl pH 7.5。
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