源自Dictyoglomus thermophilum H-6-12的嗜热β-葡萄糖苷酶Dt0262的构建、表达与表征
发布时间：2019-02-22 13:17
【摘要】：糖苷类化合物(Glycoside compounds)是一类广泛分布于自然界中的功能性分子,其在细胞识别、生长调控、信号传递、结构支持等多个方面起着重要的作用。由糖苷类化合物衍生而来的糖缀合物(Glycoconjugates),由于其高表面活性、抗菌性、高水溶性、高生物相容性等独特的理化性质,近年来受到了广泛的研究与关注。糖苷水解酶(Glycoside hydrolases,EC 3.2.1)是一类特异性水解糖苷键的水解酶类,其既可水解糖苷类化合物中糖基之间的糖苷键,又可以水解糖缀合物中糖基与非糖基之间的糖苷键。根据人们对其水解机理的研究发现,绝大多数糖苷水解酶通过控制底物与水的比例可以行使转糖苷活力。较之糖基转移酶而言,使用糖苷水解酶从糖苷类化合物出发合成新型的糖缀合物,成本更为低廉,更具有产业化价值。这其中,源自嗜热微生物的嗜热糖苷酶,由于其具有很高的热稳定性、有机溶剂耐受性,并且在高温下具有较高的反应活性,符合工业生产上的需求,与耐受性差、易失活的常温糖苷酶相比,更具有产业化优势。特别是在合成糖缀合物的过程中,高反应温度能够大大提升反应速率,降低体系粘度,避免微生物污染,因此使用嗜热糖苷酶合成糖缀合物有着十分显著的优势。分离自日本热泉中的嗜热厌氧菌Dictyoglomus thermophilum H-6-12能够在70℃的条件下利用木质纤维正常生长。其基因组中包含大量的糖苷水解酶基因,其中,基因序列ACI18764.1被认为编码GH3家族的β-葡萄糖苷酶。本课题通过Blast分析,确认该基因为GH3家族的β-葡萄糖苷酶,并通过PCR成功获得了该基因的体外扩增。通过分子生物学方法,我们将该基因转入大肠杆菌表达载体p ET-28a中,成功构建了表达工程菌,实现了该β-葡萄糖苷酶的异源可溶表达。利用p ET-28a提供的组氨酸标签,我们使用Ni-NTA层析柱通过金属离子亲和层析,获得了较纯的的蛋白。对重组β-葡萄糖苷酶Dt0262酶学性质的表征表明,该酶对人工合成底物p NPGlc与天然底物纤维二糖均具有水解效果。对p NPGlc的最适水解条件为80℃,p H 6.0;对纤维二糖的最适水解条件为75℃,p H 5.5。动力学常数测定结果表明,重组β-葡萄糖苷酶Dt0262对p NPGlc的Km与kcat分别为0.23m M与0.64s-1,对纤维二糖的Km与kcat分别为3.57m M与0.88s-1,根据kcat/Km的大小可知,该酶更偏好于水解p NPGlc。对该酶的稳定性研究表明,重组β-葡萄糖苷酶Dt0262在70℃下相对稳定,t1/2可达到92h,而在80℃与90℃下,其t1/2分别缩短至20.3h与21.8min。此外,该酶还表现出明显的热激活效果,在70℃与80℃下孵育0.5h后,酶活分别变为原来的1.32倍与1.46倍。p H稳定性研究表明,该酶能够在较为宽泛的p H条件下保持稳定。金属离子依赖性研究显示,重组β-葡萄糖苷酶Dt0262为非金属离子依赖性酶。通过对该酶的表征我们发现,Dt0262确为嗜热β-葡萄糖苷酶,具有应用于糖缀合物合成方面的潜力。
[Abstract]:Glycosides (Glycoside compounds) are a class of functional molecules widely distributed in nature. They play an important role in cell recognition, growth regulation, signal transmission, structural support and so on. The glycoconjugate (Glycoconjugates), derived from glycoside compounds has attracted extensive attention in recent years because of its unique physicochemical properties such as high surface activity, antimicrobial activity, high water solubility and high biocompatibility. Glycoside hydrolase (Glycoside hydrolases,EC 3.2.1) is a kind of hydrolytic enzymes that can hydrolyze glycoside bonds between glycosyl and non-glycosylated glycosides in glycosides. According to the study of hydrolysis mechanism, most glycoside hydrolases can exercise transglycoside activity by controlling the ratio of substrate to water. Compared with glycosyltransferase, the use of glycoside hydrolase to synthesize new glycosylated conjugates from glycosides is cheaper and more valuable for industrialization. Among them, thermophilic glycosidase from thermophilic microbes, because of its high thermal stability, organic solvent tolerance, and high reaction activity at high temperature, meet the requirements of industrial production, and poor tolerance, The inactivated glucosidase at room temperature has the advantage of industrialization. Especially in the process of synthesis of glycoconjugates, high reaction temperature can greatly increase the reaction rate, reduce the viscosity of the system, avoid microbial contamination, so the use of thermophilic glycosidase to synthesize the conjugate has a very significant advantage. The thermophilic anaerobes Dictyoglomus thermophilum H-6-12 isolated from hot springs in Japan can grow normally using wood fibers at 70 鈩，
本文编号：2428267
[Abstract]:Glycosides (Glycoside compounds) are a class of functional molecules widely distributed in nature. They play an important role in cell recognition, growth regulation, signal transmission, structural support and so on. The glycoconjugate (Glycoconjugates), derived from glycoside compounds has attracted extensive attention in recent years because of its unique physicochemical properties such as high surface activity, antimicrobial activity, high water solubility and high biocompatibility. Glycoside hydrolase (Glycoside hydrolases,EC 3.2.1) is a kind of hydrolytic enzymes that can hydrolyze glycoside bonds between glycosyl and non-glycosylated glycosides in glycosides. According to the study of hydrolysis mechanism, most glycoside hydrolases can exercise transglycoside activity by controlling the ratio of substrate to water. Compared with glycosyltransferase, the use of glycoside hydrolase to synthesize new glycosylated conjugates from glycosides is cheaper and more valuable for industrialization. Among them, thermophilic glycosidase from thermophilic microbes, because of its high thermal stability, organic solvent tolerance, and high reaction activity at high temperature, meet the requirements of industrial production, and poor tolerance, The inactivated glucosidase at room temperature has the advantage of industrialization. Especially in the process of synthesis of glycoconjugates, high reaction temperature can greatly increase the reaction rate, reduce the viscosity of the system, avoid microbial contamination, so the use of thermophilic glycosidase to synthesize the conjugate has a very significant advantage. The thermophilic anaerobes Dictyoglomus thermophilum H-6-12 isolated from hot springs in Japan can grow normally using wood fibers at 70 鈩，
本文编号：2428267
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