Thermotoga naphthopila RUK-10嗜热糖苷酶在糖缀合物合成中的应用

发布时间：2018-03-03 02:02

本文选题：糖缀合物　切入点：糖苷酶　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：糖缀合物在许多生理过程中发挥着不可或缺的作用,在食品工业,环境保护、洗涤剂工业和医药卫生等领域均有广泛应用,是非常重要的化合物。目前糖缀合物的获取方法主要是通过化学法和生物法合成,生物法中包括微生物发酵法和酶法,而酶法又占主要部分,糖苷酶在酶法合成糖缀合物中发挥重要作用。在本文中,我们使用一系列从嗜热细菌Thermotoga naphthophila RUK-10中克隆,在E.coli BL21(DE3)中表达的嗜热糖苷酶:β-葡萄糖苷酶TN0602、β-半乳糖苷酶TN1577、β-半乳糖苷酶TN0949、及TN0602的突变体,探究糖苷酶在合成不同糖缀合物,如低聚半乳糖、烷基糖苷和L-抗坏血酸半乳糖苷中的作用。低聚半乳糖作为一种益生元,其不能被人体的消化酶所消化,但可以被肠道中的益生菌所利用。目前,酶法合成低聚半乳糖大多是通过β-半乳糖苷酶以乳糖为底物通过转糖苷合成的,产物多是不同聚合度低聚半乳糖的混合物,而TN0602能够以极高的催化选择性合成低聚半乳三糖,因此该反应对单一成分低聚半乳糖的功效研究有重要作用。TN0602在p H 6.5和75°C下能以23.28 g L-1 h-1的生产率催化半乳三糖的合成,与来自米曲霉的β-半乳糖苷酶在反应动力学、酶-底物热力学结合和分子对接模拟等方面的比较研究表明,β-葡萄糖苷酶TN0602具有深且狭窄的催化口袋,可阻止乳糖和低聚半乳三糖同时进入催化位点。为了验证这一结果和降低TN0602的水解活性,我们对TN0602进行了定点突变研究,测定了突变对反应动力学、酶活性和产物组成方面的影响。结果表明,位于乳糖结合亚位点(-1)附近的414位苯丙氨酸突变为丝氨酸时,突变体通过抑制酶的水解活性增加了低聚半乳三糖的产量。不同突变体与低聚半乳三糖的分子对接结果证明了TN0602催化口袋的形状能够影响到转糖苷产物(低聚半乳糖)的组成。L-抗坏血酸是一种人体不能合成的必需营养素。由于L-抗坏血酸易降解,目前已经开发了各种L-抗坏血酸的衍生物以改善其稳定性;然而,通常合成L-抗坏血酸衍生物往往需要多步反应且产量较低。在本文中,我们开发了一种以乳糖为底物,以碳酸钠为酸碱调节剂,以TN0602为催化剂转糖苷合成L-抗坏血酸半乳糖苷的新方法。反应最佳温度和p H分别为75°C和5.0,最佳酶浓度和底物摩尔比分别为20 mg m L-1和2:1(L-抗坏血酸对乳糖)。在最优条件下,反应体系中L-抗坏血酸半乳糖苷最终浓度可达到138.88 m M。L-抗坏血酸半乳糖苷保留了L-抗坏血酸的抗氧化能力,其在氧化环境(Cu2+)中比L-抗坏血酸更稳定,因此L-抗坏血酸半乳糖苷在工业上有很大的应用潜力。烷基糖苷是一类温和、无毒、对皮肤无刺激,且易生物降解的天然表面活性剂。烷基糖苷的酶法合成主要在两相反应体系中进行,因此底物乳糖的溶解度成为制约反应速率和产率的瓶颈。本研究通过计算和筛选,发现了一种可用于TN1577催化转糖苷合成烷基半乳糖苷的离子液体——Ammoeng 102,其含有C18酰基和低聚乙二醇的四氨阳离子,能够提高辛基半乳糖苷产量2.37倍,在反应7小时后辛基半乳糖苷终浓度可达18.2 g L-1。本文在动力学研究和COSMO-RS预测中阐明了TN1577的嗜热性质。Ammoeng 102能够提高产量的主要因素有:增加底物的溶解度,抑制酶的水解活力,以及与酶的极好的生物相容性(允许TN1577最适催化温度达到95°C)。同时本研究也验证了Ammoeng 102体系的一般适用性,成功合成了正丁醇至正十四烷醇作为糖基受体,乳糖作为糖基供体的一系列烷基糖苷。许多酶促反应时间过长,降低了设备的利用效率、限制了产能;有很多报道指出,微波辐射可应用于不同的催化反应,能够大大缩短反应时间,然而,微波辐射会使酶快速失活,大多数酶催化反应不能在微波辐射下进行。本研究筛选出了一个在微波辐射下稳定的酶——TN1577,可用于烷基半乳糖苷的合成,实验发现通过微波辐射可大幅度缩短反应时间、提高烷基半乳糖苷反应的生产率、并能降低反应所需的酶量。我们系统研究了乳糖与正丁醇摩尔比(1:200-1:40),温度(65-85°C)和微波输出功率(80 W-800 W)对转糖苷产量的影响。在最佳条件下(即乳糖和正丁醇摩尔比为1:40,反应温度为75°C),仅3.5小时的反应时间就达到17.07 mg m L-1的产率。与传统加热方法相比,微波辅助法可提高生产率11倍。在反应动力学方面,微波辐射能显著增加TN1577的kcat值,但对Km值几乎没有影响。因此,微波辅助的转糖苷合成方法能够以较少的时间和较低的成本快速地生产烷基糖苷。此项工作不仅为工业生产L-抗坏血酸糖苷、烷基糖苷和高纯度低聚半乳糖提供了新选择,而且表明我们开发的嗜热糖苷酶在工业中具有巨大潜能,可以为糖缀合物的合成提供新的合成途径。
[Abstract]:Glycoconjugates play an important role in many physiological processes, protect the environment in the food industry, and are widely used for detergent industry and medical and health fields, is a very important compound. At present the glycoconjugate acquisition method is mainly through chemical and biological synthesis, including microbial fermentation and biological enzyme method method and enzyme method, and the main part, glycosidase enzymes play an important role in the enzymatic synthesis of glycoconjugates. In this paper, we use a series of thermophilic bacteria Thermotoga naphthophila clone from hot RUK-10, in E.coli BL21 (DE3) heat glycosidase expression of eosinophils: beta glucosidase TN0602. Galactosidase TN1577, beta galactosidase TN0949 and TN0602 mutants, explore the glucosidase in synthesis of different glycoconjugates, such as oligo galactose, alkyl glucoside and L- ascorbic acid galactosidase in vitro. Gos Lactose as a prebiotic, it cannot be digested by the body's digestive enzymes, but can be used by probiotics in the intestinal tract. At present, the enzymatic synthesis of oligomeric galactose mostly through beta galactosidase with lactose as substrate through transglycosylation synthesis. The product is a mixture of different degree of polymerization of oligomate and, TN0602 can be in high selective catalytic synthesis of Galacto sugar three, so the research on the effect of oligomerization of single component galactose.TN0602 has an important role in the P H 6.5 and 75 DEG C to 23.28 g L-1 by catalytic synthesis productivity of three H-1 sugar, and from Aspergillus oryzae beta galactosidase the enzyme in the reaction kinetics, comparison of enzyme substrate binding thermodynamics and molecular docking, beta glucosidase TN0602 has the catalytic pocket deep and narrow, can prevent lactose and sugar into the product by three The catalytic site. In order to verify the results and reduce the hydrolytic activity of TN0602, we studied the point mutation of TN0602 mutation on the kinetics of the determination of enzyme activity and product composition effect. The results showed that in the lactose binding subsite (-1) 414 phenylalanine near to serine mutation, mutant increased three Galacto sugar production through inhibition of enzyme hydrolysis activity. Molecular docking of different mutants and Galacto sugar three results show that the TN0602 catalytic pocket shape can affect the transglycosylation product (GOS) consisting of.L- ascorbic acid is a essential nutrients the human body can not be synthesized. Because L- ascorbic acid degradation at has developed a variety of L- ascorbic acid derivatives to improve its stability; however, usually L- synthesis of ascorbic acid derivatives often require multi-step reaction and the yield is Low. In this paper, we developed a lactose as substrate, using sodium carbonate as pH regulator, a new method of synthesis of L- ascorbic acid catalyst transglactosylaction galactoside with TN0602. The best reaction temperature and P H were 75 and 5 DEG C, the optimum enzyme concentration and substrate molar ratio were 20 mg m L-1 2:1 (L- and ascorbic acid on lactose). Under optimal conditions, the reaction system of L- ascorbic acid galactosyl final concentration can reach 138.88 m M.L- ascorbic acid galactoside retains L- ascorbic acid antioxidant capacity in the oxidizing environment (Cu2+) than L- ascorbic acid is more stable, so the potential applications of galactoside L- ascorbic acid great in the industry. APG is a kind of mild, non-toxic, no stimulation to the skin, and the natural biodegradation of surfactants. The enzymatic synthesis of alkyl polyglycoside in two-phase reaction system. Therefore, the substrate The solubility of lactose has become a bottleneck restricting the reaction rate and yield. In this study, through the calculation and selection, found a can be used for ion - Ammoeng 102 liquid TN1577 catalyzed transglycosylation synthesis of alkyl galactoside, four ammonia containing cationic C18 low acyl and polyethylene glycol, can improve the yield of octyl galactoside 2.37 times, in response to 7 hours after the octyl galactoside concentration up to 18.2 g L-1. in the dynamic research and prediction of COSMO-RS TN1577 illustrates the thermophilic nature of.Ammoeng 102 can improve the yield of the main factors are: increasing the substrate solubility, hydrolytic activity of enzyme inhibition, and excellent biocompatibility and enzyme (allowing TN1577 optimal catalytic temperature at 95 degrees C). At the same time, this study also verifies the general applicability of Ammoeng 102 system, the successful synthesis of n-butyl alcohol to fourteen alkanol as glycosyl acceptor, lactose as A series of alkyl glucoside glycosyl donor. Many enzymatic reaction time, reduces the equipment utilization efficiency and limits the capacity; there are many reports pointed out that microwave radiation can be used in various catalytic reactions, can greatly shorten the reaction time, however, microwave radiation can cause the enzyme deactivation of enzyme catalyzed reactions, most can not in the microwave radiation. This study selected a stable under the microwave irradiation enzyme, TN1577, can be used for the synthesis of alkyl galactoside, experiments can greatly shorten the reaction time by microwave radiation, improve the productivity of alkyl galactoside reaction, and can reduce the amount of enzyme required for the reaction. We study the molar ratio of lactose and n-butanol (1:200-1:40), temperature (65-85 degrees C) and microwave output power (80 W-800 W) of transglycosylation yield. Under the optimal conditions (i.e. lactose and butanol molar ratio of 1:40 And the reaction temperature is 75 DEG C), only 3.5 hours of reaction time on the yield of L-1 reached 17.07 mg m. Compared with the traditional heating methods, microwave assisted method can improve the productivity of 11 times. In the reaction kinetics, microwave radiation can significantly increase the TN1577 value of kcat, but the Km value has little effect. Therefore, synthesis of transglycosidation microwave can with less time and lower cost to the production of alkyl glucoside. This work not only for the industrial production of L- ascorbic acid glucoside, alkyl glucoside and high-purity oligomate provides a new choice, and that I have developed thermophilic glycosidase has great potential in the industry. For glycoconjugate synthesis provides a new synthetic route.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O621.3

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