本文選題：糖綴合物　切入點：糖苷酶　出處：《吉林大學》2017年博士論文　論文類型：學位論文

【摘要】：糖綴合物在許多生理過程中發(fā)揮著不可或缺的作用,在食品工業(yè),環(huán)境保護、洗滌劑工業(yè)和醫(yī)藥衛(wèi)生等領(lǐng)域均有廣泛應用,是非常重要的化合物。目前糖綴合物的獲取方法主要是通過化學法和生物法合成,生物法中包括微生物發(fā)酵法和酶法,而酶法又占主要部分,糖苷酶在酶法合成糖綴合物中發(fā)揮重要作用。在本文中,我們使用一系列從嗜熱細菌Thermotoga naphthophila RUK-10中克隆,在E.coli BL21(DE3)中表達的嗜熱糖苷酶:β-葡萄糖苷酶TN0602、β-半乳糖苷酶TN1577、β-半乳糖苷酶TN0949、及TN0602的突變體,探究糖苷酶在合成不同糖綴合物,如低聚半乳糖、烷基糖苷和L-抗壞血酸半乳糖苷中的作用。低聚半乳糖作為一種益生元,其不能被人體的消化酶所消化,但可以被腸道中的益生菌所利用。目前,酶法合成低聚半乳糖大多是通過β-半乳糖苷酶以乳糖為底物通過轉(zhuǎn)糖苷合成的,產(chǎn)物多是不同聚合度低聚半乳糖的混合物,而TN0602能夠以極高的催化選擇性合成低聚半乳三糖,因此該反應對單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的生產(chǎn)率催化半乳三糖的合成,與來自米曲霉的β-半乳糖苷酶在反應動力學、酶-底物熱力學結(jié)合和分子對接模擬等方面的比較研究表明,β-葡萄糖苷酶TN0602具有深且狹窄的催化口袋,可阻止乳糖和低聚半乳三糖同時進入催化位點。為了驗證這一結(jié)果和降低TN0602的水解活性,我們對TN0602進行了定點突變研究,測定了突變對反應動力學、酶活性和產(chǎn)物組成方面的影響。結(jié)果表明,位于乳糖結(jié)合亞位點(-1)附近的414位苯丙氨酸突變?yōu)榻z氨酸時,突變體通過抑制酶的水解活性增加了低聚半乳三糖的產(chǎn)量。不同突變體與低聚半乳三糖的分子對接結(jié)果證明了TN0602催化口袋的形狀能夠影響到轉(zhuǎn)糖苷產(chǎn)物(低聚半乳糖)的組成。L-抗壞血酸是一種人體不能合成的必需營養(yǎng)素。由于L-抗壞血酸易降解,目前已經(jīng)開發(fā)了各種L-抗壞血酸的衍生物以改善其穩(wěn)定性;然而,通常合成L-抗壞血酸衍生物往往需要多步反應且產(chǎn)量較低。在本文中,我們開發(fā)了一種以乳糖為底物,以碳酸鈉為酸堿調(diào)節(jié)劑,以TN0602為催化劑轉(zhuǎn)糖苷合成L-抗壞血酸半乳糖苷的新方法。反應最佳溫度和p H分別為75°C和5.0,最佳酶濃度和底物摩爾比分別為20 mg m L-1和2:1(L-抗壞血酸對乳糖)。在最優(yōu)條件下,反應體系中L-抗壞血酸半乳糖苷最終濃度可達到138.88 m M。L-抗壞血酸半乳糖苷保留了L-抗壞血酸的抗氧化能力,其在氧化環(huán)境(Cu2+)中比L-抗壞血酸更穩(wěn)定,因此L-抗壞血酸半乳糖苷在工業(yè)上有很大的應用潛力。烷基糖苷是一類溫和、無毒、對皮膚無刺激,且易生物降解的天然表面活性劑。烷基糖苷的酶法合成主要在兩相反應體系中進行,因此底物乳糖的溶解度成為制約反應速率和產(chǎn)率的瓶頸。本研究通過計算和篩選,發(fā)現(xiàn)了一種可用于TN1577催化轉(zhuǎn)糖苷合成烷基半乳糖苷的離子液體——Ammoeng 102,其含有C18酰基和低聚乙二醇的四氨陽離子,能夠提高辛基半乳糖苷產(chǎn)量2.37倍,在反應7小時后辛基半乳糖苷終濃度可達18.2 g L-1。本文在動力學研究和COSMO-RS預測中闡明了TN1577的嗜熱性質(zhì)。Ammoeng 102能夠提高產(chǎn)量的主要因素有:增加底物的溶解度,抑制酶的水解活力,以及與酶的極好的生物相容性(允許TN1577最適催化溫度達到95°C)。同時本研究也驗證了Ammoeng 102體系的一般適用性,成功合成了正丁醇至正十四烷醇作為糖基受體,乳糖作為糖基供體的一系列烷基糖苷。許多酶促反應時間過長,降低了設(shè)備的利用效率、限制了產(chǎn)能;有很多報道指出,微波輻射可應用于不同的催化反應,能夠大大縮短反應時間,然而,微波輻射會使酶快速失活,大多數(shù)酶催化反應不能在微波輻射下進行。本研究篩選出了一個在微波輻射下穩(wěn)定的酶——TN1577,可用于烷基半乳糖苷的合成,實驗發(fā)現(xiàn)通過微波輻射可大幅度縮短反應時間、提高烷基半乳糖苷反應的生產(chǎn)率、并能降低反應所需的酶量。我們系統(tǒng)研究了乳糖與正丁醇摩爾比(1:200-1:40),溫度(65-85°C)和微波輸出功率(80 W-800 W)對轉(zhuǎn)糖苷產(chǎn)量的影響。在最佳條件下(即乳糖和正丁醇摩爾比為1:40,反應溫度為75°C),僅3.5小時的反應時間就達到17.07 mg m L-1的產(chǎn)率。與傳統(tǒng)加熱方法相比,微波輔助法可提高生產(chǎn)率11倍。在反應動力學方面,微波輻射能顯著增加TN1577的kcat值,但對Km值幾乎沒有影響。因此,微波輔助的轉(zhuǎn)糖苷合成方法能夠以較少的時間和較低的成本快速地生產(chǎn)烷基糖苷。此項工作不僅為工業(yè)生產(chǎn)L-抗壞血酸糖苷、烷基糖苷和高純度低聚半乳糖提供了新選擇,而且表明我們開發(fā)的嗜熱糖苷酶在工業(yè)中具有巨大潛能,可以為糖綴合物的合成提供新的合成途徑。
[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.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：O621.3

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