【作者】 张健；

【导师】 高仁钧；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2021， 硕士

【摘要】 低聚半乳糖（Galactooligosaccharide,GOS）作为一种功能性益生元,可以以食品补充剂的形式掺入到各种产品中,以增强人体内多种与健康相关的生理活动。目前,低聚半乳糖的最常用的制备方法是利用β-半乳糖苷酶和β-葡萄糖苷酶的酶促合成方法。然而,由β-半乳糖苷酶催化合成的GOS,其内部具有多种糖苷键及聚合度,难以分离纯化。根据前期研究基础,我们发现来自Thermotoga naphthophila RUK10的β-葡萄糖苷酶可以特异性催化GOS3的合成。为了探索其合成机制,我们选择了位于活性口袋的一些位点进行突变,其中N222Y/F突变体的水解活力和转糖苷活力明显降低,表明该位点可能是酶催化的关键残基。同时,利用同源建模获得Tn0602的三维结构,并与底物进行分子对接,分析酶与底物之间的相互作用,确认了N222位于糖苷配基+1亚位,与活性位点E166和E351空间上靠近,再次说明可能是催化活性的关键残基。因此,选择N222位点进行饱和突变,成功构建并表达了饱和突变体。使用Ni²⁺-NTA亲和层析方法分离并纯化饱和突变体,并利用纯化酶对酶的水解活力和转糖苷活性进行表征。在野生型酶水解乳糖的最适p H条件下,所有突变体的活力均不同程度地降低。其中,与Asn具有相似侧链结构的相应氨基酸突变体（如Ser、Gln等）其活力与野生型相似;而突变为具有最小侧链的Gly、具有较大侧链的Leu和Ile、碱性氨基酸Arg和Lys,以及芳香族Phe和Tyr的对应突变体,其水解乳糖的活力降低最明显。然而,N222S突变体尽管在p H 5.0条件下,活性稍有降低,但处于p H 3.0条件中,水解乳糖活力可提高约20%。相较于野生型酶,突变体在水解乳糖的反应中,最适反应温度和最适p H存在少许偏移,并且其动力学常数表明突变体对底物的专一性降低。以上结果表明,对N222位点的突变都会导致酶水解乳糖活力的不同程度地降低,说明222位点的天冬酰胺残基是与该酶水解活性相关的关键性氨基酸残基之一,也是自然选择的最优结果。在GOS3的催化合成中,可提高合成产量的突变体包括N222W、N222D、N222Q,产率相较于野生型分别提高了23%、36%、6%。同时,突变体N222W可提高催化合成GOS4的产率,较野生酶型提高了44%。以上结果说明222位点的有些突变体可以增强酶的转糖基活力,尽管另一部分突变体未呈现增强转糖基的效果,但由于其乳糖水解活性的大幅度降低,也可以增强GOS3的时间效应积累。虽然GOS3的最高产量没有提升,但是却能够将其维持在相对较高的水平,如突变体N222G催化合成GOS3的趋势是持续增加的,在24 h时可达到野生型最高产物量的85%,在84 h时达到110%左右;突变体N222E催化合成GOS3的最大产量只比野生型低5%,8-12 h的产量可维持在较高水平,随着反应的进行,产率虽然有轻微降低,但是也可将产率维持在最大值的80%左右。以上结果说明,相较于野生型酶,部分突变体更加适合于工业生产低聚半乳糖。222位点的饱和突变不仅获得了可以提高GOS产量的突变体,同时也证明了通过合理化设计,降低酶的水解活力,同时提高转移酶/水合酶的比例是提高其应用性能的有效途径。在后续研究中,可以对具有优异性质的突变体进行叠加,以探究位点的协同作用对GOS催化合成的影响,并进一步提高突变体的应用性能。更多还原

【Abstract】 As a functional prebiotic,galactooligosaccharide（GOS）can be used as food supplements to enhance a variety of health-related physiological activities.Nowadays,the most common method to produce GOS is the enzymatic synthesis byβ-galactosidase andβ-glucosidase.However,GOS catalyzed byβ-galactosidase has complex glycosidic bonds and polymerization degree,which make it difficult to separate and purify.Based on previous research,we found thatβ-glucosidase from Thermotoga naphthophila RUK10 can specifically catalyze the synthesis of GOS3.In order to explore its mechanism,we selected some potential sites in the active pocket for mutagenesis based on related literatures.Among them,the hydrolysis and transglycosidation activity of the N222Y/F were significantly decreased,indicating that this site may be a key residue for catalysis.Meanwhile,the three-dimensional structure of Tn0602 was obtained by homology modeling,and docking was carried out with the substrate.After that,the interaction between the enzyme and the substrate was analyzed.It was confirmed that N222 was located at the aglycone+1 Subsite and was connected to the active site E166 and E351.Therefore,the N222 was selected for saturation mutation,and the saturation mutants were successfully constructed and expressed.Ni²⁺-NTA affinity chromatography was used to purify the mutants,and then the hydrolysis and transglycosidation activity were charaterized.Under the optimum p H conditions for lactose hydrolysis by wild-type,the activity of all mutants decreased to varying degrees.Among them,the corresponding mutations（such as Ser,Gln,etc.）with similar side chains to Asn have similar viability to the wild;.Besides,when it was mutated into the smallest Gly,larger Leu and Ile,alkaline Arg and Lys,as well as aromatic Phe and Tyr,the hydrolysis activity towards lactose decreased significantly.However,although the N222S mutant has a slight decrease in activity at p H 5.0,the activity can be increased by 20%at p H 3.0.Compared with the wild-type enzyme,the mutant has a slight deviation in the optimal temperature and p H in the reaction of hydrolyzing lactose,and their kinetic constants indicate lower specificities towards lactose.Taken together,the mutations at position N222 will lead to varying degrees of reduction in the hydrolysis activity towards lactose,which confirmed that Asn222 is one of key residues involved in hydrolysis process,and it is also the optimal result through natural selection.In the catalytic synthesis of GOS3,the benificial mutants include N222W,N222D,and N222Q,whose yields are increased by 23%,36%,and 6%compared with the wild.In addition,N222W can increase the yield of catalytic synthesis of GOS4,which is 44%higher than that of the wild type.The above results indicate that some mutations at position 222 can enhance the transglycosylation of the enzyme.Although the rest mutants do not show the same effect o,they can also enhance the yield of GOS3 due to their dramatic weakening in lactose hydrolysis.Although the maximum yield of GOS3 has not increased,it can be maintained at a relatively high level.For example,the level of GOS3 synthesized by the N222G increase continuously,reaching 85%of the maximum amount of wild-type product at 24 h,it reaches about110%at 84 h.The maximum yield of GOS3 catalyzed by N222E is only 5%lower than that of the wild-type.The yield can be maintained at a high level from 8 to 12 h.With the processing of the reaction,although the yield is slightly reduced,it maintains at about 80%of the maximum value.The above results indicate that compared with wild-type enzymes,some mutants are more suitable for industrial production of GOS.The saturation mutation at position 222 not only creates beneficial mutants which show higher yield of GOS,but also proves the hypothesis that reducing the hydrolysis activitywhile increasing the function of transferase/hydratase through rational design is an effective way to improve its pratical value.In following studies,mutants with excellent properties can be superimposed to explore the effect of site synergy on the catalytic synthesis of GOS,and further improveits application potential.更多还原