【作者】 曹昊；

【导师】 张应玖；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 糖苷水解酶家族13是一类专门作用于α糖苷键的酶，也称作α淀粉酶家族，这个家族拥有数量庞大的成员，它们具有不同类型的催化性质，包括α-1，4糖苷键和α-1，6糖苷键的水解和转糖苷活性。其中，糖苷水解酶家族13中的第20亚家族（新普鲁蓝酶亚家族）因为同时具有上述2种或4种催化活性，而被作为多功能淀粉酶成为研究热点。我们之前发现了一株来自于Bacillus sp.ZW2531-1菌株的嗜热多功能淀粉酶，命名为OPMA-N，它可以经水解和转糖苷反应将廉价的淀粉催化生成麦芽糖，麦芽三糖，异麦芽三糖和异麦芽四糖，而异麦芽低聚糖作为一种双歧因子具有较高的经济价值和药用价值。近年来，随着对蛋白质结构信息了解的加深，以及计算机软硬件技术的发展，蛋白质的合理设计技术得到了较快发展和应用。结合蛋白质工程手段对蛋白质进行改造，不光有利于得到功能更优的蛋白质（酶），还可以更深入地研究蛋白质结构和功能之间的内在关系。在本文的研究中，我们首先利用同源建模的方法得到了OPMA-N的初始结构模型，然后利用分子力学和分子动力学方法对初始模型进行了优化，多种评估软件证明了OPMA-N模型的合理性。通过对模型的分析，并结合家族基因的同源序列的比对，本文选定了两个重要的氨基酸残基（Trp358，Val328）进行定点突变，并选择了以N端结构域为核心模块的多种重组酶进行结构域重组以及模块组合的研究。本研究首先模拟构建了358位点不同氨基酸的20种3D结构模型，并把它们与底物分子进行对接，结合分子对接结果和氨基酸侧链极性、电荷、位阻等因素，将这些氨基酸分为5类，进而选取了7种突变株进行研究，它们分别是W358K，W358R，W358N，W358T，W358G，W358D，W358E。在利用分子生物学手段构建突变载体，表达纯化得到7种突变株的纯酶之后，我们通过对它们进行性质测定发现，将此位点的W突变成具有正电荷侧链的K和R之后，它们的产物以葡萄糖为主，其比例达到了83.6%和80.2%，同时伴有少量的麦芽糖，而野生型和其它突变性的催化产物是没有葡萄糖的，这个结果说明W358K和W358R已经丧失了绝大多数转糖苷活性，成为了完全的淀粉水解酶。相反，W358N/T，W358D/E的产物中低聚异麦芽糖比例有提高，说明它们的转糖苷活性得到了提高。另外一个比较明显的变化是，W358K/R的酶活力提升了近40%，其Km值较野生型下降，同时kcat值升高，这样的突变提升了酶与底物的亲和力以及酶的催化效率。突变体W358E的酶活力比野生型提高了近一半，而其Km值和kcat值均升高，说明这样的突变在提升酶催化效率的同时却降低了酶与底物的结合能力。W358N/T的活力、动力学数值与野生型相近，而W358G在催化效率上远低于野生型OPMA-N。W358K/R和W358D/E突变体的热稳定性要略好于野生型，可能是这些引入的带电荷且具有一定空间位阻的氨基酸残基增加了此位点与附近氨基酸残基的相互作用，从而增加了酶分子的稳定性。酶分子寡聚化的实验结果显示，不同的突变体，从单体到五聚体的比例也不相同，尤其是单体与二聚体的比例、单体与二聚体之和所占的比例有可能影响着酶分子的催化形式和性质。最后，我们试图通过研究计算机模拟分子对接结果来找到突变酶催化性质变化的分子机理，野生型中W358残基在底物结合口袋+2亚位点处形成了很强的空间位阻，这样的结构导致了OPMA-N水解和转糖苷活性的平衡，而突变体中底物结合的方向和位置都发生了改变，N/T358和D/E358与底物+3和+2位点分别形成了氢键，它们与底物以及E356的相互作用也发生了很大变化。而K/R358与底物以及催化残基E356形成了很强的相互作用网络，G358的突变则丧失了此位点的原有位阻和与周围残基的相互作用。本文选择的另一个重要氨基酸突变靶点是V328，同时根据已有的研究报道，我们想研究一下此位点氨基酸侧链位阻的大小是否决定多功能淀粉酶OPMA-N的催化类型，因此设计了三种突变体分别是V328I，V328A和V328G。实验结果显示，此位点处过大的侧链I和过小的侧链G都会显著降低催化活性。而当V突变成大侧链的I后，产物中异麦芽低聚糖的含量明显下降，而反之V328A和V328G中其含量略有升高。另外一个比较显著的变化是，将V突变成A之后，此酶对β-CD有了一定的催化活性，产物以麦芽糖和麦芽三糖居多。此位点突变对酶的pH、温度性质影响不大，而对酶分子的寡聚化程度有影响。分子对接结果显示，此位点对邻近的N330、受体糖结合位点E331及催化残基D327的位置和取向有一定影响，尤其是V328A突变体中N330与底物+2亚位点形成两根氢键，明显将底物拉向受体糖结合口袋方向，或许这就是它底物特异性和产物比例变化的原因。另一方面，本文围绕新普鲁兰酶亚家族的核心N端结构域构建了OPMA-NN、OPMA-TNT、OPMA-NTN克隆，加上原有的OPMA-N和OPMA-NT，我们对不同结构域重组体以及不同模块的等分子数组合进行酶学性质检测发现，它们的产物比例并不发生明显变化。模块组合的活力和组合之前基本差别不大，OPMA-TNT的活力略高于野生型。一个比较明显的结果是，具有两个N端结构域的重组体OPMA-NTN的热稳定性较野生型提高了20℃，结合OPMA-N和OPMA-NT的对比，我们发现N端结构域对酶的热稳定性有着很大贡献。综上，本文通过对多功能淀粉酶OPMA-N关键氨基酸残基的合理性定点突变实验，及以N端结构域为核心的模块重组实验，并结合计算机模拟分子对接结果，在一定程度上揭示了OPMA-N催化功能集成性的机制。同时得到了一些具有工业应用价值和潜力的突变酶如OPMA-N/W358N，OPMA-N/V328A，OPMA-NTN。更多还原

【Abstract】 Glycoside hydrolase family13（GH13） has a large number of members andwhich shows a special effect on the α-glycosidic linkages, and it also calledα-amylase family. They show different types of catalytic properties, includinghydrolysis or transglycosylation for the α-（1,4） and α-（1,6）-glycosidic linkages. The20subfamily of the GH13（neopullulanase） is capable of catalyzing all or two ofabove types of reactions, it is also though of as a multi-functional amylase andbecame a hotspot for researching in nowadays. We have reported that theoligosaccharide-producing multifunctional amylase-N （OPMA-N） is a novel type ofstarch degrading enzyme secreted from Bacillus sp. ZW2531-1, it can degradedstarch to maltose, maltotriose, isomaltotriose and isomaltotetraose via reactions ofhydrolysis and transglycosylation, the isomaltooligosaccharides is known as bifidusfactors and has a great economic and medicinal value.In recent years, with the deepening understanding of protein structureinformation, as well as the development of computer technology, the technology ofreasonable design for protein has a rapid development and application. Combinewith protein engineering, we will not only to get better protein （or enzyme） forapplication, but also can deeply study the intrinsic relationship between proteinstructure and function. In this study, we first built the initial structure model ofOPMA-N by homology modeling, and then used the energy optimization andmolecular dynamics simulation to optimize the initial model, at last, multipleassessment software were adopted to prove the rationality of OPMA-N model.Combined with the analysis for model, and the homologous sequence alignment ofthe GH13family, two important residues （Trp358, Val328） were chosen forsite-directed mutagenesis, and recombinant enzymes with multi-N-domain ormulti-catalytic domain were built to study.We first constructed all of the possible3D structures with20different aminoacids at this site by homology modeling. After careful analysis, taking into theaccount factors such as sterics, polarity, and charge, the seven mutants W358K, W358R, W358N,W358T, W358G, W358D, andW358E were selected to representthe diversity of this site. After constructed mutant plasmids, we got pure enzymesafter expression and purification, and then their nature were determined, we foundthat when W mutated into a positively charged side chain of K/R, their productswere given priority to glucose, the ratio reached to83.6%and80.2%, at the sametime with a little maltose, while the products of wild type and other mutants had noglucose, the results show that W358K and W358R had lost most of thetransglycosylation activity, and became the starch hydrolyzing enzymes. In contrast,isomaltooligosaccharides in the products of W358N/T, W358D/E had increased, weillustrated that their transglycosylation activity had improved. Another significantchange was the activities of W358K/R had increased nearly40%, their Km valueshad decreased while their kcat values had increased compared to the wild type,mutation like them might increased affinity between enzyme with substrate and theircatalytic efficiencies. The enzyme activities of W358D/E were half higher than thewild type, while the Km and kcat values were increased, indicating that mutationslike them could improved catalytic efficiencies but also reduced the binding abilitybetween enzyme with substrate. The numerical of W358N/T were similar to the wildtype, while the W358G was far lower than that of the wild type OPMA-N incatalytic efficiency. The thermal stability of mutants W358K/R and W358D/E wasslightly better than the wild-type, we inferred that the introduction of the chargedand sterically hindered amino acid residues might increased the interaction with theiraround residues. Oligomerization results showed that different mutants, formeddifferent oligomerization which from monomer to pentamer, it （especially the ratioof monomer and dimer, amount of monomer and dimmer） might influenced thecatalytic properties of the enzyme molecules form and nature. Finally, through thecomputer simulation of molecular docking results, we wanted to find the molecularmechanism of the changes in enzymes’catalytic properties, wild type Trp358locatedin the substrate binding pocket+2sub-site and it formed very strong steric hindrance,this structure formed the activity balance between hydrolysis and transglycosylationfor OPMA-N, and it had changed in the mutants, there were newly formed hydrogen bonds between Asn/Thr358and Asp/Glu358with+3/+2sub-sites of substraterespectively, their interactions between them with substrate and Glu356had greatchanges. The strong interactions were formed between Lys/Arg358with substrateand catalytic residue Glu356. Mutant Gly358had lost the original steric interactionand the interaction with the surrounding residues.Val328was another important amino acids the mutation target that selected,according to research reported at the same time, we wanted to explore whether thesize of the residue’s side chain determines the catalytic type of multifunctionalamylase OPMA-N in this site, so three mutants V328I, V328A and V328G weredesigned respectively. Results showed that, in this site the bigger side chain of Ileand smaller side chains of Gly significantly decreased catalytic activity. When Valmutated into Ile, its product of isomaltooligosaccharides was significantly decreased,whereas the contents of V328A and V328G had increased slightly. Anothersignificant change was this enzyme had certain activity for β-CD when the mutationof Val into Ala, its products were mainly maltose and maltotriose. The mutation ofthis site had little effect on pH, temperature properties of enzymes, but influencedthe degree of oligomerization. Molecular docking results showed this site had acertain effect on the adjacent Asn330, receptor sugar binding site Glu331and theposition and orientation of the catalytic residues of Asp327, especially there weretwo newly hydrogen bonds were formed between Asn330with the+2sub-site ofsubstrate in mutant V328A, the bonds obviously pulled the substrate to the receptorsugar binding pocket direction, perhaps this was the reason of the changes in itssubstrate specificity and the product ratios.This article, on the other hand, focuses on the core of N-domain of theGH13₂0subfamily, the reconstruction vectors OPMA-NN, OPMA-TNT,OPMA-NTN were constructed, together with the original OPMA-N and OPMA-NT,the study on enzymatic properties detection of different recombinants and differentmodules combinations were found that their product proportion did not changesignificantly, the activities between the combination module and those beforecombination had littler difference, the activity of OPMA-TNT was slightly higher than that of the wild type. An obvious result was that the thermal stability ofrecombinant OPMA-NTN with two N-domain was increased by20℃comparedwith the wild type, combined with OPMA-N and OPMA-NT, we demonstrated thatN-domain had a great contribution for the thermal stability of OPMA-N.In conclusion, through the rationality mutation study of multifunctional amylaseOPMA-N, and the restructuring module, combined with molecular docking results,to a certain extent, revealed the mechanism of the function integration and catalyticactivity for OPMA-N. At the same time, we got some mutant enzymes such asOPMA-N/W358N, OPMA-N/V328A, OPMA-NTN which shown potential value inindustrial application.更多还原