【作者】 郁凯；

【导师】 梅乐和；

【作者基本信息】 浙江大学 ， 生物化工， 2013， 博士

【摘要】 谷氨酸脱羧酶(glutamate decarboxylase,简称GAD)广泛存在于动物、植物、微生物,是Y.氨基丁酸(γ-aminobutyric acid,简称GAB A)生物合成过程中的关键酶。GABA具有延缓大脑衰老、降低血压、镇定安神等生理活性,作为一种新型的功能性因子,在食品、医疗、农业等行业具有广泛的应用前景。目前,GAD在大规模生物合成GABA的应用方面主要有两个限制因素：第一,GAD的最适pH通常为酸性,而在酸性pH下底物L-谷氨酸的溶解度非常小；第二,酶蛋白极易热失活,不能通过提高温度的方式加快反应速率。基于短乳杆菌GAD在GABA生物合成领域的应用前景,以突破天然酶的瓶颈、获得可商业用于高效生物制备GABA的理想催化剂为目标,论文以短乳杆菌GAD为亲本,采用同源模建、分子对接以及酶分子设计的生物信息学手段对GAD进行了分子改造。论文首先建立了一种可用于GAD酶活性高通量筛选的显色检测法。根据GAD催化脱羧反应时会不断消耗质子这一特点,采用pKa值基本一致的溴甲酚绿指示剂和醋酸缓冲液体系,将因质子消耗引起的体系pH值升高通过指示剂颜色的变化反映出来,颜色的变化进一步用620nm波长处的吸光度变化得以量化表征,从而实现酶促反应速率与吸光度变化速率之间的线性关联。最终得到的酶促反应速率计算公式为第二,以E. coli GadB为模板,采用同源模建技术构建了短乳杆菌GAD的结构模型,比较了MODLLER和Swiss Model这两种不同方式对模建结果的影响,并用PROCHECK、ERRAT及ProSA程序对所得模型进行构象和能量评价。以GAD结构模型为依据,对酶活性中心进行分析后发现一些重要的功能残基：Phe65和Thr215构成了疏水的底物入口,决定着底物分子进入活性中心时的取向；Lys279、Asp248对酶的催化过程有直接的贡献；Ser127、Asp248、His278、Lys279以及PLP磷酸基团附近的α-螺旋对辅酶PLP在活性中心的定位和正确取向有着非常重要的作用。第三,以MODELLER构建的GAD模型为蛋白质受体,在GAD催化机制的指导下构建了模拟反应过渡态的偕二胺中间体模型,然后采用ROSETTALIGAND程序将该底物模型对接进入酶的活性中心。通过对接分析,发现Gln166和Thr64与底物分子形成氢键作用,是维持底物在酶活性中心正确位置和取向的关键。此外,L-谷氨酸的α-羧基基本处于与PLP吡啶环垂直的方向,PLP的吡啶环以磷酸基团为轴翻转了约6。,体现了脱羧反应的特点。这些结果不仅揭示了L.谷氨酸在GAD活性中心的结合模式以及活性中心重要功能残基的分布,而且也为进一步研究GAD结构和功能的关系以及酶的分子设计提供了结构模型。第四,观察短乳杆菌GAD的结构模型,发现酶的C末端在中性pH下阻挡了底物入口,从而抑制了酶活性。据此,构建了C末端缺失14个氨基酸的突变体GADAC。经过改造,GADAC变体酶在pH6.0下催化活性得到提高,反应2h后的GABA产量为野生型酶的4.8倍。紫外/可见光谱、荧光光谱和圆二色谱分析也显示C末端的切除改变了酶活性中心的微环境,解除了原先C末端对活性口袋的“封闭”效应。该突变酶在生物转化法连续制备GABA方面有良好的应用前景,同时也进一步阐明了短乳杆菌GAD的C末端的在酶的pH-活性调控方面作用,为GAD结构和功能的研究提供了新的线索。最后,为提高酶的热稳定性,提高GAD在工业上的应用价值,我们采用RosettaDesign程序,用Monte Carlo算法搜索蛋白质的序列空间,通过求取全局最小值来预测有望提高GAD热稳定性的突变位点。经过计算共得到了20个建议的突变：A63N、T64Q、C66S、I87W、I98W、I105W、K138G、M185Y、 M212W、Y216W、L226G、V229G、P240G、S249W、F256Y、V283G、 W292G、C379V、K402H、413G。用定点突变方法构建这些突变体,通过显色法初筛和HPLC法复筛,最终得到了突变酶C379V,它的半失活温度T1/2比亲本酶提高了5℃,并且酶的比活力比亲本提高了19%。疏水作用的增加是酶性能得以提高的主要原因。该研究不仅构建了更适合用于制备GABA的生物催化剂,同时也为其他酶热稳定变体的设计与改造提供了理论基础和方法保障。更多还原

【Abstract】 Glutamate decarboxylase (GAD) is an essential enzyme widely distributed in nature from microorganisms to plants and animals. It is the key enzyme for the biosynthesis of GABA, which is useful as a functional bioactive component in food and pharmaceutical, for its anti-hypertensive and analgesic properties as well as calming effects.Currently the mass production of GABA in bioreactors is still limited due to the bottleneck caused by the low substrate solubility at the acidic pH optimum of GAD and the inherently thermal unstable nature of this enzyme. Based on the prospects in utilizing Lactobacillus brevis GAD for the biosynthesis of GABA, we employed bioinformatics methods such as homology modeling, molecular docking and computational enzyme design to create a more efficient biocatalyst for GABA preparation, thus breaking through the above mentioned bottleneck.Firstly, a pH-sensitive colorimetric assay was established to quantitatively measure GAD activity in bacterial cell extracts using a microplate format. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pKa to the acetate buffer used. The corresponding absorbance change at620nm was recorded with a microplate reader as the reaction proceeded. The enzymatic reaction rate could be calculated using the formula:v=2.4×106×dA/dt (μmol·min-1). This is a simple, economical assay that can be carried out in robotic high-throughput devices in directed evolution experiments for the rapid determination of GAD activity.Secondly, we constructed the homology model of Lactobacillus brevis GAD with E. coli GadB as the template using both MODELLER and Swiss Model programs. Then PROCHECK, ERRAT and ProSA were used to evaluate the quality of resulting models. The analysis of enzyme active site revealed the following functional residues:Phe65and Thr215constituted the hydrophobic substrate entrance; Asp248and Lys279directly contributed the catalysis; Serl27, Asp248, His278, Lys279and an a-helix near the PLP phosphate group were responsible for keeping the favorable position and orientation of the cofactor PLP in the active center.Thirdly, we modeled the geminal diamine intermediate consisting of the substrate, PLP and Lys279according to the reaction mechanism, and then docked it into GAD homo logy model using ROSETTALIGAND. A total of5000docking trajectories were generated and the best scored enzyme-substrate complex showed that the hydrogen bonds from Gln166and Thr64were of vital importance in substrate binding. Besides, the positon of a-carboxylate group of the substrate was almost pepenticular to the pyridine ring of PLP, which was rotated by approximately6°with respect to its original position, with the PLP phosphate moiety acting as an anchor. These geometries are in accord with the characteristics often observed in PLP dependent decarboxylases. This work not only revealed the binding mode of substrate in the GAD active site and the distribution of functional residues, but also provided a structure model for further study on the enzyme structure-function relationship as well as enzyme design.Fourthly, we speculated that the substrate entrance was probably blocked by a C-terminal tail of GAD based on the homology model, which could be the reason for no detectable enzymatic activity at neutral pH. Site-directed mutagenesis was performed to delete14C-terminal residues to generate a mutant, designated as GAD△C, which exhibited extended activity at pH6.0compared to the wild type enzyme. Comparison of the UV-visible, fluorescence and circular dichroism spectra of the mutant with those of the wild type revealed that the microenvironment of the active site had been changed and the "blocking" effect might be eliminated. These results provided evidence for the important role of C-terminal region in the pH-dependent regulation of enzymatic activity, and the resulting mutant would be useful in a bioreactor for continuous production of GABA.Lastly, RosettaDesign algorithm was employed with the aim of improving the thermostability of GAD. Sequence space was searched with an iterative Monte Carlo procedure, replacing a single amino acid rotamer at a time, and reevaluating the energy. With global enregy minimum found, twenty point mutations were suggested by the program:A63N, T64Q, C66S, I87W, I98W, I105W, K138G, M185Y, M212W, Y216W, L226G, V229G, P240G, S249W, F256Y, V283G, W292G, C379V, K402H and K413G. Site-directed mutagenesis was used to generate each of these mutant enzymes. After an initial screening for the enzymatic activity and a further thermal denaturation experiment, the variant C379V was selected out, which increased the T1/2by5℃, and the catalytic efficiency was enhanced by19%compared with those of the wild-type GAD. Increased hydrophobic interactions brought about by this mutation was speculated as the main reason for the improved properties. This work created a more efficient biocatalyst for GABA preparation and built the basis for the computational thermostabilization of other enzymes for industrial use.更多还原