芽孢杆菌属来源1,3-1,4-β-葡聚糖酶的热稳定性研究

发布时间：2018-04-04 01:19

本文选题：1　切入点：3-1　出处：《江南大学》2017年博士论文

【摘要】：1,3-1,4-β-葡聚糖酶,以下简称β-葡聚糖酶,是一种重要的工业用酶。β-葡聚糖酶通过专一性切割1,4-β-糖苷键可以有效降解禾本科植物细胞壁中的高分子量β-葡聚糖。在啤酒酿造行业中,外源添加β-葡聚糖酶可以有效加快麦汁过滤速度,提高麦汁浸出率和成品啤酒的非生物稳定性。在饲料行业中,添加外源β-葡聚糖酶可以消除β-葡聚糖的“抗营养因子”效应从而提高禽畜对麦类饲料的吸收效率,同时维护禽畜的肠道健康。然而目前β-葡聚糖酶依然存在热稳定性差、催化效率低等问题,其在麦芽制备、麦汁糖化和颗粒饲料制备过程中完全失活。因此,提高β-葡聚糖酶的热稳定性具有重要工业应用价值。本论文通过表面赖氨酸改造、二硫键引入和突变热点区域分析法等理性/半理性手段成功提高了芽孢杆菌来源β-葡聚糖酶的热稳定性并在枯草芽孢杆菌WB600中实现表达,最后将高热稳定性β-葡聚糖酶在麦汁协定糖化过程中进行应用。主要结论如下:(1)对芽孢杆菌来源β-葡聚糖酶表面赖氨酸在其热稳定性中作用进行分析。首先采用亚硝酸对β-葡聚糖酶表面赖氨酸进行化学修饰,发现赖氨酸ε-氨基基团的修饰有效提高了酶热稳定性。和野生酶相比,修饰酶的T50值提高了2.5℃,在50℃和60℃的半衰期分别提高了56%和76.8%。基于化学修饰研究,将特基拉芽孢杆菌来源β-葡聚糖酶表面赖氨酸选择性突变为丝氨酸。以蛋白质总能量值、氢键数量和比活力值为标准对热稳定性有利突变进行筛选,发现突变酶K20S、K117S和K165S的热稳定性优于野生酶。经过组合突变,组合突变酶K20S/K117S/K165S的最适温度和T50值和野生酶相比分别提高了15℃和14℃,而其在50℃和60℃的半衰期分别延长了170.9%和81.5%。与此同时,组合突变酶的催化性质优于野生酶。蛋白质结构分析发现在组合突变酶中形成了更多有序二级结构及更多氢键,这可能是赖氨酸突变后β-葡聚糖酶热稳定性提高的原因。(2)采用基于蛋白质结构分析及柔性分析的三步筛选法对β-葡聚糖酶热稳定性有利二硫键引入位点进行筛选。根据筛选结果,在β-葡聚糖酶中引入二硫键N31C-T187C和P102C-N125C可以降低β-葡聚糖酶整体/区域柔性且不影响酶催化性质。结果显示,引入二硫键后突变酶N31C-T187C和P102C-N125C的溶解温度和野生酶相比分别提高1.4℃和2.3℃。组合突变酶N31C-T187C/P102C-N125C的溶解温度和野生酶相比提高了4.1℃,而其在60℃的半衰期也明显延长。组合突变酶的催化性质并没有改变,而其最适p H值从p H6.5降至p H6.0。蛋白质结构分析显示预期二硫键的引入及突变区域内新氢键的形成可能是组合突变酶热稳定性提升的原因。(3)采用基于同源蛋白质氨基酸序列比对、结构空间区域化和分子动力学模拟的突变热点区域分析法对微生物来源β-葡聚糖酶热稳定性关键位点进行预测。结果显示,β-葡聚糖酶中钙离子结合区域和酶热稳定性有较高相关性。将钙离子结合区域中6个非保守氨基酸残基进行改造,发现40位、43位、46位和205位氨基酸的突变有效提高了β-葡聚糖酶的热稳定性。采用迭代饱和突变法对该四个位点进行突变,得到突变酶E46P/S43E/H205P/S40E的最适温度、T50值和溶解温度和野生酶相比分别提高了20℃、14.5℃和13.8℃。突变酶在60℃和70℃的半衰期是野生酶的3.86倍和7.13倍。蛋白质结构分析显示突变酶的钙离子结合区域带有更多负电荷,其与钙离子结合更为紧密。与此同时,突变酶整体结构有序性和野生酶相比有大幅度提高,这可能是突变酶热稳定性提升的原因。(4)将理性/非理性方法得到的β-葡聚糖酶热稳定性有利突变进行组合突变,并在枯草芽孢杆菌WB600中实现表达。组合突变酶的最适温度、T50值和溶解温度为70℃、81.7℃和56.2℃,和野生酶相比分别提高了25℃、19.7℃和15.9℃。组合突变酶在60℃和70℃的半衰期达到153.2 min和99.6 min,分别是野生酶的4.71倍和9.05倍。组合突变酶的最适p H值和野生酶相比降低了0.5个p H,而其在酸性环境下的稳定性也有大幅度提高。与此同时,组合突变酶的比活力值和kcat值和野生酶相比分别提高72.4%和37.5%。重组枯草芽孢杆菌经过发酵条件优化后分泌β-葡聚糖酶最高酶活达到4840.4U·m L-1,是野生酶酶活和重组大肠杆菌发酵酶活的3.31倍和2.03倍。将组合突变酶添在麦汁协定糖化过程中进行应用,发现其能将麦汁过滤时间和麦汁黏度分别降低29.7%和12.3%,效果优于两种市售酶。
[Abstract]:1,3-1,4- p-glucanase, hereinafter referred to as p-glucanase, is an important industrial enzyme. P-glucanase by specific cleavage of 1,4- beta glycosidic bond can effectively degrade the high molecular weight beta gramineous plant cell wall glucan. In the beer brewing industry, exogenous addition of beta glucanase can effectively accelerate the wort filtration rate, improve the non biological stability of beer and wort leaching rate. In the feed industry, the addition of exogenous beta glucanase can eliminate beta glucan "anti nutritional factors" effect on wheat to improve livestock feed absorption efficiency, while maintaining livestock intestinal health. However, beta glucan the enzyme still has poor thermal stability, low catalytic efficiency, the preparation of malt, wort saccharification and pellet preparation process completely inactivated. Therefore, to improve the thermal stability of p-glucanase is important The industrial application value. This paper surface lysine modification by two disulfide bonds, the introduction and analysis of regional hot spot mutation method rational / semi rational means of success to improve the thermal stability of Bacillus derived beta glucan enzyme and expression in Bacillus subtilis WB600, the thermal stability of beta glucan enzyme was used in the process wort saccharification agreement. The main conclusions are as follows: (1) the source of Bacillus p-glucanase surface lysine effect in the thermal stability analysis. The nitrite of p-glucanase surface chemical modification of lysine, found that the modified lysine amino groups improved the enzyme thermal stability. Compared with the wild-type enzyme, enzymatic modification of T50 is increased by 2.5 degrees, 56% and 76.8%. respectively based on the chemical modification of 50 degrees and 60 degrees increase in the half-life, tequila Bacillus source of beta glucan sugar The surface of the enzyme lysine selectivity to serine mutation based on total protein and energy value, the hydrogen bond number and activity value as the standard of good thermal stability of mutation screening, found that the mutant enzyme K20S, K117S and K165S thermal stability is better than the wild-type enzyme. Through the combination of mutation, combination of the mutant enzyme K20S/K117S/K165S the optimum temperature and T50 value and wild compared to the enzyme were increased by 15 degrees and 14 degrees, and in the 50 degrees and 60 degrees of the half-life increased by 170.9% and 81.5%. at the same time, the combination of the mutant enzyme catalytic properties is better than that of the wild-type enzyme. The analysis found that the combination of sudden changes in enzyme in the formation of more ordered two more hydrogen bond structure and protein structure, which is probably the reason why lysine mutant beta glucan enzyme thermal stability increased. (2) the protein structure analysis and flexible analysis of three step method of p-glucanase favorable thermal stability into two disulfide bonds based on Screening was carried out. According to the screening results, the introduction of the two disulfide bonds in N31C-T187C and P102C-N125C in the beta glucan enzyme can reduce the p-glucanase overall regional flexible and does not affect the enzyme catalytic properties. The results showed that the dissolution temperature and the wild-type enzyme introduced two disulfide bonds after the mutant enzyme N31C-T187C and P102C-N125C were improved by 1.4 and 2.3 DEG C C. The melting temperature and wild enzyme combinations of the mutant enzyme N31C-T187C/P102C-N125C was increased by 4.1 degrees, and the half-life of 60 C was significantly prolonged. The catalytic properties of the enzyme combination mutation did not change, and the optimum p value from the H P H6.5 to analysis show the expected introduction of two disulfide bonds and the formation of new mutations within the region hydrogen bonds may be a combination of reason to enhance the thermal stability of the mutant enzyme P H6.0. protein structure. (3) by homologous protein amino acid sequence alignment based on the simulation of molecular dynamics and spatial structure of regional Mutation hotspot region analysis method to predict the microbial source of beta glucan enzyme thermal stability of key sites. The results showed that beta glucan enzyme in the calcium binding domain and enzyme thermal stability have higher correlation. The calcium binding 6 non conserved amino acid residues in the area of transformation, found 40, 43, 46 mutations and 205 amino acid can improve the thermal stability of p-glucanase. Using iterative saturation mutagenesis mutations of the four loci, the optimal temperature of the mutant enzyme E46P/S43E/H205P/S40E, T50 value of 20 C increased compared with the wild-type enzyme and solution temperature respectively, 14.5 degrees and 13.8 degrees. The half-life of the enzyme in the mutant 60 degrees and 70 degrees is 3.86 times and 7.13 times of the wild-type enzyme. Analysis showed that the mutant enzyme calcium binding region with more negative charge of the protein structure, and calcium binding more closely. At the same time, the mutant enzyme The structure of order and has greatly improved compared to the wild-type enzyme, which may be the reason to enhance the thermal stability of the mutant enzyme. (4) the rational / irrational method obtained p-glucanase favorable thermal stability and realize combination of mutation and mutation, expression in Bacillus subtilis WB600. The optimum temperature of group mutation enzyme the T50 value and dissolved temperature is 70 C, 81.7 C and 56.2 C, and compared the wild-type enzyme were increased by 25 degrees, 19.7 degrees and 15.9 degrees. The combination of the mutant enzyme reached 153.2 min and 99.6 min at 60 degrees and 70 degrees of the half-life were 4.71 times and 9.05 times of the wild enzyme combination. The mutant enzyme optimum p value of H and the wild-type enzyme decreased by 0.5 compared to P H, and its stability in acid environment was improved greatly. At the same time, a combination of mutations in the specific activity of the enzyme and kcat values and compared with the wild-type enzyme were increased by 72.4% and 37.5%. by fermentation of recombinant Bacillus subtilis Glucanase enzyme activity reached the highest 4840.4U m L-1 secretion conditions were optimized, wild enzyme and fermentation of recombinant Escherichia coli enzyme activity 3.31 times and 2.03 times. The mutant enzyme used in the process of adding wort saccharification in the agreement, the wort filtration time and wort viscosity were decreased by 29.7% and 12.3%, the sale of two kinds of enzyme is better than the city.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ925

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