本文選題：1　切入點(diǎn)：3-1　出處：《江南大學(xué)》2017年博士論文

【摘要】：1,3-1,4-β-葡聚糖酶,以下簡稱β-葡聚糖酶,是一種重要的工業(yè)用酶。β-葡聚糖酶通過專一性切割1,4-β-糖苷鍵可以有效降解禾本科植物細(xì)胞壁中的高分子量β-葡聚糖。在啤酒釀造行業(yè)中,外源添加β-葡聚糖酶可以有效加快麥汁過濾速度,提高麥汁浸出率和成品啤酒的非生物穩(wěn)定性。在飼料行業(yè)中,添加外源β-葡聚糖酶可以消除β-葡聚糖的“抗?fàn)I養(yǎng)因子”效應(yīng)從而提高禽畜對麥類飼料的吸收效率,同時(shí)維護(hù)禽畜的腸道健康。然而目前β-葡聚糖酶依然存在熱穩(wěn)定性差、催化效率低等問題,其在麥芽制備、麥汁糖化和顆粒飼料制備過程中完全失活。因此,提高β-葡聚糖酶的熱穩(wěn)定性具有重要工業(yè)應(yīng)用價(jià)值。本論文通過表面賴氨酸改造、二硫鍵引入和突變熱點(diǎn)區(qū)域分析法等理性/半理性手段成功提高了芽孢桿菌來源β-葡聚糖酶的熱穩(wěn)定性并在枯草芽孢桿菌WB600中實(shí)現(xiàn)表達(dá),最后將高熱穩(wěn)定性β-葡聚糖酶在麥汁協(xié)定糖化過程中進(jìn)行應(yīng)用。主要結(jié)論如下:(1)對芽孢桿菌來源β-葡聚糖酶表面賴氨酸在其熱穩(wěn)定性中作用進(jìn)行分析。首先采用亞硝酸對β-葡聚糖酶表面賴氨酸進(jìn)行化學(xué)修飾,發(fā)現(xiàn)賴氨酸ε-氨基基團(tuán)的修飾有效提高了酶熱穩(wěn)定性。和野生酶相比,修飾酶的T50值提高了2.5℃,在50℃和60℃的半衰期分別提高了56%和76.8%�；诨瘜W(xué)修飾研究,將特基拉芽孢桿菌來源β-葡聚糖酶表面賴氨酸選擇性突變?yōu)榻z氨酸。以蛋白質(zhì)總能量值、氫鍵數(shù)量和比活力值為標(biāo)準(zhǔn)對熱穩(wěn)定性有利突變進(jìn)行篩選,發(fā)現(xiàn)突變酶K20S、K117S和K165S的熱穩(wěn)定性優(yōu)于野生酶。經(jīng)過組合突變,組合突變酶K20S/K117S/K165S的最適溫度和T50值和野生酶相比分別提高了15℃和14℃,而其在50℃和60℃的半衰期分別延長了170.9%和81.5%。與此同時(shí),組合突變酶的催化性質(zhì)優(yōu)于野生酶。蛋白質(zhì)結(jié)構(gòu)分析發(fā)現(xiàn)在組合突變酶中形成了更多有序二級結(jié)構(gòu)及更多氫鍵,這可能是賴氨酸突變后β-葡聚糖酶熱穩(wěn)定性提高的原因。(2)采用基于蛋白質(zhì)結(jié)構(gòu)分析及柔性分析的三步篩選法對β-葡聚糖酶熱穩(wěn)定性有利二硫鍵引入位點(diǎn)進(jìn)行篩選。根據(jù)篩選結(jié)果,在β-葡聚糖酶中引入二硫鍵N31C-T187C和P102C-N125C可以降低β-葡聚糖酶整體/區(qū)域柔性且不影響酶催化性質(zhì)。結(jié)果顯示,引入二硫鍵后突變酶N31C-T187C和P102C-N125C的溶解溫度和野生酶相比分別提高1.4℃和2.3℃。組合突變酶N31C-T187C/P102C-N125C的溶解溫度和野生酶相比提高了4.1℃,而其在60℃的半衰期也明顯延長。組合突變酶的催化性質(zhì)并沒有改變,而其最適p H值從p H6.5降至p H6.0。蛋白質(zhì)結(jié)構(gòu)分析顯示預(yù)期二硫鍵的引入及突變區(qū)域內(nèi)新氫鍵的形成可能是組合突變酶熱穩(wěn)定性提升的原因。(3)采用基于同源蛋白質(zhì)氨基酸序列比對、結(jié)構(gòu)空間區(qū)域化和分子動力學(xué)模擬的突變熱點(diǎn)區(qū)域分析法對微生物來源β-葡聚糖酶熱穩(wěn)定性關(guān)鍵位點(diǎn)進(jìn)行預(yù)測。結(jié)果顯示,β-葡聚糖酶中鈣離子結(jié)合區(qū)域和酶熱穩(wěn)定性有較高相關(guān)性。將鈣離子結(jié)合區(qū)域中6個(gè)非保守氨基酸殘基進(jìn)行改造,發(fā)現(xiàn)40位、43位、46位和205位氨基酸的突變有效提高了β-葡聚糖酶的熱穩(wěn)定性。采用迭代飽和突變法對該四個(gè)位點(diǎn)進(jìn)行突變,得到突變酶E46P/S43E/H205P/S40E的最適溫度、T50值和溶解溫度和野生酶相比分別提高了20℃、14.5℃和13.8℃。突變酶在60℃和70℃的半衰期是野生酶的3.86倍和7.13倍。蛋白質(zhì)結(jié)構(gòu)分析顯示突變酶的鈣離子結(jié)合區(qū)域帶有更多負(fù)電荷,其與鈣離子結(jié)合更為緊密。與此同時(shí),突變酶整體結(jié)構(gòu)有序性和野生酶相比有大幅度提高,這可能是突變酶熱穩(wěn)定性提升的原因。(4)將理性/非理性方法得到的β-葡聚糖酶熱穩(wěn)定性有利突變進(jìn)行組合突變,并在枯草芽孢桿菌WB600中實(shí)現(xiàn)表達(dá)。組合突變酶的最適溫度、T50值和溶解溫度為70℃、81.7℃和56.2℃,和野生酶相比分別提高了25℃、19.7℃和15.9℃。組合突變酶在60℃和70℃的半衰期達(dá)到153.2 min和99.6 min,分別是野生酶的4.71倍和9.05倍。組合突變酶的最適p H值和野生酶相比降低了0.5個(gè)p H,而其在酸性環(huán)境下的穩(wěn)定性也有大幅度提高。與此同時(shí),組合突變酶的比活力值和kcat值和野生酶相比分別提高72.4%和37.5%。重組枯草芽孢桿菌經(jīng)過發(fā)酵條件優(yōu)化后分泌β-葡聚糖酶最高酶活達(dá)到4840.4U·m L-1,是野生酶酶活和重組大腸桿菌發(fā)酵酶活的3.31倍和2.03倍。將組合突變酶添在麥汁協(xié)定糖化過程中進(jìn)行應(yīng)用,發(fā)現(xiàn)其能將麥汁過濾時(shí)間和麥汁黏度分別降低29.7%和12.3%,效果優(yōu)于兩種市售酶。
[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.

【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ925

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