解淀粉芽孢杆菌阿魏酸酯酶的基因克

发布时间：2018-03-09 10:02

  本文选题：芽孢杆菌　切入点：阿魏酸酯酶　出处：《江南大学》2017年硕士论文　论文类型：学位论文

【摘要】：阿魏酸酯酶(FAEs,EC 3.1.1.73)属于羧酸酯水解酶的一个亚纲,其能够水解植物细胞壁中阿魏酸与多糖之间的酯键,从而将阿魏酸释放出来。阿魏酸酯酶在食品、饲料、医药、造纸、化妆品、生物能源、生物合成等领域均有广泛的应用前景。目前,来源于细菌的阿魏酸酯酶数目较少。本论文首次对芽孢杆菌阿魏酸酯酶进行了异源表达和酶学性质的研究。主要研究结果如下:1)将实验室保藏的122株芽孢杆菌置于含有阿魏酸乙酯作为碳源的筛选培养基中进行筛选,通过比较菌株产生的透明圈大小得到了一株阿魏酸酯酶活性较强的菌株:解淀粉芽孢杆菌(Bacillus amyloliquefaciens H47)。2)根据对B amyloliquefaciens H47的全基因组序列分析预测了10个可能的阿魏酸酯酶基因,它们分别为Ba Fae01(906 bp)、Ba Fae02(780 bp)、Ba Fae03(774 bp)、Ba Fae08(696 bp)、Ba Fae09(741 bp)和Ba Fae10(822 bp)。对这10个基因进行了克隆表达,酶活验证发现只有Ba Fae04、Ba Fae06和Ba Fae09具有阿魏酸酯酶的活性,它们的蛋白分子量分别约为30 k Da、29 k Da和31 k Da。与其他细菌阿魏酸酯酶相比,这3个新型的阿魏酸酯酶展现了很低的序列相似度(均小于30%)。3)对这3个阿魏酸酯酶进行了酶学性质的研究。Ba Fae04、Ba Fae06和Ba Fae09的最适反应p H均为8,最适反应温度分别为30℃、35℃和40℃。Ba Fae04在25-60℃条件下保温1 h展现了较好的稳定性,在60℃下仍能保持59%的活性。52种酯底物水解实验结果显示3个阿魏酸酯酶都拥有广泛的底物水解范围,并且它们能够高效地水解阿魏酸酯酶常见底物。另外,这3个阿魏酸酯酶可以水解去淀粉麦麸的细胞壁并将其中的阿魏酸释放出来。3个酶对阿魏酸甲酯、阿魏酸乙酯、对香豆酸甲酯和绿原酸的动力学参数如下:对于Ba Fae04,K_m值分别为1.14、2.15、0.78和6.08 mmol·L~(-1),k_(cat)/K_m值分别为10.57、11.72、11.40和45.31 L?mmol~(-1)·s~(-1);对于Ba Fae06,K_m值分别为1.99、0.58、1.42和1.51 mmol·L~(-1),k_(cat)/K_m值分别为4.32、5.48、30.90和4.32 L·mmol~(-1)·s~(-1);对于Ba Fae09,K_m值分别为0.49、0.86、1.81和4.79 mmol·L~(-1),k_(cat)/K_m值分别为0.69、1.23、1.21和2.52 L·mmol~(-1)·s~(-1)。
[Abstract]:Ferulic acid esterase (FAESS EC 3.1.1.73) belongs to a subclass of carboxylate hydrolase, which can hydrolyze the ester bond between ferulic acid and polysaccharide in plant cell wall, thus releasing ferulic acid. Ferulic acid esterase is used in food, feed, medicine, paper making, cosmetics, etc. Bioenergy, biosynthesis and other fields have a wide range of applications. At present, The number of ferulic esterase derived from bacteria is relatively small. In this paper, the heterologous expression and enzymatic properties of ferulic esterase from Bacillus sp. Were studied for the first time. The main results are as follows: 1) 122 strains of Bacillus spp. In the screening medium containing ethyl ferulate as carbon source, A strain with strong ferulic acid esterase activity was obtained by comparing the size of transparent circle produced by the strain: Bacillus amyloliquefaciens H47.2. 10 possible ferulic esterase genes were predicted based on the whole genome sequence analysis of B amyloliquefaciens H47. They are Ba Fae01(906 Fae02(780 bpnb, Ba Fae03(774 Fae03(774 bpnb, Ba Fae08(696 BpPU, Ba Fae09(741 BpP) and Ba Fae10(822 BpP, respectively. The 10 genes were cloned and expressed. Only Ba Fae04Ba Fae06 and Ba Fae09 have ferulic esterase activity, the results of enzyme activity test show that Ba Fae 04 Ba Fae06 and Ba Fae09 have ferulic esterase activity. The results show that Ba Fae 04 Ba Fae06 and Ba Fae09 have ferulic esterase activity. The molecular weights of the proteins were about 30 kDa 29kDa and 31 kDa. compared with other bacteria, ferulic acid esterase. The three new ferulic esterases showed very low sequence similarity (all less than 30%. 3). The enzymatic properties of the three ferulic esterases were studied. The optimum reaction pH of Ba Fae04 Ba Fae06 and Ba Fae09 were both 8, and the optimum reaction temperature was 8, respectively. For 30 鈩，

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