适冷性酯酶基因的克
发布时间：2019-01-22 16:35
【摘要】：酯酶(Esterase,EC 3.1.1.X)是水解酶的一种,能够催化酯键断裂生成醇类和酸类产物,也可以催化酯键的形成。酯酶由于能够催化酯水解反应和转酯反应,广泛应用于工业、农业、洗涤业以及制药业等领域。冷活性酶具有在低温下催化效率高,可以节约能源和特殊化合物的合成。因此,本研究针对酯酶EstA进行了研究,主要结果如下:从肠肝菌Enterobacter sp.中克隆到一个新的酯酶基因estA,大小为717bp,编码238个氨基酸,分子量为25 kDa。序列比对结果发现EstA没有被报道过。另外,序列分析表明EstA序列具有水解酶典型的结构催化三联体Ser-Asp-His及包含Ser的保守结构Gly-Ala-Ser-Met-Gly。酯酶EstA在大肠杆菌Escherichia coli BL21(DE3)中进行异源表达和纯化后,对酶学性质进行了分析,结果表明:在不同侧链长度的对硝基苯酯类底物中,酯酶EstA对对-硝基基苯乙酸酯活性最高,活性随着底物侧链长度增加而降低,对对硝基苯基棕榈酸酯(p-Nitrophenyl palmitate)的活性最低。最适反应温度是40°C,最适反应pH值为9.0。以对硝基苯乙酸酯为底物,测得EstA的动力学常数Km为393.75μmol/L,Kcat为905.6 min~(-1),Kcat/Km为2.30×103 min~(-1)?μM~(-1)。在0°C到20°C范围之间,EstA仍保持70%以上的活性,说明酯酶EstA具有较好的冷活性。在3M NaCl存在下,EstA保留60%左右的活性,而且经过4M NaCl溶液4°C处理24 h,EstA仍保留100%活性,说明酯酶具有一定程度的耐盐性。乙二醇、30%的异丙醇和30%的丙酮对EstA有明显的抑制作用,10%、20%的异丙醇、丙酮、乙醇、甲醇和二甲基亚砜对EstA活性影响较小,说明EstA具有有机溶剂耐受性。EstA在0.5%和1%Triton~(-1)00、tween-20、tween-80 CTAB及10%的Triton~(-1)00、tween-20去污剂中保留60%以上的活性。EstA热稳定性差,45°C处理120 min或50°C处理30 min,酶活几乎丧失。为了提高EstA的热稳定性,通过随机突变得到一个热稳定性提高的突变体A92P,45°C处理120 min,仍保留95%的活性,是野生型的19倍;A92P在50°C处理30 min,保留55%的活性,是野生型EstA的2倍;为了进一步探讨92位氨基酸对热稳定性的影响,对92位位点进行饱和突变,结果表明:突变体A92D热稳定性显著提高,45°C处理120 min对其活性几乎没有影响,50°C处理30 min,仍保留85%左右的活性,是野生型的3.4倍。为了探究92位突变位点对酶热稳定的影响机理,对EstA及突变体进行同源模拟及92位点附近空间结构进行了分析,结果显示:突变体A92D的92位疏水性氨基酸(Ala)被亲水性氨基酸(Asp)取代后,其与溶液之间及周围亲疏水结构之间形成了更有利于蛋白稳定的结构;而突变体A92P热稳定提高可能是由于脯氨酸Pro具有限制蛋白二级结构的旋转,增加蛋白的刚性,从而推测可能是提高热稳定性的主要原因。这些结果为进一步研究冷活性酶结构和功能关系及热稳定性研究提供了重要信息。
[Abstract]:Esterase (Esterase,EC 3.1.1.X) is one of the hydrolases, which can catalyze the breakage of ester bonds to produce alcohols and acids, and also catalyze the formation of ester bonds. Esterase is widely used in industrial, agricultural, washing and pharmaceutical industries due to its catalytic hydrolysis and transesterification. Cold active enzymes have high catalytic efficiency at low temperature, which can save energy and synthesis of special compounds. Therefore, the esterase EstA was studied in this study. The main results are as follows: Enterobacter sp. from Enterobacter hepatica A novel esterase gene estA, encoding 238 amino acids with a molecular weight of 25 kDa. was cloned Sequence alignment results showed that EstA had not been reported. In addition, sequence analysis showed that the EstA sequence had the typical structure of hydrolase catalytic triad Ser-Asp-His and conserved Gly-Ala-Ser-Met-Gly. containing Ser. Esterase EstA was heterologous expressed and purified in Escherichia coli BL21 (DE3), and the enzymatic properties were analyzed. The results showed that: in the substrates of p-nitrobenzene esters with different side chain lengths, The activity of esterase EstA was the highest for p-nitrophenylacetate, and decreased with the increase of side chain length of substrate, and the activity for p-nitrophenyl palmitate (p-Nitrophenyl palmitate) was the lowest. The optimum reaction temperature is 40 掳C and the optimum pH value is 9.0. The kinetic constants of EstA were determined to be 393.75 渭 mol/L,Kcat, 905.6 min~ (-1) and 2.30 脳 10 ~ 3 min~ (-1)? 渭 M ~ (-1), respectively, using p-nitrophenylacetate as substrate. In the range of 0 掳C to 20 掳C, EstA remained more than 70% activity, indicating that esterase EstA had better cold activity. In the presence of 3M NaCl, the activity of EstA was about 60%, and the activity of Esterase was 100% after 4 掳C treatment of 4m NaCl solution for 24 h, which indicated that the esterase had a certain degree of salt tolerance. Ethylene glycol, 30% isopropanol and 30% acetone had obvious inhibitory effect on EstA, 10% isopropanol, acetone, ethanol, methanol and dimethyl sulfoxide had little effect on EstA activity. The results showed that EstA had organic solvent tolerance, EstA retained more than 60% activity in 0.5% and 1 Triton ~ (-1) 00Tween-20 tween-80 CTAB and 10% Triton~ (-1) 00Tween-20 detergent, and EstA had poor thermal stability. The enzyme activity was almost lost at 45 掳C for 120 min or 50 掳C for 30 min,. In order to improve the thermal stability of EstA, a mutant A92PX 45 掳C with increased thermal stability was obtained by random mutation, and the activity of A92PX 45 掳C was kept at 95% for 120 min, which was 19 times higher than that of wild type. A92P retained 55% activity at 50 掳C for 30 min, twice as much as wild-type EstA. In order to further study the effect of 92 amino acids on thermal stability, saturation mutation at 92 site was carried out. The results showed that the thermal stability of the mutant A92D was significantly improved, the activity of A92D was almost unchanged after 45 掳C treatment for 120 min, and that of 50 掳C treatment was 30 min,. The activity was still about 85%, 3.4 times of that of the wild type. In order to explore the mechanism of the effect of 92 mutation site on enzyme thermal stability, homologous simulation of EstA and mutant and analysis of spatial structure near 92 site were carried out. The results showed that when the 92 hydrophobic amino acid (Ala) of mutant A92D was replaced by hydrophilic amino acid (Asp), it formed a more stable protein structure between solution and surrounding hydrophobic structure. The increase of thermal stability of the mutant A92P may be due to the rotation of the protein secondary structure limited by proline Pro and the increase of protein rigidity, which may be the main reason for improving the thermal stability of the mutant A92P. These results provide important information for further study on the structure and function relationship and thermal stability of cold active enzymes.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q55
本文编号：2413386
[Abstract]:Esterase (Esterase,EC 3.1.1.X) is one of the hydrolases, which can catalyze the breakage of ester bonds to produce alcohols and acids, and also catalyze the formation of ester bonds. Esterase is widely used in industrial, agricultural, washing and pharmaceutical industries due to its catalytic hydrolysis and transesterification. Cold active enzymes have high catalytic efficiency at low temperature, which can save energy and synthesis of special compounds. Therefore, the esterase EstA was studied in this study. The main results are as follows: Enterobacter sp. from Enterobacter hepatica A novel esterase gene estA, encoding 238 amino acids with a molecular weight of 25 kDa. was cloned Sequence alignment results showed that EstA had not been reported. In addition, sequence analysis showed that the EstA sequence had the typical structure of hydrolase catalytic triad Ser-Asp-His and conserved Gly-Ala-Ser-Met-Gly. containing Ser. Esterase EstA was heterologous expressed and purified in Escherichia coli BL21 (DE3), and the enzymatic properties were analyzed. The results showed that: in the substrates of p-nitrobenzene esters with different side chain lengths, The activity of esterase EstA was the highest for p-nitrophenylacetate, and decreased with the increase of side chain length of substrate, and the activity for p-nitrophenyl palmitate (p-Nitrophenyl palmitate) was the lowest. The optimum reaction temperature is 40 掳C and the optimum pH value is 9.0. The kinetic constants of EstA were determined to be 393.75 渭 mol/L,Kcat, 905.6 min~ (-1) and 2.30 脳 10 ~ 3 min~ (-1)? 渭 M ~ (-1), respectively, using p-nitrophenylacetate as substrate. In the range of 0 掳C to 20 掳C, EstA remained more than 70% activity, indicating that esterase EstA had better cold activity. In the presence of 3M NaCl, the activity of EstA was about 60%, and the activity of Esterase was 100% after 4 掳C treatment of 4m NaCl solution for 24 h, which indicated that the esterase had a certain degree of salt tolerance. Ethylene glycol, 30% isopropanol and 30% acetone had obvious inhibitory effect on EstA, 10% isopropanol, acetone, ethanol, methanol and dimethyl sulfoxide had little effect on EstA activity. The results showed that EstA had organic solvent tolerance, EstA retained more than 60% activity in 0.5% and 1 Triton ~ (-1) 00Tween-20 tween-80 CTAB and 10% Triton~ (-1) 00Tween-20 detergent, and EstA had poor thermal stability. The enzyme activity was almost lost at 45 掳C for 120 min or 50 掳C for 30 min,. In order to improve the thermal stability of EstA, a mutant A92PX 45 掳C with increased thermal stability was obtained by random mutation, and the activity of A92PX 45 掳C was kept at 95% for 120 min, which was 19 times higher than that of wild type. A92P retained 55% activity at 50 掳C for 30 min, twice as much as wild-type EstA. In order to further study the effect of 92 amino acids on thermal stability, saturation mutation at 92 site was carried out. The results showed that the thermal stability of the mutant A92D was significantly improved, the activity of A92D was almost unchanged after 45 掳C treatment for 120 min, and that of 50 掳C treatment was 30 min,. The activity was still about 85%, 3.4 times of that of the wild type. In order to explore the mechanism of the effect of 92 mutation site on enzyme thermal stability, homologous simulation of EstA and mutant and analysis of spatial structure near 92 site were carried out. The results showed that when the 92 hydrophobic amino acid (Ala) of mutant A92D was replaced by hydrophilic amino acid (Asp), it formed a more stable protein structure between solution and surrounding hydrophobic structure. The increase of thermal stability of the mutant A92P may be due to the rotation of the protein secondary structure limited by proline Pro and the increase of protein rigidity, which may be the main reason for improving the thermal stability of the mutant A92P. These results provide important information for further study on the structure and function relationship and thermal stability of cold active enzymes.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q55
【参考文献】
相关期刊论文 前2条
1 杨华;娄永江;;国产碱性脂肪酶的测定方法及特性研究[J];中国食品学报;2006年03期
2 辛明秀,周培瑾;冷活性酶的结构柔韧性[J];生命的化学;2005年01期
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