【摘要】：酯酶(Esterase,EC 3.1.1.X)是水解酶的一種,能夠催化酯鍵斷裂生成醇類和酸類產(chǎn)物,也可以催化酯鍵的形成。酯酶由于能夠催化酯水解反應(yīng)和轉(zhuǎn)酯反應(yīng),廣泛應(yīng)用于工業(yè)、農(nóng)業(yè)、洗滌業(yè)以及制藥業(yè)等領(lǐng)域。冷活性酶具有在低溫下催化效率高,可以節(jié)約能源和特殊化合物的合成。因此,本研究針對酯酶EstA進(jìn)行了研究,主要結(jié)果如下:從腸肝菌Enterobacter sp.中克隆到一個新的酯酶基因estA,大小為717bp,編碼238個氨基酸,分子量為25 kDa。序列比對結(jié)果發(fā)現(xiàn)EstA沒有被報道過。另外,序列分析表明EstA序列具有水解酶典型的結(jié)構(gòu)催化三聯(lián)體Ser-Asp-His及包含Ser的保守結(jié)構(gòu)Gly-Ala-Ser-Met-Gly。酯酶EstA在大腸桿菌Escherichia coli BL21(DE3)中進(jìn)行異源表達(dá)和純化后,對酶學(xué)性質(zhì)進(jìn)行了分析,結(jié)果表明:在不同側(cè)鏈長度的對硝基苯酯類底物中,酯酶EstA對對-硝基基苯乙酸酯活性最高,活性隨著底物側(cè)鏈長度增加而降低,對對硝基苯基棕櫚酸酯(p-Nitrophenyl palmitate)的活性最低。最適反應(yīng)溫度是40°C,最適反應(yīng)pH值為9.0。以對硝基苯乙酸酯為底物,測得EstA的動力學(xué)常數(shù)Km為393.75μmol/L,Kcat為905.6 min~(-1),Kcat/Km為2.30×103 min~(-1)?μM~(-1)。在0°C到20°C范圍之間,EstA仍保持70%以上的活性,說明酯酶EstA具有較好的冷活性。在3M NaCl存在下,EstA保留60%左右的活性,而且經(jīng)過4M NaCl溶液4°C處理24 h,EstA仍保留100%活性,說明酯酶具有一定程度的耐鹽性。乙二醇、30%的異丙醇和30%的丙酮對EstA有明顯的抑制作用,10%、20%的異丙醇、丙酮、乙醇、甲醇和二甲基亞砜對EstA活性影響較小,說明EstA具有有機(jī)溶劑耐受性。EstA在0.5%和1%Triton~(-1)00、tween-20、tween-80 CTAB及10%的Triton~(-1)00、tween-20去污劑中保留60%以上的活性。EstA熱穩(wěn)定性差,45°C處理120 min或50°C處理30 min,酶活幾乎喪失。為了提高EstA的熱穩(wěn)定性,通過隨機(jī)突變得到一個熱穩(wěn)定性提高的突變體A92P,45°C處理120 min,仍保留95%的活性,是野生型的19倍;A92P在50°C處理30 min,保留55%的活性,是野生型EstA的2倍;為了進(jìn)一步探討92位氨基酸對熱穩(wěn)定性的影響,對92位位點進(jìn)行飽和突變,結(jié)果表明:突變體A92D熱穩(wěn)定性顯著提高,45°C處理120 min對其活性幾乎沒有影響,50°C處理30 min,仍保留85%左右的活性,是野生型的3.4倍。為了探究92位突變位點對酶熱穩(wěn)定的影響機(jī)理,對EstA及突變體進(jìn)行同源模擬及92位點附近空間結(jié)構(gòu)進(jìn)行了分析,結(jié)果顯示:突變體A92D的92位疏水性氨基酸(Ala)被親水性氨基酸(Asp)取代后,其與溶液之間及周圍親疏水結(jié)構(gòu)之間形成了更有利于蛋白穩(wěn)定的結(jié)構(gòu);而突變體A92P熱穩(wěn)定提高可能是由于脯氨酸Pro具有限制蛋白二級結(jié)構(gòu)的旋轉(zhuǎn),增加蛋白的剛性,從而推測可能是提高熱穩(wěn)定性的主要原因。這些結(jié)果為進(jìn)一步研究冷活性酶結(jié)構(gòu)和功能關(guān)系及熱穩(wěn)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 擄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 擄C to 20 擄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 擄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 擄C for 120 min or 50 擄C for 30 min,. In order to improve the thermal stability of EstA, a mutant A92PX 45 擄C with increased thermal stability was obtained by random mutation, and the activity of A92PX 45 擄C was kept at 95% for 120 min, which was 19 times higher than that of wild type. A92P retained 55% activity at 50 擄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 擄C treatment for 120 min, and that of 50 擄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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78;Q55

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本文編號：2413386