热纤梭菌阿魏酸酯酶的酶学特性分析及其植物高效表达载体构建
发布时间：2018-12-17 22:39
【摘要】：阿魏酸和半纤维素、木质素共价交联形成木质素—阿魏酸—阿拉伯木聚糖复合物,是禾本科植物细胞壁形成坚固抗降解屏障的重要分子基础。在植物中异源表达阿魏酸酯酶(Ferulic acid esterase,FAE),可水解细胞壁中的阿魏酸酯键,促进细胞壁解聚,有效降低木质纤维素降解转化的成本,但目前仍面临转基因的常温FAE酶活性干扰宿主植物生长发育、降低抗逆性等技术挑战,而采用嗜热FAE酶基因进行遗传转化是一种重要的替代策略。本文对一种来源于热纤梭菌(Clostridium thermocellum)的嗜热FAE酶的酶学特性开展了系统的研究,并构建了该嗜热FAE酶编码基因的植物高效表达载体,为培育木质纤维素易降解的新型能源植物奠定了基础,其主要研究结果如下:1)分别克隆了热纤梭菌C.thermocellum XynZ的阿魏酸酯酶催化域(FAE)及该阿魏酸酯酶催化域和碳水化合物结合域(FAE-CBM6)编码基因,与pET22b连接分别构建了原核表达重组质粒pET22b-FAE和pET22b-FAE-CBM6,并在大肠杆菌BL21(DE-3)中实现异源表达,分别获得分子量约为29.0 kDa和45.0 kDa的重组蛋白产物。2)分析比较了温度、pH、底物、金属离子等因子对FAE和FAE-CBM6这两种重组嗜热阿魏酸酯酶活性的影响。结果表明,它们的最适温度分别为60℃和70℃,最适pH值分别为6.0和7.0。FAE酶在pH 5.0-pH 9.0范围内比较稳定,而FAE-CBM6酶在pH 4.0-pH 9.0范围内比较稳定;FAE酶在70℃或75℃下孵育2 h后其相对酶活仍能维持在60%以上,而FAE-CBM6酶在70℃孵育2 h后仍能维持80%以上的酶活。Mn2+和Zn2+对于FAE酶的酶活有促进作用,但Mg2+、Cu2+、Ni2+有抑制作用;而Cu2+和Zn2+对FAE-CBM6酶活有明显抑制作用。在同一反应条件下,FAE-CBM6的酶活一般比FAE的高,说明CBM6结合域的存在对该嗜热阿魏酸酯酶活性有促进作用。3)根据禾本科植物玉米(Zea mays)密码子偏好性对嗜热阿魏酸酯酶的编码基因序列进行了优化,并人工合成了优化后的基因序列,在此基础之上,通过与单子叶植物Ubiquitin强启动子、增强子Ω序列以及不同亚细胞定位信号肽(质外体或内质网定位信号肽)编码序列进行组合,构建了该嗜热阿魏酸酯酶基因的多种植物高效表达载体。
[Abstract]:Ferulic acid and hemicellulose, lignin co-crosslinked to form lignin-ferulic acid-arabinoglycan complex, is an important molecular basis for the formation of solid anti-degradation barrier in the cell walls of Gramineae plants. Heterologous expression of ferulic esterase (Ferulic acid esterase,FAE) in plants can hydrolyze ferulic acid ester bond in cell wall, promote cell wall depolymerization, and effectively reduce the cost of lignocellulose degradation and transformation. However, at present, the transgenic FAE enzyme activity at room temperature interferes with the growth and development of host plants and reduces the resistance to stress. Therefore, the genetic transformation of thermophilic FAE enzyme gene is an important alternative strategy. In this paper, the enzymatic properties of a thermophilic FAE enzyme derived from (Clostridium thermocellum) of Clostridium thermophilus were systematically studied, and the plant expression vector encoding the thermophilic FAE enzyme gene was constructed. For the cultivation of lignocellulose degradable new energy plants laid the foundation, The main results are as follows: 1) the ferulic esterase catalytic domain (FAE), ferulic esterase catalytic domain (FAE-CBM6) and carbohydrate binding domain (FAE-CBM6) coding genes of C.thermocellum XynZ were cloned, respectively. The prokaryotic expression plasmids pET22b-FAE and pET22b-FAE-CBM6, were constructed by ligation with pET22b, and heterologous expression was realized in Escherichia coli BL21 (DE-3). The recombinant protein products with molecular weight of 29.0 kDa and 45.0 kDa were obtained respectively. 2) the effects of temperature, pH, substrate and metal ions on the activity of FAE and FAE-CBM6 were compared. The results showed that their optimum temperature was 60 鈩，
本文编号：2384897
[Abstract]:Ferulic acid and hemicellulose, lignin co-crosslinked to form lignin-ferulic acid-arabinoglycan complex, is an important molecular basis for the formation of solid anti-degradation barrier in the cell walls of Gramineae plants. Heterologous expression of ferulic esterase (Ferulic acid esterase,FAE) in plants can hydrolyze ferulic acid ester bond in cell wall, promote cell wall depolymerization, and effectively reduce the cost of lignocellulose degradation and transformation. However, at present, the transgenic FAE enzyme activity at room temperature interferes with the growth and development of host plants and reduces the resistance to stress. Therefore, the genetic transformation of thermophilic FAE enzyme gene is an important alternative strategy. In this paper, the enzymatic properties of a thermophilic FAE enzyme derived from (Clostridium thermocellum) of Clostridium thermophilus were systematically studied, and the plant expression vector encoding the thermophilic FAE enzyme gene was constructed. For the cultivation of lignocellulose degradable new energy plants laid the foundation, The main results are as follows: 1) the ferulic esterase catalytic domain (FAE), ferulic esterase catalytic domain (FAE-CBM6) and carbohydrate binding domain (FAE-CBM6) coding genes of C.thermocellum XynZ were cloned, respectively. The prokaryotic expression plasmids pET22b-FAE and pET22b-FAE-CBM6, were constructed by ligation with pET22b, and heterologous expression was realized in Escherichia coli BL21 (DE-3). The recombinant protein products with molecular weight of 29.0 kDa and 45.0 kDa were obtained respectively. 2) the effects of temperature, pH, substrate and metal ions on the activity of FAE and FAE-CBM6 were compared. The results showed that their optimum temperature was 60 鈩，
本文编号：2384897
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