熱纖梭菌阿魏酸酯酶的酶學特性分析及其植物高效表達載體構(gòu)建
發(fā)布時間：2018-12-17 22:39
【摘要】：阿魏酸和半纖維素、木質(zhì)素共價交聯(lián)形成木質(zhì)素—阿魏酸—阿拉伯木聚糖復合物,是禾本科植物細胞壁形成堅固抗降解屏障的重要分子基礎(chǔ)。在植物中異源表達阿魏酸酯酶(Ferulic acid esterase,FAE),可水解細胞壁中的阿魏酸酯鍵,促進細胞壁解聚,有效降低木質(zhì)纖維素降解轉(zhuǎn)化的成本,但目前仍面臨轉(zhuǎn)基因的常溫FAE酶活性干擾宿主植物生長發(fā)育、降低抗逆性等技術(shù)挑戰(zhàn),而采用嗜熱FAE酶基因進行遺傳轉(zhuǎn)化是一種重要的替代策略。本文對一種來源于熱纖梭菌(Clostridium thermocellum)的嗜熱FAE酶的酶學特性開展了系統(tǒng)的研究,并構(gòu)建了該嗜熱FAE酶編碼基因的植物高效表達載體,為培育木質(zhì)纖維素易降解的新型能源植物奠定了基礎(chǔ),其主要研究結(jié)果如下:1)分別克隆了熱纖梭菌C.thermocellum XynZ的阿魏酸酯酶催化域(FAE)及該阿魏酸酯酶催化域和碳水化合物結(jié)合域(FAE-CBM6)編碼基因,與pET22b連接分別構(gòu)建了原核表達重組質(zhì)粒pET22b-FAE和pET22b-FAE-CBM6,并在大腸桿菌BL21(DE-3)中實現(xiàn)異源表達,分別獲得分子量約為29.0 kDa和45.0 kDa的重組蛋白產(chǎn)物。2)分析比較了溫度、pH、底物、金屬離子等因子對FAE和FAE-CBM6這兩種重組嗜熱阿魏酸酯酶活性的影響。結(jié)果表明,它們的最適溫度分別為60℃和70℃,最適pH值分別為6.0和7.0。FAE酶在pH 5.0-pH 9.0范圍內(nèi)比較穩(wěn)定,而FAE-CBM6酶在pH 4.0-pH 9.0范圍內(nèi)比較穩(wěn)定;FAE酶在70℃或75℃下孵育2 h后其相對酶活仍能維持在60%以上,而FAE-CBM6酶在70℃孵育2 h后仍能維持80%以上的酶活。Mn2+和Zn2+對于FAE酶的酶活有促進作用,但Mg2+、Cu2+、Ni2+有抑制作用;而Cu2+和Zn2+對FAE-CBM6酶活有明顯抑制作用。在同一反應條件下,FAE-CBM6的酶活一般比FAE的高,說明CBM6結(jié)合域的存在對該嗜熱阿魏酸酯酶活性有促進作用。3)根據(jù)禾本科植物玉米(Zea mays)密碼子偏好性對嗜熱阿魏酸酯酶的編碼基因序列進行了優(yōu)化,并人工合成了優(yōu)化后的基因序列,在此基礎(chǔ)之上,通過與單子葉植物Ubiquitin強啟動子、增強子Ω序列以及不同亞細胞定位信號肽(質(zhì)外體或內(nèi)質(zhì)網(wǎng)定位信號肽)編碼序列進行組合,構(gòu)建了該嗜熱阿魏酸酯酶基因的多種植物高效表達載體。
[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，
本文編號：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，
本文編號：2384897
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