發(fā)布時間：2018-03-09 10:02

  本文選題：芽孢桿菌　切入點：阿魏酸酯酶　出處：《江南大學》2017年碩士論文　論文類型：學位論文

【摘要】：阿魏酸酯酶(FAEs,EC 3.1.1.73)屬于羧酸酯水解酶的一個亞綱,其能夠水解植物細胞壁中阿魏酸與多糖之間的酯鍵,從而將阿魏酸釋放出來。阿魏酸酯酶在食品、飼料、醫(yī)藥、造紙、化妝品、生物能源、生物合成等領域均有廣泛的應用前景。目前,來源于細菌的阿魏酸酯酶數(shù)目較少。本論文首次對芽孢桿菌阿魏酸酯酶進行了異源表達和酶學性質(zhì)的研究。主要研究結(jié)果如下:1)將實驗室保藏的122株芽孢桿菌置于含有阿魏酸乙酯作為碳源的篩選培養(yǎng)基中進行篩選,通過比較菌株產(chǎn)生的透明圈大小得到了一株阿魏酸酯酶活性較強的菌株:解淀粉芽孢桿菌(Bacillus amyloliquefaciens H47)。2)根據(jù)對B amyloliquefaciens H47的全基因組序列分析預測了10個可能的阿魏酸酯酶基因,它們分別為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)。對這10個基因進行了克隆表達,酶活驗證發(fā)現(xiàn)只有Ba Fae04、Ba Fae06和Ba Fae09具有阿魏酸酯酶的活性,它們的蛋白分子量分別約為30 k Da、29 k Da和31 k Da。與其他細菌阿魏酸酯酶相比,這3個新型的阿魏酸酯酶展現(xiàn)了很低的序列相似度(均小于30%)。3)對這3個阿魏酸酯酶進行了酶學性質(zhì)的研究。Ba Fae04、Ba Fae06和Ba Fae09的最適反應p H均為8,最適反應溫度分別為30℃、35℃和40℃。Ba Fae04在25-60℃條件下保溫1 h展現(xiàn)了較好的穩(wěn)定性,在60℃下仍能保持59%的活性。52種酯底物水解實驗結(jié)果顯示3個阿魏酸酯酶都擁有廣泛的底物水解范圍,并且它們能夠高效地水解阿魏酸酯酶常見底物。另外,這3個阿魏酸酯酶可以水解去淀粉麥麩的細胞壁并將其中的阿魏酸釋放出來。3個酶對阿魏酸甲酯、阿魏酸乙酯、對香豆酸甲酯和綠原酸的動力學參數(shù)如下:對于Ba Fae04,K_m值分別為1.14、2.15、0.78和6.08 mmol·L~(-1),k_(cat)/K_m值分別為10.57、11.72、11.40和45.31 L?mmol~(-1)·s~(-1);對于Ba Fae06,K_m值分別為1.99、0.58、1.42和1.51 mmol·L~(-1),k_(cat)/K_m值分別為4.32、5.48、30.90和4.32 L·mmol~(-1)·s~(-1);對于Ba Fae09,K_m值分別為0.49、0.86、1.81和4.79 mmol·L~(-1),k_(cat)/K_m值分別為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，

本文編號：1588057