论文部分内容阅读
阿魏酸酯酶广泛应用于食品、药品、饲料及造纸等行业, 挖掘适用于工业化的阿魏酸酯酶至关重要.从嗜热真菌Thermoascus crustaceus JCM12803中克隆得到一个阿魏酸酯酶基因FAE-2515, 经基因序列分析, FAE-2515 c DNA全长为1 581 bp, 编码526个氨基酸和1个终止子, 理论分子量大小为57 k Da, 等电点为5.08.将FAE-2515成功地在毕赤酵母中实现高效异源表达, 通过对重组蛋白进行酶活测定, 比活为 (53.653±3.451) U/mg, Kcat/Km值为1.423, 最适pH为6.0, 最适温度为55℃, 60℃处理1 h之后还能保持60％的酶活, 热稳定性较已报道的同类酶更为稳定.以阿魏酸甲酯为底物时酶活表现为最高, 以硝基苯棕榈酸酯为底物时几乎没有酶活.金属离子K+、Ca2+、Na+对该酶有显著的促进作用, Fe3+、Zn2+对该酶有轻微抑制作用, Mn2+、Cu2+对该酶有显著的抑制作用.综上, 该酶在造纸工业和动物饲料制备工业中具有一定优势.“,”Ferulic acid esterase is widely used in food, pharmacy and paper-making industries, etc. It is of great importance to excavate feruloyl esterase suitably applied in industrialization. This study cloned a ferulic acid esterase gene FAE-2515 from thermophilic fingi Thermoascus crustaceus JCM12803. According to gene sequence analysis, FAE-2515 was 1 581 bp in length, code 526 amino acids and one terminator. Its theoretical molecular weight was57 k Da, isoelectric point was 5. 08. After successfully heterologous expression of Pichia pastoris of FAE-2515 and measuring enzyme in recombinant proteins, the ratio of living was ( 53. 653 ± 3. 451) U/mg, and Kcat/Kmvalue was1.423. The optimum pH was 6.0 and optimum temperature was 55℃. Enzyme activity could still maintain 60％ after60℃ treatment for 1 h. Thermal stability was more stable than that of the reported similar enzymes. The enzyme activity was the highest in the methyl feruate, and there was little enzymatic activity in the base of 4-nitrobenzene palmitate. Metallic ion K+, Ca2+, Na+had significant facilitation on this enzyme; while Fe3+, Zn2+had slight inhibition on it. Moreover, Mn2+, Cu2+had obvious inhibition effect on feruloyl esterase. In conclusion, FAE-2515 possessed certain advantages in paper-making industry and livestock feed preparation industry.