Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola
Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola
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DOI：
10.1007/s00253-010-2533-3
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发表时间：
2010-07-01
影响因子：
5
通讯作者：
Hauer, Bernhard
中科院分区：
文献类型：
--
作者：
Koschorreck, Katja;Liu, Danni;Hauer, Bernhard
角质酶 CUTAB1 是从芸苔链格孢的角质诱导培养物中克隆出来的，并在甲醇诱导型 AOX1 启动子的控制下在毕赤酵母中异源表达。从400ml培养物中获得36mg纯化的重组酶。生化表征表明，使用棕榈酸对硝基苯酯 (p-NPP) 作为底物，该酶在 40A 摄氏度和 pH 7-9 条件下具有最高催化活性。在测试的几种脂肪酸甲酯和乙酯、甘油酯和对硝基苯酯中，CUTAB1 对三丁酸甘油酯表现出最高的活性（3,302 A +/- 160 U mg(-1)），并且该活性随着所研究的酯链长度的增加而降低。 p-NPP 的活性最低。用 CUTAB1 疏水结合环中较小的丙氨酸分别替换 Leu80、Leu181 和 Ile183，大大降低了酶的整体活性。另一方面，位于 CUTAB1 小螺旋瓣中的突变 A84F 显着增加了酶对长链底物（如 p-NPP）的活性。
The cutinase CUTAB1 was cloned from a cutin induced culture of Alternaria brassicicola and heterologously expressed in Pichia pastoris under the control of the methanol-inducible AOX1 promoter. From a 400-ml culture, 36 mg of purified recombinant enzyme were obtained. Biochemical characterization revealed highest catalytic activity of the enzyme at 40A degrees C and pH 7-9 using p-nitrophenyl palmitate (p-NPP) as substrate. Among several fatty acid methyl and ethyl esters, glycerol esters and p-nitrophenyl esters tested, CUTAB1 showed highest activity towards tributyrin (3,302 A +/- 160 U mg(-1)) and the activity decreased with increase in chain length of the investigated esters. Lowest activity was found for p-NPP. Replacing Leu80, Leu181 and Ile183, respectively, by the smaller alanine in the hydrophobic binding loop of CUTAB1, drastically reduced the overall activity of the enzyme. On the other hand, mutation A84F located in the small helical flap of CUTAB1 significantly increased the activity of the enzyme towards longer chain substrates like p-NPP.