Department of Microbiology, University of Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain,
Appl Microbiol Biotechnol. 2014 May;98(10):4479-90. doi: 10.1007/s00253-013-5458-9. Epub 2014 Jan 10.
Lipases and esterases are important biocatalysts for synthetic organic fine chemistry. An esterase from Bacillus sp. BP-7 (EstBP7) bears in its amino acid sequence a rare GGG(A)X oxyanion hole motif, where an uncommon threonine (T) is found at the third position. Detection of this pattern motivated evaluation of the ability of EstBP7 for conversion of tertiary alcohols. The enzyme was engineered in order to optimize its performance to provide important chiral building blocks: five variants with mutations in the oxyanion hole motif were created to investigate the influence on activity and enantioselectivity in the kinetic resolution of eight acetates of tertiary alcohols. Wild-type enzyme converted all esters of tertiary alcohols assayed with low enantioselectivity, whereas some of the mutants displayed significantly increased E-values. One of the mutants (EstBP7-AGA; Mut 5) showed an E >100 towards a complex tertiary alcohol acetate (2-(4-pyridyl)but-3-yn-2-yl acetate) at low reaction temperature (4 °C). Therefore, the catalytic toolbox was expanded for biocatalysis of optically pure tertiary alcohols valuable for the pharmaceutical industry.
脂肪酶和酯酶是合成有机精细化学的重要生物催化剂。芽孢杆菌 BP-7（EstBP7）的酯酶在其氨基酸序列中具有一个罕见的GGG(A)X 氧阴离子穴模体，其中第三个位置发现了一种不常见的苏氨酸（T）。检测到这种模式促使我们评估 EstBP7 转化叔醇的能力。为了优化其性能以提供重要的手性构建块，对该酶进行了工程改造：创建了五个突变体，用于研究氧阴离子穴模体中的突变对动力学拆分八种叔醇的乙酸酯的活性和对映选择性的影响。野生型酶对所有测定的叔醇酯的转化率都很低，而有些突变体的对映选择性明显提高。其中一个突变体（EstBP7-AGA；Mut5）在低反应温度（4°C）下对复杂的叔醇乙酸酯（2-（4-吡啶基）丁-3-炔-2-基乙酸酯）表现出 E > 100，因此，为制药工业有价值的光学纯叔醇的生物催化扩展了催化工具箱。
