Sehgal Amitabh C, Kelly Robert M
North Carolina State University, Department of Chemical Engineering, Raleigh, North Carolina 27695-7905, USA.
Biotechnol Prog. 2003 Sep-Oct;19(5):1410-6. doi: 10.1021/bp034032c.
Homologues to Carboxylesterase NP and Candida rugosa lipase, used for the chiral separation of racemic mixtures of 2-arylpropionic methyl esters, were identified by BLAST searches of available genome sequences for hyperthermophilic microorganisms. Two potential candidates were identified: a putative lysophospholipase from Pyrococcus furiosus (Pfu-LPL) and a carboxylesterase from Sulfolobus solfataricus P1 (Sso-EST1). Although both enzymes showed hydrolytic preference toward the (S) methyl ester, only Sso-EST1 yielded highly optically pure (S) naproxen (%ee(p) >/= 90) and was thus further investigated. Changes in pH or reaction time showed little improvement in %ee(p) or E values with Sso-EST1. However, the addition of 25% methanol resulted in a 25% increase in E. The effect of various cosolvents on the enantiomeric ratio showed no correlation with the log P or dielectric constant values of the solvent. However, an inverse relationship between E and the denaturation capacity (DC) of the water miscible cosolvents was observed. This was attributed to an increase in enzyme flexibility with increasing solvent DC values leading to a concomitant reduction in the resolving power of Sso-EST1. The results here show that although bioinformatics tools can be used to select candidate biocatalysts for chiral resolution of 2-arylpropionic esters, biochemical characterization is needed to definitively determine functional characteristics.
通过对嗜热微生物现有基因组序列进行BLAST搜索，鉴定出了与羧酸酯酶NP和皱褶假丝酵母脂肪酶同源的、用于手性拆分2-芳基丙酸甲酯外消旋混合物的酶。鉴定出了两个潜在候选酶：来自激烈热球菌的一种假定溶血磷脂酶（Pfu-LPL）和来自嗜热栖热菌P1的一种羧酸酯酶（Sso-EST1）。尽管这两种酶对（S）甲酯均表现出水解偏好性，但只有Sso-EST1能产生高光学纯度的（S）萘普生（%ee(p)≥90），因此对其进行了进一步研究。pH或反应时间的变化对Sso-EST1的%ee(p)或E值几乎没有改善作用。然而，添加25%的甲醇会使E增加25%。各种助溶剂对映体比例的影响与溶剂的log P或介电常数无关。然而，观察到E与与水混溶的助溶剂的变性能力（DC）之间呈反比关系。这归因于随着溶剂DC值的增加，酶的柔韧性增加，导致Sso-EST1的拆分能力随之降低。此处结果表明，尽管生物信息学工具可用于选择用于手性拆分2-芳基丙酸酯的候选生物催化剂，但仍需要进行生化表征以明确确定其功能特性。
