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Enhancement of the Organic Solvent-Stability of the LST-03 Lipase by Directed Evolution

机译：通过定向进化增强LST-03脂肪酶的有机溶剂稳定性

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LST-03 lipase from an organic solvent-tolerant Pseudomonas aeruginosa LST-03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent-stability of LST-03 lipase was attempted by directed evolution. The structural gene of the LST-03 lipase was amplified by the error prone-PCR method. Organic solvent-stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri-n-butyrin and which overlaid a plate medium. And the organic solvent-stability was also confirmed by measuring the half-life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent-stability in the presence of DMSO. The organic solvent-stabilities of mutated LST-03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half-lives of the LST-03-R65 lipase in the presence of cyclohexane and n-decane were about 9 to 11-fold longer than those of the wild-type lipase, respectively. Some substituted amino acid residues of mutated LST-03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues.
机译：来自耐受有机溶剂的铜绿假单胞菌的LST-03脂肪酶LST-03在各种有机溶剂存在下具有很高的稳定性和活性。在这项研究中，试图通过定向进化来增强LST-03脂肪酶的有机溶剂稳定性。 LST-03脂肪酶的结构基因通过易错PCR方法扩增。突变的脂肪酶的有机溶剂稳定性通过形成琼脂的透明区域来测定，该透明区域包含二甲基亚砜（DMSO）和三正丁酸，并覆盖平板培养基。并且通过测量在DMSO存在下的活性的半衰期来确认有机溶剂的稳定性。基于在DMSO存在下的高有机溶剂稳定性，选择了四种突变酶。测量了在各种有机溶剂存在下突变的LST-03脂肪酶的有机溶剂稳定性，并鉴定了其突变的氨基酸残基。在存在环己烷和正癸烷的情况下，LST-03-R65脂肪酶的半衰期分别比野生型脂肪酶的半衰期长约9至11倍。突变的LST-03脂肪酶的一些取代的氨基酸残基已位于酶分子的表面，而其他一些氨基酸的残基已从中性变为碱性残基。

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