通过在钙结合位点突变 T118A 提高家族 I.3 脂肪酶 LipSR1 的酶热稳定性。
Enhanced enzyme thermostability of a family I.3 lipase LipSR1 by T118A mutation at the calcium-binding site.
机构信息
School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, China.
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
出版信息
Biotechnol Lett. 2023 Sep;45(9):1199-1207. doi: 10.1007/s10529-023-03413-8. Epub 2023 Jul 13.
OBJECTIVES
The lipase gene lipSR1 isolated from oil-contaminated soil exhibits high hydrolytic activity for short-chain fatty acid substrates. A single calcium ion is required to anchor the lid of LipSR1 in an open conformation by coordination with two aspartate residues and three other residues in the lid. The lid of LipSR1 is anchored by Ca, which is coordinated by side-chain carboxyl oxygens of Asp153 and Asp157, carbonyl oxygens of Thr118 and Ser144, and the side chain of Gln120.
RESULTS
D157A, D153R, Q120A, S144A, and T118A mutants were produced by site-directed mutagenesis in this study. Analyses of hydrolytic activity and thermostability showed that the properties of D157A, D153R, Q120A, and S144A were almost lost, suggesting that Asp157, Asp153, Gln120, and Ser144 are important residues for LipSR1. However, the catalytic performance of T118A was clearly maintained. Moreover, the thermostability of mutant T118A was higher than that of wild-type LipSR1.
CONCLUSIONS
These results indicated that mutation of threonine at position 118 improved the stability of the enzyme at high temperature.
目的
从受油污染的土壤中分离出的脂肪酶基因 lipSR1 对短链脂肪酸底物表现出很高的水解活性。一个钙离子通过与 lid 中的两个天冬氨酸残基和其他三个残基配位来锚定 LipSR1 的 lid 处于开放构象。LipSR1 的 lid 由 Ca 锚定，由 Asp153 和 Asp157 的侧链羧基氧、 Thr118 和 Ser144 的羰基氧以及 Gln120 的侧链配位。
结果
本研究通过定点突变产生了 D157A、D153R、Q120A、S144A 和 T118A 突变体。水解活性和热稳定性分析表明，D157A、D153R、Q120A 和 S144A 的特性几乎丧失，表明 Asp157、Asp153、Gln120 和 Ser144 是 LipSR1 的重要残基。然而，T118A 的催化性能明显得到保持。此外，突变体 T118A 的热稳定性高于野生型 LipSR1。
结论
这些结果表明，第 118 位苏氨酸的突变提高了酶在高温下的稳定性。
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