热稳定性伯克霍尔德菌脂肪酶A突变体的筛选

Screening for mutants with thermostabe lipase A from Burkholderia sp. ZYB002

【目的】为更好地提高伯克霍尔德菌ZYB002脂肪酶LipA在TMP纸浆造纸工艺中的应用,有必要利用蛋白质工程技术,提高其热稳定性。【方法】基于B-factor值筛选LipA多肽链中潜在的突变位点,利用迭代饱和诱变技术,构建突变文库,筛选热稳定性提高的突变体。【结果】利用上述方法,从4个突变文库中分别筛选到在55℃下,半衰期较野生型脂肪酶LipA分别提高了1.8倍、3倍、2.2倍和1.7倍的脂肪酶突变体。【结论】基于B-factor值选择突变位点,利用迭代饱和突变技术,快速筛选到热稳定性有显著提高的突变体。

[Objective]We improved the thermostability of Lip A from Burkholderia cecapia ZYB002 by protein engineering technology to expand the application of lipase Lip A.[Method] On the basis of B-factor value of lipase Lip A,series of potential mutation hotspots were selected for iterative saturation mutagenesis and the corresponding small mutation gene libraries were then constructed to screen the hyperthermal variants.[Results] From the above mutation libraries,we obtained a series of mutants whose enzyme half-life at 55℃ increased by 1.7 to 2.2-fold.[Conclusion]B-factor iterative test（B-FIT） is feasible to mutate thermostable strains.