理性设计二硫键提高华根霉脂肪酶热稳定性

Rationale design of disulfide bond in Rhizopus chinensis lipase to improve thermostability

摘要:

【背景】华根霉脂肪酶的工业应用前景广泛，但是受到酶热稳定性较差的限制。【目的】对华根霉Rhizopus chinensis CCTCC M201021脂肪酶r27RCL分子结构进行理性设计，以提高该酶热稳定性。【方法】以Disulfide by design软件筛选r27RCL分子表面能够形成二硫键的突变位点，共得到7对二硫键突变。利用全质粒PCR进行定点突变，并在毕赤酵母中表达获得突变酶。【结果】最佳突变酶m9/10 (S85C-Q145C)与野生型酶r27RCL相比，60 °C下的半衰期分别提高了4.5倍，Tm值提高了4.2 °C，而催化活性保持不变。蛋白质晶体结构模拟显示，位于β2折叠上的85C和位于α4螺旋上的145C可形成二硫键，从而提高酶的热稳定性。【结论】酶分子中引入新增二硫键可以显著提升酶的热稳定性。

关键词:华根霉脂肪酶，热稳定性，二硫键，理性设计

Abstract:

[Background] Poor thermostability of Rhizopus chinensis lipase limits its potential in industrial applications. [Objective] Rationale molecular design was used to improve the thermostability of r27RCL (from Rhizopus chinensis lipase CCTCC M201021). [Methods] Seven eligible cysteine pair mutations were selected according to the prediction of the Disulfide by design algorithm. Mutant gene was produced by whole-plasmid PCR and then expressed in Pichia pastoris to obtain mutant lipases. [Results] The optimal mutant enzyme m9/10 (S85C-Q145C) showed improved thermostability with a 4.5-fold increase in t1/2 at 60 °C and a 4.2 °C increase of Tm compared with the parent enzyme, without compromising the catalytic efficiency. A disulfide bond between 85 cysteine residue (located in 2nd β-sheet) and 145 cysteine residue (located in 4th α-helix) was introduced into the lipase according to the cystal structure, leading to the improved thermostability of lipase. [Conclusion] The thermostability of fungal lipase could be greatly improved by newly introduced disulfide bond.

Key words:Rhizopus chinensis lipase, thermostability, disulfide bond, rational prediction