半理性改造提升细菌漆酶Lac15的催化活性

Semi-rational engineering of microbial laccase Lac15 for enhanced activity

摘要:

【目的】漆酶可氧化各种底物,在多个工业领域有很好的潜在应用价值。Lac15是一种微生物漆酶,已表现出可观的应用潜能,可望通过蛋白质工程改造提升和拓展其应用。【方法】通过基于结构分析的半理性改造策略,选取推测与电子/质子转移或底物结合直接或间接相关的位点进行定点突变,并测定突变酶对各种底物的活性及酶学性质。【结果】部分突变体对某些底物的活性显著提升,其中D216N对ABTS的催化效率提高了1.0倍,R178V和K433T对多巴胺的催化效率分别增加了1.2和11.1倍。同时,这些突变体保留了野生型酶在应用中的优点如中性最适pH和盐促活等性质。【结论】利用基于结构的半理性改造方法,可改善漆酶Lac15对某些底物的活性。漆酶中的一个保守Asp,对应于Lac15中的D216,可作为提升以ABTS为底物或介体时漆酶活性的一个共同改造目标。

关键词:细菌漆酶;Lac15;半理性改造;活性;特异性

Abstract:

[Objective] Laccases can oxidize a wide range of compounds and have great application value in diverse industries.Lac15,a microbial laccase with considerable application potential,can be modified by protein engineering to expand the application range.[Methods] By structure-based analysis,we chose some residues supposed to contribute directly or indirectly to electron/proton transfer or substrate interaction for site-directed mutagenesis,and measured the activities of the mutants for various substrates and the enzymatic properties.[Results] Some of the mutants showed significantly enhanced activity towards particular substrates.To be specific,the catalysis efficiency of mutant D216N toward 2,2'-azino-bis (3-ethylbenzthiazoline-6-suphonic acid)(ABTS) was 1.0 fold higher,and that of mutants R178V and K433T toward dopamine was 1.2 and 11.1 folds,respectively,higher than that of Lac15.Moreover,the mutants maintained the advantages of Lac15 in application,such as the neutral optimum pH and salt activation.[Conclusion] By structure-based semi-rational engineering,the specific activities of Lac15 toward some substrates can be improved.A conserved Asp in laccases,corresponding to D216 in Lac15,can be a common engineering target for enhancing the activity of the enzymes with ABTS as substrate or mediator.

Key words:microbial laccase;Lac15;semi-rational engineering;activity;specificity