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稳定和放荡的银河糖氧化酶是通过定向进化,原子设计和祖先序列重建来设计的

Merve Keser¹, Ivan Mateljak², Roman Kittl³

¹Department of Biocatalysis, Institute of Catalysis, ICP-CSIC, 28049 Madrid, Spain.

ACS synthetic biology

|December 13, 2024

概括

改造的银河糖氧化酶 (GOase) 突变体在酵母中表现出增强的热稳定性和高产量生产. 这些改进的酶显示了可持续化学应用的潜在活动.

科学领域:

生物催化和酶工程生物催化和酶工程
合成化学合成化学
可持续的化学可持续的化学

背景情况:

银河酸氧化酶 (GOase) 是一种多功能生物催化剂,在合成化学和生物电化学设备中具有应用.
之前的工程产生了M-RQW突变,具有新的C6-OH氧化活性在葡萄糖上和二次醇的杂交性.
突变M-RQW的骨干促进了更广泛的基质接受,为进一步的酶优化铺平了道路.

研究的目的:

在酵母中设计具有高产量标位的高热稳定性银河糖氧化酶 (GOase) 变体.
开发具有潜在活动的酶,适合可持续化学.
利用定向进化与计算方法相结合,以快速改善酶.

主要方法:

利用M-RQW GOase突变体作为进一步工程的起点.
整合了一次性计算变异发生 (PROSS算法) 和祖先序列重建到指导进化工作流程中.
雇佣了Pichia pastoris用于高位料批量生物反应器生产工程GOases.

主要成果:

在Pichia pastoris.中实现了工程GOas的g/L生产水平.
设计的GOases对pH值和高温具有显著的抵抗力,热稳定性 (T50) 比M-RQW母体增加到27°C.
设计的GOases对葡萄糖和各种大量的二次芳香醇表现出潜在的活性.

结论:

定向进化和计算突变发生的协同方法迅速产生了高度稳定和可生产的GOase变体.
这些工程酶具有潜在的活动,对可持续的化学合成有价值.
开发的GOases为未来的工业酶工程工作提供了坚实的基础.

关键词:

普罗斯的原子设计.祖先的序列重建的重建.指导进化是指导进化的.表达方式表达方式表达方式银河糖氧化酶是一种银河糖氧化酶.乱伦乱伦乱伦乱伦热稳定性热稳定性

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