Enhanced cofactor recycling and accelerated reaction rate via liquid-liquid phase separation in dual-enzyme condensates  

液-液相分离介导的双酶凝聚体增强辅因子循环效率与反应速率

作　　者：Jiaxu Liu Jiaxin Chen Xiaoyan Zhang Daidi Fan Yunpeng Bai 刘佳旭;陈佳鑫;张晓彦;范代娣;白云鹏(华东理工大学,生物反应器工程国家重点实验室,上海生物制造产业省部共建协同创新中心,上海200237;西北大学化工学院,陕西省生物材料与发酵工程技术研究中心,陕西西安710069)

机构地区：[1]State Key Laboratory of Bioreactor Engineering,Shanghai Collaborative Innovation Center for Biomanufacturing,East China University of Science and Technology,Shanghai 200237,China [2]Shaanxi R&D Centre of Biomaterials and Fermentation Engineering,School of Chemical Engineering,Northwest University,Xi’an 710069,Shaanxi,China

出　　处：《Chinese Journal of Catalysis》2025年第2期135-148,共14页催化学报(英文)

基　　金：国家重点研发计划(2023YFA0913600);国家自然科学基金(22378120,22078096)。

摘　　要：Enzyme catalysis is a promising way to produce chiral products in a green and sustainable way.However,the high cost of cofactors and their relatively low recycling efficiency hinder the widespread application of enzyme catalysis in industry.In contrast,cofactor regeneration and recycling in cells is highly efficient,mainly due to physical effects caused by the ordered spatial organization of enzymes in vivo.The construction of similar catalytic systems with high cofactor recycling in vitro remains challenging.Here,we present a facile method to generate dual enzyme condensates in vitro based on intrinsically disordered region-mediated liquid-liquid phase separation.Typically,a carbonyl reductase from Serratia marcescens(SmCR_(V4))and a glucose dehydrogenase from Bacillus megaterium(BmGDH)were co-localized in the condensates.This resulted in an up to 20-fold increase in cofactor recycling efficiency(substrate converted per cofactor per unit time),and a 3.4-fold increase in space-time yield compared to the free enzyme system.The reaction enhancement was shown to be highly correlated with the degree of condensation of the dual enzymes.Fluorescence confocal microscopy showed that the cofactor,nicotinamide adenine dinucleotide phosphate(NADPH),was enriched between neighboring enzymes during the reaction due to the proximity effect,facilitating its regeneration and recycling within the condensate.In a scaled-up synthesis,the consumption of NADPH was reduced 50-fold compared to industrial biocatalytic standards,while the condensate still maintained efficient product synthesis.Concentrating multiple enzymes in a nano-and micro-condensate to increase the reaction rate may provide a general and inexpensive method for improving cofactor-involved enzymatic reactions.酶催化作为一种绿色的生物制造方法,凭借其高选择性和在温和反应条件下工作的特性,成为了合成手性化学品的重要且优选的方法.近年来,酶催化技术取得了显著进展,但是许多反应需要昂贵的辅因子,较低的辅因子循环效率导致反应成本高、速率低,在工业催化中的应用受到限制.细胞中辅因子的再生和循环过程非常高效,这是因为细胞内部具有高度有序的微纳结构和酶的空间组织.然而,在体外构建具有类似辅因子高效循环效率的催化系统仍然是一个巨大的挑战。本文报道了一种基于蛋白质内在无序区域(IDRs)介导的液-液相分离(LLPS)制备双酶凝聚体的策略.IDRs分别与负责催化反应的羰基还原酶(SmCR_(v4))和负责生成辅因子的葡萄糖脱氢酶(BmGDH)融合表达,获得既具有催化功能又可以自发发生液-液相分离的融合蛋白.对四种不同来源但均能在体外发生LLPS的IDRs进行了筛选,得到酶活力最高的组蛋白H3K27去甲基化酶X-连锁基因突变蛋白融合酶.共聚焦显微镜表征结果表明,融合蛋白形成液液相分离的凝聚体,且蛋白质浓度越高,相分离程度越大凝聚体对pH的耐受性高,在pH=5.0-9.0均可发生LLPS.荧光漂白实验证明凝聚体内部流动性良好,分子扩散不受限制.共聚焦图像分析结果证明两种融合蛋白共定位在同一个凝聚体内.辅因子使用荧光标签标记后,共聚焦显微镜下观察到辅因子在双酶凝聚体中富集,表明邻近效应促进了辅因子在凝聚体内部和周围再生,从而提升辅因子循环的效率.催化4-氧代癸酸甲酯不对称还原的实验结果表明,双酶凝聚体体系中辅因子循环效率是游离酶体系的20倍,时空产率是3.4倍。在辅因子浓度低至1μmol/L时,双酶凝聚体可以在50min内催化16μumol底物完全转化.双酶凝聚体展现出了良好的稳定性,在连续反应12h后,凝聚体的形貌并未改变.在放大合成实验中,与

关 键 词：Cofactor recycling CONDENSATE Proximity effect Intrinsically disordered region Enzyme catalysis 

分 类 号：O64[理学—物理化学] O643.32[理学—化学]