文献类型：期刊文献

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

作者：Liu, Jiaxu[1];Chen, Jiaxin[1];Zhang, Xiaoyan[1];Fan, Daidi[2];Bai, Yunpeng[1,2]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Northwest Univ, Shaanxi R&D Ctr Biomat & Fermentat Engn, Sch Chem Engn, Xian 710069, Shaanxi, Peoples R China

年份：2025

卷号：69

起止页码：135

外文期刊名：CHINESE JOURNAL OF CATALYSIS

收录：;EI(收录号：20251017984121);WOS:【SCI-EXPANDED(收录号:WOS:001440626300001)】；

基金：This work was supported by the National Key R&D Program of China (2023YFA0913600) , and the National Natural Science Foundation of China (22378120, 22078096) .

语种：英文

外文关键词：Cofactor recycling; Condensate; Proximity effect; Intrinsically disordered region; Enzyme catalysis

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 (SmCRV4) 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. Published by Elsevier B.V. All rights reserved.