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From enzyme dispersion to purification: Optimizing biocatalytic membranes for high-purity ginsenoside Rd production

Wenqing Zhang,Lu Yin,Qiuyu Han,Jinhui Xu,Yanyu Xiang,Ziyang Zhang,Liping Fan,Pingyuan Ge,Yufan Shang,Xinyuan Wang,Yue Zhang,Zhishu Tang,Mianhua Wu,Bo Li,Huaxu Zhu

This study presents a novel biocatalytic membrane (BM) system for the continuous production of high-purity ginsenoside Rd. Recognizing that enzymes concentrated on one side of BM caused by reverse filtration hinder substrate access and catalytic efficiency, polyethyleneimine (PEI) and polyallylamine hydrochloride (PAH) were added during dopamine (DA) deposition. They effectively disrupted the clustering of enzyme molecules, enabling a more homogeneous enzyme distribution within BMs, as confirmed by microscopy, activity assays, and simulations. This controlled dispersion, facilitated by Protein-Polyelectrolyte Complexes (PPCs) formation, significantly improved substrate accessibility and catalytic performance. Exceptional catalytic performance was exhibited by the optimized PDA/PEI BM (using 1800 Da PEI), enabling highly efficient conversion of Rb1 to Rd. This high conversion efficiency, coupled with the inherent temperature-dependent solubility of Rd, enables a remarkably simple purification process. By exploiting Rd precipitation upon cooling, high-purity (93.1 %) Rd was achieved at a 55.6 % recovery from a 70 % purity Rb1 feed using a simple solid–liquid separation, eliminating the need for other separation techniques. This study provides new insights into the preparation of BMs and offers innovative strategies for the transformation and purification of ginsenosides.