One-step purification of transglutaminase with high purity using a thermoseparating ethylene oxide/propylene oxide copolymer.

Due to the ability to catalyze the formation of covalent cross-links between proteins, microbial transglutaminase (MTGase) has been widely applied in the fields of food industry, biomedicine and biotechnology. However, the industrial-scale purification of MTGase is often limited by the complexity and low efficiency of traditional chromatographic techniques. Herein, we introduce an economical and handy method utilizing a recyclable thermoseparating ethylene oxide-propylene oxide (EOPO) random copolymer (EO20PO80) for the direct and efficient precipitation of MTGase from fermentation broth. Through systematic optimization of copolymer concentration, pH, ionic strength and subsequent wash step with ethanol, we obtained MTGase powder after freeze drying with high product yield and enzyme activity. The purity of was detected through SDS-PAGE and the specific activity increased by 63 times compared with the of MTGase in fermentation broth. The MTGase purified by this process had an enzyme activity up to 46411.88 U/g with the enzyme activity recovery rate reaching up to 99% while over 98% of precipitant EO20PO80 can be recovered through only temperature adjustment. The molecular dynamic simulation was adopted to visualize the process of MTGase precipitation and interaction between MTGase and EOPO. The work establishes a simple, scalable, and efficient purification strategy for MTGase, highlighting the potential of smart copolymers as versatile tools for downstream processing in industrial enzyme production.