Preparation of cross-linked cell aggregates (CLCAs) of recombinant E. coli harboring glutamate dehydrogenase and glucose dehydrogenase for efficient asymmetric synthesis of l-phosphinothricin

Abstract

The enzymatic asymmetric synthesis of L-phosphinothricin (L-PPT) from 2-carboxyl-4-(hydroxymethylphosporyl) butyric acid (PPO) is a green and efficient method. In our previous works, we constructed a glutamate dehydrogenase (GluDH) and glucose dehydrogenase (GDH) co-expression strain, Escherichia coli BL21 (DE3)/pETDuet-gludh-gdh, which can asymmetrically reduce PPO to L-PPT. To improve the stability and recyclability of the biocatalyst, we developed a novel method for whole-cell immobilization with cross-linked cell aggregates (CLCAs) and employed immobilized cells in L-PPT production. Montmorillonite was used as a carrier, and dextran polyaldehyde (DPA) was developed as a new cross-linker for cell immobilization. The highest activity recovery (79.2%) was obtained under the following conditions: 20 g/L cell concentration, 0.9% montmorillonite concentration, 1.2% polyethyleneimine (PEI) concentration, and 1.2% DPA concentration. The half-lives of CLCAs were 169.10 h (35 °C), 17.39 h (45 °C), and 10.65 h (55 °C), which were 1.87-, 3.70-, and 10.78-fold than that of free cells, respectively. The CLCAs showed good reusability, retaining over 85% of their initial activities after 10 cycles of utilization. The total yield of L-PPT reached 667 mmol/g cells, and the enantiomeric excess value was more than 99%. The immobilized whole-cell in the form of CLCAs is a valuable biocatalyst for industrial applications.