In this work, an engineered ketoreductase, apKRED-9, derived from Acetobacter pasteurianus 386B was successfully immobilized on two platforms, namely, glutaraldehyde-activated amino polymer beads, LX1000 HA, and cofactor enriched poly­(ethylenimine) (CEP) mediated coaggregation followed by glutaraldehyde cross-linking, respectively. The enzyme apKRED-9 immobilized on LX1000HA was evaluated in a packed bed reactor (PBR) for continuous-flow synthesis of (R)-tetrahydrothiophene-3-ol from 3-keto tetrahydrothiophene in an aqueous-isopropanol mixture, while the enzyme apKRED-9 immobilized on CEP was tested in batch mode until pilot scale for the same reaction. The long-term operational stability of the enzyme in both continuous-flow and batch modes was demonstrated, with high conversion of >99.0% and ee > 99.5% in both the cases. From the pilot-scale application of apKRED-9-CEP, (R)-tetrahydrothiophene-3-ol was obtained (118.0 g, GC purity 99.9%, chiral purity ee 99.9% and yield 76.3%). In the PBR flow reactor, the productivity in terms of space time yield (STY) 729 g L–1 d–1 was achieved with 64 h of continuous usage. Based on performance metrics, both platforms are scalable and reproducible, while CEP offers additional advantages on effective cost and adaptability to other enzymes.

d-Amino acids are important intermediates for the synthesis of β-lactam antibiotics and other vital pharmaceuticals, which can be synthesized by various methods including biocatalysis. In this work, we have demonstrated using immobilized multienzyme cofactor-driven cascade reaction for the synthesis of a model d-amino acid, (R)-2-amino-3-(2-bromophenyl)propanoic acid. In the present study, three enzymes, namely, d-amino acid amino transaminase from Bacillus cereus (bcDAAT), a d-lactate dehydrogenase (lhD-LDH) from Lactobacillus helveticus, and a formate dehydrogenase (cbFDH) from Candida boidinii, were successfully demonstrated in a practical and scalable immobilization protocol on glutaraldehyde-activated amino polymer beads LX1000HA. From the results, it was evident that the sequentially co-immobilized cbFDH along with the other two enzymes exhibited excellent stability at >90% for 10 cycles (150 h). Pilot-scale batches conducted at 50 g scale using the above immobilized multienzyme resulted in an overall isolated yield of 65% of (R)-2-amino-3-(2-bromophenyl)propanoic acid (∼33 g white powder; HPLC purity, >99%; ee, 99.0%). The application of the immobilized enzyme was also evaluated in PBR continuous-flow reaction, in which a stable conversion of >95% for 36 h was achieved (space–time yield, 323.3 g L–1 day–1]. d-Amino acids are vital building blocks used in pharmaceuticals and fine chemicals. Hence, we believe that immobilized multienzyme cofactor-driven cascade reaction could be a potential manufacturing platform for d-amino acids.