Optimization of Metal Affinity Ketoreductase Immobilization for Application in Batch and Flow Processes

Protein isolation from fermentation broth or protein mixtures by using cost-effective, scalable, regenerable resins offers a significant increase in value and productivity; hence, many recombinant proteins contain a fused histidine tag to facilitate immobilized metal affinity chromatography purification. A newly developed methacrylic iminodiacetic acid-functionalized resin, capable of attaining metal ion loadings of 0.25 mmol metal/g and high affinity toward His-tagged protein, was studied in the immobilization of a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent His-tagged ketoreductase and in the coimmobilization of the ketoreductase and glucose dehydrogenase. The affinity immobilization was compared to various other resin-enzyme interactions, that is, covalent, ionic, and hydrophobic, showing that the His-tag immobilization provides up to 4 times increase in specific activity due to the high immobilization selectivity and suitable enzyme orientation. Biocatalyst recycling in batch showed excellent stability over 10 cycles in aqueous and 5 cycles in organic media. NADPH cofactor recycling was studied in batch and continuous flow using cosubstrate 2-propanol as well as separately immobilized and coimmobilized ketoreductase and glucose dehydrogenase, in the presence of glucose. The best biotransformation with cofactor regeneration was in the presence of the coimmobilized ketoreductase and glucose dehydrogenase, while the lowest conversion was obtained by using the two enzymes separately immobilized. These studies showed that, by using cofactor regeneration systems, an NADPH concentration 1000 times lower than the substrate concentration can be used while maintaining the enzyme productivity.