Semi-Continuous Flow Biocatalysis with Affinity Co-Immobilized Ketoreductase and Glucose Dehydrogenase.

Michal Plž,Martin Rebroš,Tatiana Petrovičová

doi:10.3390/molecules25184278

Michal Plž, Martin Rebroš + Show 1 more

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https://doi.org/10.3390/molecules25184278

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Abstract

The co-immobilization of ketoreductase (KRED) and glucose dehydrogenase (GDH) on highly cross-linked agarose (sepharose) was studied. Immobilization of these two enzymes was performed via affinity interaction between His-tagged enzymes (six histidine residues on the N-terminus of the protein) and agarose matrix charged with nickel (Ni2+ ions). Immobilized enzymes were applied in a semicontinuous flow reactor to convert the model substrate; α-hydroxy ketone. A series of biotransformation reactions with a substrate conversion of >95% were performed. Immobilization reduced the requirement for cofactor (NADP+) and allowed the use of higher substrate concentration in comparison with free enzymes. The immobilized system was also tested on bulky ketones and a significant enhancement in comparison with free enzymes was achieved.

Highlights

Enzyme based biocatalyst plays an important role in organic synthesis and in the establishment of many chemical industries, e.g., fine chemicals, food or energy, textiles, agricultural, cosmeceutical, medicinal, and pharmaceutical industries [1].The use of ketoreductases is well recognized for enantioselective reduction of prochiral ketones into stereo defined chiral alcohols [2,3]
Ketoreductases from wild strains are usually expressed in E.coli for effective carbonyl reduction, e.g., ketoreductase from Pichia glucozyma [7], and ketoreductase from Hansenula polymorpha [8]
Kanamycin was purchased from Gibco® (Life Technologies, Glasgow, UK), ethyl-2-methylacetoacetate was purchased from Sigma-Aldrich

Summary

Introduction

The use of ketoreductases is well recognized for enantioselective reduction of prochiral ketones into stereo defined chiral alcohols [2,3]. Many wild strains of microbes can effectively reduce ketones, e.g., Saccharomyces cerevisiae, Candida magnoliae [4], Sporobolomyces salmonicolor [5], and Pichia minuta IAM 12215 [6]. Ketoreductases from wild strains are usually expressed in E.coli for effective carbonyl reduction, e.g., ketoreductase from Pichia glucozyma [7], and ketoreductase from Hansenula polymorpha [8]. In an effective carbonyl reduction, there is a need for increased productivity and biocatalyst stability in terms of repeated use and process capacity. Besides expressing ketoreductases in E. coli, enzyme immobilization can be beneficial for the reduction process allowing the repeated and continuous use.

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