Engineering Promiscuous Alcohol Dehydrogenase Activity of a Reductive Aminase AspRedAm for Selective Reduction of Biobased Furans.

Catalytic promiscuity is a promising starting point for improving the existing enzymes and even creating novel enzymes. In this work, site-directed mutagenesis was performed to improve promiscuous alcohol dehydrogenase activity of reductive aminase from Aspergillus oryzae (AspRedAm). AspRedAm showed the cofactor preference toward NADPH in reductive aminations, while it favored NADH in the reduction reactions. Some key amino acid residues such as N93, I118, M119, and D169 were identified for mutagenesis by molecular docking. Variant N93A showed the optimal pH and temperature of 8 and 30°C, respectively, in the reduction of 5-hydroxymethylfurfural (HMF). The thermostability was enhanced upon mutation of N93 to alanine. The catalytic efficiency of variant N93A (k cat/K m, 23.6 mM-1 s-1) was approximately 2-fold higher compared to that of the wild-type (WT) enzyme (13.1 mM-1 s-1). The improved catalytic efficiency of this variant may be attributed to the reduced steric hindrance that stems from the smaller side chain of alanine in the substrate-binding pocket. Both the WT enzyme and variant N93A had broad substrate specificity. Escherichia coli (E. coli) cells harboring plain vector enabled selective reduction of biobased furans to target alcohols, with the conversions of 35-95% and the selectivities of >93%. The introduction of variant N93A to E. coli resulted in improved substrate conversions (>98%) and selectivities (>99%).

Bioinspired Cooperative Photobiocatalytic Regeneration of Oxidized Nicotinamide Cofactors for Catalytic Oxidations.

Invited for this month's cover is the group of Ning Li at South China University of Technology. The image shows an efficient photobiocatalytic system to regenerate oxidized nicotinamide cofactors for dehydrogenase-mediated oxidations. The Communication itself is available at 10.1002/cssc.202100184.

Bioinspired Cooperative Photobiocatalytic Regeneration of Oxidized Nicotinamide Cofactors for Catalytic Oxidations.

Inspired by water-forming NAD(P)H oxidases, a cooperative photobiocatalytic system has been designed to aerobically regenerate the oxidized nicotinamide cofactors. Photocatalysts enable NAD(P)H oxidation with O2 under visible-light irradiation, producing H2 O2 as a byproduct, which is subsequently used as an oxidant by the horseradish peroxidase mediator system (PMS) to oxidize NAD(P)H. The photobiocatalytic system shows a turnover frequency of 8800 min-1 in the oxidation of NAD(P)H. Photobiocatalytic NAD(P)H oxidation proceeds smoothly at pH 6-9. In addition to natural NAD(P)H, synthetic biomimetics are also good substrates for this regeneration system. Total turnover numbers of up to 180000 are obtained for the cofactor when the photobiocatalytic regeneration system is coupled with dehydrogenase-catalyzed oxidations. It may be a promising protocol to recycle the oxidized cofactors for catalytic oxidations.

One-Pot Enzyme Cascade for Controlled Synthesis of Furancarboxylic Acids from 5-Hydroxymethylfurfural by H2 O2 Internal Recycling.

Furancarboxylic acids are promising biobased building blocks in pharmaceutical and polymer industries. In this work, dual-enzyme cascade systems composed of galactose oxidase (GOase) and alcohol dehydrogenases (ADHs) are constructed for controlled synthesis of 5-formyl-2-furancarboxylic acid (FFCA) and 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF), based on the catalytic promiscuity of ADHs. The byproduct H2 O2 , which is produced in GOase-catalyzed oxidation of HMF to 2,5-diformylfuran (DFF), is used for horseradish peroxidase (HRP)-mediated regeneration of the oxidized nicotinamide cofactors for subsequent oxidation of DFF promoted by an ADH, thus implementing H2 O2 internal recycling. The desired products FFCA and FDCA are obtained with yields of more than 95 %.

Dehydrogenase-Catalyzed Oxidation of Furanics: Exploitation of Hemoglobin Catalytic Promiscuity.

The catalytic promiscuity of hemoglobin (Hb) was explored for regenerating oxidized nicotinamide cofactors [NAD(P)+ ]. With H2 O2 as oxidant, Hb efficiently oxidized NAD(P)H into NAD(P)+ within 30 min. The new NAD(P)+ regeneration system was coupled with horse liver alcohol dehydrogenase (HLADH) for the oxidation of bio-based furanics such as furfural and 5-hydroxymethylfurfural (HMF). The target acids (e.g., 2,5-furandicarboxylic acid, FDCA) were prepared with moderate-to-good yields. The enzymatic regeneration method was applied in l-glutamic dehydrogenase (DH)-mediated oxidative deamination of lglutamate and for l-lactic-DH-mediated oxidation of l-lactate, which furnished α-ketoglutarate and pyruvate in yields of 97 % and 81 %, respectively. A total turnover number (TTON) of up to approximately 5000 for cofactor and an E factor of less than 110 were obtained in the bi-enzymatic cascade synthesis of α-ketoglutarate. Overall, a proof-of-concept based on catalytic promiscuity of Hb was provided for in situ regeneration of NAD(P)+ in DH-catalyzed oxidation reactions.