Formate dehydrogenase (Candida boidinii)

The pressure to optimize enzymatic rate accelerations has driven the evolution of the induced-fit mechanism for enzyme catalysts where the binding interactions of nonreacting phosphodianion or adenosyl substrate pieces drive enzyme conformational changes to form protein substrate cages that are activated for catalysis. We report the results of experiments to test the hypothesis that utilization of the binding energy of the adenosine 5′-diphosphate ribose (ADP-ribose) fragment of the NAD cofactor to drive a protein conformational change activates Candida boidinii formate dehydrogenase (CbFDH) for catalysis of hydride transfer from formate to NAD⁺. The ADP-ribose fragment provides a >14 kcal/mol stabilization of the transition state for CbFDH-catalyzed hydride transfer from formate to NAD⁺. This is larger than the ca. 6 kcal/mol stabilization of the ground-state Michaelis complex between CbFDH and NAD⁺ (K_NAD = 0.032 mM). The ADP, AMP, and ribose 5′-phosphate fragments of NAD⁺ activate CbFDH for catalysis of hydride transfer from formate to nicotinamide riboside (NR). At a 1.0 M standard state, these activators stabilize the hydride transfer transition states by ≈5.5 (ADP), 5.5 (AMP), and 4.4 (ribose 5′-phosphate) kcal/mol. We propose that activation by these cofactor fragments is partly or entirely due to the ion-pair interaction between the guanidino side chain cation of R174 and the activator phosphate anion. This substitutes for the interaction between the α-adenosyl pyrophosphate anion of the whole NAD⁺ cofactor that holds CbFDH in the catalytically active closed conformation.

This study optimized the simultaneous saccharification and citric acid (CA) production from banana pseudostem (BP). Thereafter, kinetic assessment of Aspergillus brasiliensis growth and CA production were determined for the optimum conditions using fresh water (SSF_optimizedFW) or dairy wastewater (SSF_DWW) and compared to Sabouraud Dextrose Emmon’s medium modified with BP (SSF_SDEmodified). The optimized conditions gave a CA concentration of 14.408 g/L. Kinetic assessment revealed the same maximum specific growth rates (μ_max) (0.05 h⁻¹) for all three bioprocesses, while the SSF_SDEmodified process resulted in the highest maximum potential CA concentration (P_m) (13.991 g/L) in comparison to the SSF_DWW (P_m = 13.095 g/L) and SSF_optimizedFW (P_m = 12.967 g/L) systems. Findings from this study facilitates the implementation of waste-based lignocellulosic bioprocesses that may eradicate the use of expensive pretreatment chemicals, fermentation medium constituents, and resources, in keeping with the water, energy and food nexus towards developing a circular bioeconomy.

α-hydroxy ketones (HK) and 1,2-diols are important building blocks for fine chemical synthesis. Here, we describe the R-selective 2,3-butanediol dehydrogenase from B. clausii DSM 8716^T (BcBDH) that belongs to the metal-dependent medium chain dehydrogenases/reductases family (MDR) and catalyzes the selective asymmetric reduction of prochiral 1,2-diketones to the corresponding HK and, in some cases, the reduction of the same to the corresponding 1,2-diols. Aliphatic diketones, like 2,3-pentanedione, 2,3-hexanedione, 5-methyl-2,3-hexanedione, 3,4-hexanedione and 2,3-heptanedione are well transformed. In addition, surprisingly alkyl phenyl dicarbonyls, like 2-hydroxy-1-phenylpropan-1-one and phenylglyoxal are accepted, whereas their derivatives with two phenyl groups are not substrates. Supplementation of Mn²⁺ (1 mM) increases BcBDH's activity in biotransformations. Furthermore, the biocatalytic reduction of 5-methyl-2,3-hexanedione to mainly 5-methyl-3-hydroxy-2-hexanone with only small amounts of 5-methyl-2-hydroxy-3-hexanone within an enzyme membrane reactor is demonstrated.

Formic acid (FA) is a promising reservoir for hydrogen storage and distribution. Its dehydrogenation releases CO₂ as a by-product, which limits its practical application. A proof of concept for a bio-catalytic system that simultaneously combines the dehydrogenation of formic acid for H₂, in-situ capture of CO₂ and its re-hydrogenation to reform formic acid is demonstrated. Enzymatic reactions catalyzed by carbonic anhydrase (CA) and formate dehydrogenase (FDH) under ambient condition are applied for in-situ CO₂ capture and re-hydrogenation, respectively, to develop a sustainable system. Continuous production of FA from stripped CO₂ was achieved at a rate of 40% using FDH combined with sustainable co-factor regeneration achieved by electrochemistry. In this study, the complete cycle of FA dehydrogenation, CO₂ capture, and re-hydrogenation of CO₂ to FA has been demonstrated in a single system. The proposed bio-catalytic system has the potential to reduce emissions of CO₂ during H₂ production from FA by effectively using it to recycle FA for continuous energy supply.

The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716^T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33-43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R, R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12 U/mg and 23 U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115 U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (V_max 74 U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD⁺ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development.