Purification and Characterization of (2R,3R)-2,3-Butanediol Dehydrogenase of the Human Pathogen Neisseria gonorrhoeae FA1090 Produced in Escherichia coli.

2,3-Butanediol dehydrogenase (BDH), also known as acetoin/diacetyl reductase, is a pivotal enzyme for the formation of 2,3-butanediol (2,3-BD), a chiral compound with potential roles in the virulence of certain pathogens. Here, a NAD(H)-dependent (2R,3R)-BDH from Neisseria gonorrhoeae FA1090 (NgBDH), the causative agent of gonorrhoea, was functionally characterized. Sequence analysis indicated that it belongs to zinc-containing medium-chain dehydrogenase/reductase family. The recombinant NgBDH migrated as a single band with a size of around 45 kDa on SDS-PAGE and could be confirmed by Western blotting and mass spectrometry. For the oxidation of either (2R,3R)-2,3-BD or meso-2,3-BD, the enzyme exhibited a broad pH optimum between pH 9.5 to 11.5. For the reduction of (3R/3S)-acetoin, the pH optimum was around 6.5. The enzyme could catalyze the stereospecific oxidation of (2R,3R)-2,3-BD (Km = 0.16 mM, kcat/Km = 673 s-1 · mM-1) and meso-BD (Km = 0.72 mM, kcat/Km = 165 s-1 · mM-1). Moreover, it could also reduce (3R/3S)-acetoin with a Km of 0.14 mM and a kcat/Km of 885 s-1 · mM-1. The results presented here contribute to understand the 2,3-BD metabolism in N. gonorrhoeae and pave the way for studying the influence of 2,3-BD metabolism on the virulence of this pathogen in the future.

Alcohol Oxidase from the Methylotrophic Yeast Ogataea polymorpha: Isolation, Purification, and Bioanalytical Application.

Alcohol oxidase (EC 1.1.3.13; AOX) is a flavoprotein that catalyzes the oxidation of primary short-chain alcohols to corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. It is a key enzyme of methanol metabolism in methylotrophic yeasts, catalyzing the first step of methanol oxidation to formaldehyde.Here we describe the isolation and purification of AOX from the thermotolerant methylotrophic yeast Ogataea (Hansenula) polymorpha, and using this enzyme in enzymatic assay of ethanol, simultaneous analysis of methanol and formaldehyde, and in construction of amperometric biosensors selective to primary alcohols and formaldehyde.

Crystal structure of acetoacetyl-CoA reductase from Rickettsia felis.

Rickettsia felis, a Gram-negative bacterium that causes spotted fever, is of increasing interest as an emerging human pathogen. R. felis and several other Rickettsia strains are classed as National Institute of Allergy and Infectious Diseases priority pathogens. In recent years, R. felis has been shown to be adaptable to a wide range of hosts, and many fevers of unknown origin are now being attributed to this infectious agent. Here, the structure of acetoacetyl-CoA reductase from R. felis is reported at a resolution of 2.0 Å. While R. felis acetoacetyl-CoA reductase shares less than 50% sequence identity with its closest homologs, it adopts a fold common to other short-chain dehydrogenase/reductase (SDR) family members, such as the fatty-acid synthesis II enzyme FabG from the prominent pathogens Staphylococcus aureus and Bacillus anthracis. Continued characterization of the Rickettsia proteome may prove to be an effective means of finding new avenues of treatment through comparative structural studies.

Identification and characterization of a novel 2R, 3R-Butanediol dehydrogenase from Bacillus sp. DL01

BACKGROUND: 2R,3R-butanediol dehydrogenase (R-BDH) and other BDHs contribute to metabolism of 3R/3S-Acetoin (3R/3S-AC) and 2,3-butanediol (2,3-BD), which are important bulk chemicals used in different industries. R-BDH is responsible for oxidizing the hydroxyl group at their (R) configuration. Bacillus species is a promising producer of 3R/3S-AC and 2,3-BD. In this study, R-bdh gene encoding R-BDH from Bacillus sp. DL01 was isolated, expressed and identified. RESULTS: R-BDH exerted reducing activities towards Diacetyl (DA) and 3R/3S-AC using NADH, and oxidizing activities towards 2R,3R-BD and Meso-BD using NAD+ , while no activity was detected with 2S,3S-BD. The R-BDH showed its activity at a wide range of temperature (25 C to 65 C) and pH (5.0­8.0). The R-BDH activity was increased significantly by Cd2+ when DA, 3R/3S-AC, and Meso-BD were used as substrates, while Fe2+ enhanced the activity remarkably at 2R,3R-BD oxidation. Kinetic parameters of the R-BDH from Bacillus sp. DL01 showed the lowest Km, the highest Vmax, and the highest Kcat towards the racemic 3R/3S-AC substrate, also displayed low Km towards 2R,3R-BD and Meso-BD when compared with other reported R-BDHs. CONCLUSIONS: The R-BDH from Bacillus sp. DL01 was characterized as a novel R-BDH with high enantioselectivity for R-configuration. It considered NAD+ and Zn2+ dependant enzyme, with a significant affinity towards 3R/3S-AC, 2R,3R-BD, and Meso-BD substrates. Thus, R-BDH is providing an approach to regulate the production of 3R/3S-AC or 2,3-BD from Bacillus sp. DL01.

Kinetic and Bioinformatic Characterization of d-2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1.

d-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (PaD2HGDH) catalyzes the oxidation of d-2-hydroxyglutarate to 2-ketoglutarate, which is a necessary step in the serine biosynthetic pathway. The dependence of P. aeruginosa on PaD2HGDH makes the enzyme a potential therapeutic target against P. aeruginosa. In this study, recombinant His-tagged PaD2HGDH was expressed and purified to high levels from gene PA0317, which was previously annotated as an FAD-binding PCMH-type domain-containing protein. The enzyme cofactor was identified as FAD with fluorescence emission after phosphodiesterase treatment and with mass spectrometry analysis. PaD2HGDH had a kcat value of 11 s-1 and a Km value of 60 µM with d-2-hydroxyglutarate at pH 7.4 and 25 °C. The enzyme was also active with d-malate but did not react with molecular oxygen. Steady-state kinetics with d-malate and phenazine methosulfate as an electron acceptor established a mechanism that was consistent with ping-pong bi-bi steady-state kinetics at pH 7.4. A comparison of the kcat/Km values with d-2-hydroxyglutarate and d-malate suggested that the C5 carboxylate of d-2-hydroxyglutarate is important for the substrate specificity of the enzyme. Other homologues of the enzyme have been previously grouped in the VAO/PMCH family of flavoproteins. PaD2HGDH shares fully conserved residues with other α-hydroxy acid oxidizing enzymes, and these conserved residues are found in the active site of the PaD2HDGH homology model. An Enzyme Function Initiative-Enzyme Similarity Tool Sequence Similarity Network analysis suggests a functional difference between PaD2HGDH and human D2HGDH, and no relationship with VAO. A phylogenetic tree analysis of PaD2HGDH, VAO, and human D2HGDH establishes genetic diversity among these enzymes.

Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1.

Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble recombinant BDH1 was converted into a soluble form by adding glycerol in LB medium. The kcat and kcat/Km values of soluble form BDH1 for (+)-borneol turned out to be about 34-fold and 45-fold higher, respectively, than those of the refolded enzyme. On the other hand, a gene knockout mutant, TCU-HL1Δbdh, was constructed to investigate the possible presence of a second copy of the bdh gene in TCU-HL1 genome. A new gene, bdh2, encoding a BDH isozyme, was identified, and the recombinant BDH2 protein was produced in a soluble form. Both bdh1 and bdh2 genes are expressed in the crude extract of wild type TCU-HL1, as shown by RT-qPCR results. Both BDH isozymes prefer to degrade (+)-borneol, rather than (-)-borneol, probably because (+)-camphor is the main form present in nature.

Purification and characterization of formaldehyde dismutases of Methylobacterium sp. FD1.

In the present study, we purified and characterized three formaldehyde dismutases (Fdms) (EC 1.2.98.1) (Fdm1, Fdm2, and Fdm3) of Methylobacterium sp. FD1. These Fdms (with His-tag) were produced in the recombinant E. coli and purified by immobilized metal affinity chromatography from the E. coli extracts. In each of the three Fdms, the enzyme-bound coenzyme was nicotinamide adenine dinucleotide (NAD(H)) and the enzyme-bound metal was zinc. The quaternary structures of these Fdms were estimated as homotetrameric. The optimal pHs and temperatures of Fdm1, Fdm2, and Fdm3 were approximately 6.5, 6.0, and 6.0, and 35°C, 25°C, and 30°C, respectively. The Km values of Fdm1, Fdm2, and Fdm3 were 621, 865, and 414 mM, respectively. These results were similar to the properties of already-known Fdms. However, each of the Fdms of FD1 had methanol:p-nitroso-N,N-dimethylaniline oxidoreductase activity that is not found in already-known Fdms.

Effects on hyphal morphology and development by the putative copper radical oxidase glx1 in Trichoderma virens suggest a novel role as a cell wall associated enzyme.

Trichoderma spp. have been characterized for their capacity to act as biological control agents against several pathogens through the activity of secondary metabolites and cell wall degrading enzymes. However, only T. reesei has been widely studied for the ability to assimilate lignocellulose substrates. Protein analysis by SDS-PAGE of culture filtrate of T. virens revealed the presence of an unknown ∼77 kDa band protein (GLX1) that showed sequence homology to glyoxal-like oxidase genes involved in lignin degradation. The analysis and biochemical characterization of the 1,119 amino acid coded protein showed the presence of five carbohydrate-binding modules (CBMs) with affinity for colloidal chitin, and a functional glyoxal oxidase catalytic domain that is involved in the production of hydrogen peroxide when methylglyoxal was used as a substrate. The silencing of the glx1 gene resulted in mutants with more than 90% expression reduction and the absence of glyoxal oxidase catalytic activity. These mutants showed delayed hyphal growth, reduced colony and conidial hydrophobicity, but showed no changes in their biocontrol ability. Most significantly, mutants exhibited a loss of growth directionality resulting in a curled phenotype that was eliminated in the presence of exogenous H2O2. Here we present evidence that in T. virens, glx1 is not involved in the breakdown of lignin but instead is responsible for normal hyphal growth and morphology and likely does this through free radical production within the fungal cell wall. This is the first time that a glyoxal oxidase protein has been isolated and characterized in ascomycete fungi.

Expression of engineered carbonyl reductase from Ogataea minuta in Rhodococcus opacus and its application to whole-cell bioconversion in anhydrous solvents.

The carbonyl reductase from the methylotrophic yeast Ogataea minuta can catalyze the regio- and enantio-selective reduction of prochiral ketones to chiral alcohols, and is available for industrial manufacturing of statin drugs. We previously conducted a directed evolution experiment of the enzyme, and obtained a mutant (OCR_V166A) with improved tolerance to organic solvents. This expanded the applicability of the enzyme to the bioconversion of water-insoluble compounds (Honda et al., J. Biosci. Bioeng., 123, 673-678, 2017). In the present study, we expressed OCR_V166A in Rhodococcus opacus cells, which have a highly lipophilic surface structure and are dispersible in anhydrous organic solvents, and developed a whole-cell biocatalyst which can function in an organic-solvent-based reaction medium. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeADH) was employed as an NADPH-regenerating enzyme and co-expressed with OCR_V166A in R. opacus. The whole-cell bioconversion of 2,2,2-trifluoroacetophenone to α-(trifluoromethyl)benzyl alcohol was performed in organic solvents, including isopropanol, isobutanol, and cyclohexanol, which served both as reaction media and as substrates for TeADH. The type of organic solvents markedly affected not only the product titer but also the enantio-purity of the product. When isobutanol was used as the reaction medium, the whole-cell biocatalyst showed higher stability than the isolated enzyme. Consequently, a high concentration (1 M) of the substrate was converted to the product with an overall conversion yield of 81% (mol/mol) in 24 h.

Cloning and coexpression of recombinant N-demethylase B and Glycolate oxidase genes in Escherichia coli.

NdmB genes from Pseudomonas putida CBB5 and GO genes from spinach, which encode N-demethylase B (NdmB) and Glycolate oxidase (GO) respectively, were separately ligated into expression vectors of pACYCDuet-1 and pET32a to construct recombinant plasmids of pACYCDuet-1-ndmBHis (pBH) and pET32a-GOHis (pGOH). Then the two plasmids were both transformed in Escherichia coli (E. coli) strain BL21 (DE3) and screening the recombinants (pBHGOH) using ampicillin and chloramphonicol as two antibiotics in Luria-Bertani medium. After induction with IPTG, both recombinant ndmB and GO genes were coexpressed in E. coli. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the estimated molecular weight of NdmB and GO was 35 kDa and 40 kDa, respectively. By two-step purification of Ni affinity chromatography and Q-Sepharose chromatography, the coexpressed NdmB and GO were separated and resulted in a 15.8-fold purification with 8.7% yield and 12.8-fold purification with 7.2% yield, respectively.

Expression, Purification, and Activity Analysis of Chlorophyllide Oxidoreductase and Ni2+-Sirohydrochlorin a,c-Diamide Reductase.

Nitrogenase-like enzymes play a vital role in the reduction of the conjugated ring systems of diverse tetrapyrrole molecules. The biosynthesis of all bacteriochlorophylls involves the two-electron reduction of the C7-C8 double bond of the green pigment chlorophyllide, which is catalyzed by the nitrogenase-like two-component metalloenzyme chlorophyllide oxidoreductase (COR); whereas in all methanogenic microbes, another nitrogenase-like system, CfbC/D, is responsible for the sophisticated six-electron reduction of Ni2+-sirohydrochlorin a,c-diamide in the course of coenzyme F430 biosynthesis. The first part of this chapter describes the production and purification of the COR components (BchY/BchZ)2 and BchX2, the measurement of COR activity, and the trapping of the ternary COR complex; and the second part describes the strategy for obtaining homogenous and catalytically active preparations of CfbC2 and CfbD2 and a suitable method for extracting the reaction product Ni2+-hexahydrosirohydrochlorin a,c-diamide.

[Identification of Heparin-binding Proteins on the Cell Surface of Cryptococcus neoformans].

Interactions between virulence factors of pathogens and host responses play an important role in the establishment of infection by microbes. We focused on interactions between Cryptococcus neoformans proteins and heparin, which is abundant on host epithelial cells. Surface proteins were extracted and analyzed. Fractions from anion-exchange column chromatography interacted with heparin in surface plasmon resonance analyses. Heparin-binding proteins were purified and then separated by gel electrophoresis; and were identified as transaldolase, glutathione-disulfide reductase, and glyoxal oxidase. These results imply that multifunctional molecules on C. neoformans cells, such as those involved in heparin binding, may play roles in adhesion that trigger responses in the host.

Impact of the lanthanide contraction on the activity of a lanthanide-dependent methanol dehydrogenase - a kinetic and DFT study.

Interest in the bioinorganic chemistry of lanthanides is growing rapidly as more and more lanthanide-dependent bacteria are being discovered. Especially the earlier lanthanides have been shown to be preferentially utilized by bacteria that need these Lewis acids as cofactors in their alcohol dehydrogenase enzymes. Here, we investigate the impact of the lanthanide ions lanthanum(iii) to lutetium(iii) (excluding Pm) on the catalytic parameters (vmax, KM, kcat/KM) of a methanol dehydrogenase (MDH) isolated from Methylacidiphilum fumariolicum SolV. Kinetic experiments and DFT calculations were used to discuss why only the earlier lanthanides (La-Gd) promote high MDH activity. Impact of Lewis acidity, coordination number preferences, stability constants and other properties that are a direct result of the lanthanide contraction are discussed in light of the two proposed mechanisms for MDH.

Purification, characterization, and stabilization of alcohol oxidase from Ogataea thermomethanolica.

Alcohol oxidase (AOX) functions in oxidation of primary alcohols into the corresponding aldehydes with potential on catalyzing synthesis reactions in chemical industry. In this study, AOX from a thermotolerant methylotrophic yeast, Ogataea thermomethanolica (OthAOX) was purified to high homogeneity using a single step chromatographic separation on a DEAE-Sepharose column. The purified OthAOX had a specific activity of 15.34 U/mg with 77.5% recovery yield. The enzyme worked optimally at 50 °C in an alkaline range (pH 9.0). According to kinetic analysis, OthAOX showed a higher affinity toward short-chain aliphatic primary alcohol with the Vmax, Km, and kcat of 0.24 nmol/min, 0.27 mM, and 3628.8 min-1, respectively against methanol. Addition of alginic acid (0.35%) showed a protective effect on enhancing thermal stability of the enzyme, resulting in 72% increase in its half-life at 40 °C under the operational conditions. This enzyme represents a promising candidate for conversion of bioethanol to acetaldehyde as secondary chemical in biorefinery.

The crystal structure of methanol dehydrogenase, a quinoprotein from the marine methylotrophic bacterium Methylophaga aminisulfidivorans MPT.

The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MPT (MDH Mas ), was determined at 1.7 Å resolution. The active form of MDH Mas (or MDHI Mas ) is a heterotetrameric α2ß2, where each ß-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two ß-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a ß-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg2+ ion, instead of the Ca2+ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the ß-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHI Mas integrity in the sea water environment to provide a firm basis for complex formation with MxaJ Mas or Cyt cL. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

Characterization of two carbonyl reductases from Ogataea polymorpha NBRC 0799.

The enzyme responsible for the enantioselective production of (S)-1,1,1-trifluoro-2-propanol ((S)-TFP) from 1,1,1-trifluoroacetone (TFA) has been identified in Ogataea polymorpha NBRC 0799. We purified two carbonyl reductases, OpCRD-A and OpCRD-B from this strain, and revealed their characteristics. Both enzymes were specific to NADH, but the following characteristics were different: The molecular mass of subunit OpCRD-A was 40 kDa and that of OpCRD-B was 43 kDa. Amino acid sequences of both enzymes were only 21% identical. OpCRD-B contained 4 mol of zinc per mole of enzyme, but OpCRD-A did not. The optimal pH, temperature, pH stability, thermostability, and inhibitor specificity were also remarkably different. With regard to substrate specificity, both enzymes exhibited high reductase activity toward a wide variety of ketones, aldehydes and fluoroketones, and dehydrogenase activity toward 2-propanol and 2-butanol. The reductase activity was much higher than the dehydrogenase activity at acidic pH. OpCRD-A enantioselectively produced (S)-TFP from TFA, but OpCRD-B preferentially produced (R)-TFP. Thus, we concluded that OpCRD-A plays the main role in the production of (S)-TFP by a reaction of O. polymorpha NBRC 0799 cells and that OpCRD-A has great potential for efficient production of (S)-TFP, as it is an S-specific enzyme and does not catalyze the dehydrogenation of (S)-TFP.

3-Hydroxyisobutyrate Dehydrogenase Is Involved in Both, Valine and Isoleucine Degradation in Arabidopsis thaliana.

In plants, amino acid catabolism is especially relevant in metabolic stress situations (e.g. limited carbohydrate availability during extended darkness). Under these conditions, amino acids are used as alternative substrates for respiration. Complete oxidation of the branched-chain amino acids (BCAAs) leucine, isoleucine (Ile), and valine (Val) in the mitochondria efficiently allows the formation of ATP by oxidative phosphorylation. However, the metabolic pathways for BCAA breakdown are largely unknown so far in plants. A systematic search for Arabidopsis (Arabidopsis thaliana) genes encoding proteins resembling enzymes involved in BCAA catabolism in animals, fungi, and bacteria as well as proteomic analyses of mitochondrial fractions from Arabidopsis allowed the identification of a putative 3-hydroxyisobutyrate dehydrogenase, AtHDH1 (At4g20930), involved in Val degradation. Systematic substrate screening analyses revealed that the protein uses 3-hydroxyisobutyrate but additionally 3-hydroxypropionate as substrates. This points to a role of the enzyme not only in Val but possibly also in Ile metabolism. At4g20930 knockdown plants were characterized to test this conclusion. Root toxicity assays revealed increased root growth inhibition of the mutants if cultivated in the presence of Val or Ile but not in the presence of leucine. We conclude that AtHDH1 has a dual role in BCAA metabolism in plants.

Design and synthesis of affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase.

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.

Development of a simplified purification method for a novel formaldehyde dismutase variant from Pseudomonas putida J3.

Formaldehyde dismutase (FDM) is a very interesting enzyme, due to the fact that it comprises an internal cofactor regeneration mechanism. The FDM, therefore, is able to catalyze redox reactions independent of exogenous cofactor addition, rendering the enzyme powerful for industrial applications. Currently, only one enzyme of this type has been characterized enzymatically. Furthermore, only one additional DNA-sequence with high homology to FDM has been published. In this work, we identified a new variant of a formaldehyde dismutase gene (fdm) in the Pseudomonas putida J3 strain. To isolate and characterize the enzyme, we developed a simplified method for its purification. This purification is based on a C-terminal 6xHis-tag, which enables functional expression of the enzyme in E. coli and a one-step purification method. In addition, we tested several expression systems for optimal yields and combined this with co-expression of the chaperonins GroESL. Using this simplified and rapid method, we are now able to produce sufficient material in reproducible quality and quantity for application tests with the enzyme. The newly identified enzyme will be applied in a redox cascade for biomethanol production from biogas and shows potential for further industrial biotransformation with integrated cofactor recycling.