Cofactor Specificity Engineering of Streptococcus Mutans Nadh Oxidase 2 for Nad(p) + Regeneration in Biocatalytic Oxidations

Petschacher, Barbara; Staunig, Nicole; Müller, Monika; Schürmann, Martin; Mink, Daniel; Wildeman, S. De; Gruber, Karl; Glieder, Anton

doi:10.5936/csbj.201402005

Cited by 50 publications

(58 citation statements)

References 71 publications

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“…Regarding the NADPH, since the cofactor dependence is a drawback that hampers the use of alcohol dehydrogenases at large scale, an NADH oxidase from Streptococcus mutans (NOX) (EC 1.6.3.4) engineered to accept NADPH, was used as a cofactor regeneration enzyme ( Figure 1). [28][29][30] The immobilization of NOX is not presented here due to the fact that previous results obtained with this enzyme, did not show any significant operational improvement (data not shown).…”

Section: Introductionmentioning

confidence: 87%

Ketoisophorone Synthesis with an Immobilized Alcohol Dehydrogenase

Solé

Brummund²,

Caminal

et al. 2019

ChemCatChem

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The monoterpenoid α-isophorone is sourced from the available and renewable plant dry matter, as well as a waste recovery operation from acetone. This compound, can be hydroxylated to 4-hydroxy-isophorone which is the main precursor for the synthesis of ketoisophorone. On its turn, ketoisophorone is a key intermediate for the production of carotenoids and Vitamin E. Here, the enzymatic oxidation of 4-hydroxy-isophorone to ketoisophorone is demonstrated employing an alcohol dehydrogenase (ADHaa) from Artemisia annua and a NADPH oxidase (NOX), as a cofactor regeneration enzyme. After 24 h of reaction and an initial substrate concentration of 50 mM, 95.7 % yield and a space time yield of 6.52 g L À 1 day À 1 could be obtained. Furthermore, the immobilization of the alcohol dehydrogenase was studied on 17 different supports. An epoxy-functionalized agarose resulted in the highest metrics, 100 � 0% immobilization yield and 58.2 � 3.5 % retained activity. Finally, the immobilized ADHaa was successfully implemented in 4 reaction cycles (96 h operation) presenting a biocatalyst yield of 23.4 g product g À 1 of enzyme. It represents a 2.5-fold increase compared with the reaction with soluble enzymes.

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Section: Introductionmentioning

confidence: 87%

Ketoisophorone Synthesis with an Immobilized Alcohol Dehydrogenase

Solé

Brummund²,

Caminal

et al. 2019

ChemCatChem

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“…The water forming Streptococcus mutans NADH oxidase 2 ( Sm NOX2) is specific for NADH. Petschacher and co‐workers however engineered Sm NOX2 as a universal regeneration system to regenerate both NAD + and NADP + . The Sm NOX2_V193R_V194H mutant (Mut10) was shown to accept both NAD + and NADP + with kinetic studies revealing similar affinities and catalytic efficiencies for both cofactors.…”

Section: Resultsmentioning

confidence: 99%

An Alcohol Dehydrogenase from the Short‐Chain Dehydrogenase/Reductase Family of Enzymes for the Lactonization of Hexane‐1,6‐diol

et al. 2018

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Biocatalytic production of lactones, and in particular ϵ‐caprolactone (CL), have gained increasing interest as a greener route to polymer building blocks, especially through the use of Baeyer–Villiger monooxygenases (BVMOs). Despite several advances in the field, BVMOs, however, still suffer several practical limitations. Alcohol dehydrogenase (ADH)‐mediated lactonization of diols in turn has received far less attention and very few enzymes have been identified for the conversion of diols to lactones, with horse‐liver ADH (HLADH) remaining the catalyst of choice. Screening of a diverse panel of ADHs, AaSDR‐1, a member of the short‐chain dehydrogenase/reductase family, was found to produce ϵ‐caprolactone from hexane‐1,6‐diol. Moreover, cofactor regeneration by an NADH oxidase eliminated the requirement of co‐substrates, yielding water as the sole by‐product. Despite lower turnover frequencies as compared to HLADH, higher selectivity was found for the production of CL, with HLADH forming significant amounts of 6‐hydroxyhexanoic acid and adipic acid through aldehyde dehydrogenation/oxidation of the gem‐diol intermediates. Also, CL yield were shown to be dependent on buffer choice, as structural elucidation of a Tris adduct confirmed the buffer amine to react with aliphatic aldehydes forming a Schiff‐base intermediate which through further ADH oxidation, forms a tricyclic acetal product.

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“…[ 12 ] Thermodynamic and kinetic requirements as well as cost considerations had to be taken into account, as regeneration of NAD + is not well established, compared to NADH. [ 13 ] We applied a system originally described by Pival et al, in which Ct XR reduces 9,10-phenanthrenequinone (PQ) to 9,10-phenanthrene hydroquinone (PQH 2 ). [ 12 ] The NADH produced in UDP-Glc oxidation is recycled to NAD + during PQ reduction.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

Eixelsberger

Nidetzky

2014

Adv Synth Catal

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Synthetic ways towards uridine 5′-diphosphate (UDP)-xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP-glucose 6-dehydrogenase (hUGDH) and UDP-xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP-α-xylose from UDP-glucose. In a mimic of the natural biosynthetic route, UDP-glucose is converted to UDP-glucuronic acid by hUGDH, followed by subsequent formation of UDP-xylose by hUXS. The nicotinamide adenine dinucleotide (NAD+) required in the hUGDH reaction is continuously regenerated in a three-step chemo-enzymatic cascade. In the first step, reduced NAD+ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10-phenanthrenequinone (PQ). Radical chemical re-oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H2O2) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo-enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP-xylose as well as an adequate oxygen supply for PQ re-oxidation as major bottlenecks of effective performance of the overall multi-step reaction system. Net oxidation of UDP-glucose to UDP-xylose by hydrogen peroxide (H2O2) could thus be achieved when using an in situ oxygen supply through periodic external feed of H2O2 during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g l−1) UDP-α-xylose. After two-step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi-step redox cascades in the synthesis of nucleotide sugar products.

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Cofactor Specificity Engineering of Streptococcus Mutans Nadh Oxidase 2 for Nad(p) + Regeneration in Biocatalytic Oxidations

Cited by 50 publications

References 71 publications

Ketoisophorone Synthesis with an Immobilized Alcohol Dehydrogenase

Ketoisophorone Synthesis with an Immobilized Alcohol Dehydrogenase

An Alcohol Dehydrogenase from the Short‐Chain Dehydrogenase/Reductase Family of Enzymes for the Lactonization of Hexane‐1,6‐diol

Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

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