Asymmetric Reduction of α-Keto Esters with Thermus thermophilus NADH-Dependent Carbonyl Reductase using Glucose Dehydrogenase and Alcohol Dehydrogenase for Cofactor Regeneration

2011 ◽  
Vol 2011 (23) ◽  
pp. 4361-4366 ◽  
Author(s):  
Angela Pennacchio ◽  
Assunta Giordano ◽  
Mosè Rossi ◽  
Carlo A. Raia
Keyword(s):  
Alcohol Dehydrogenase ◽  
Asymmetric Reduction ◽  
Glucose Dehydrogenase ◽  
Thermus Thermophilus ◽  
Carbonyl Reductase ◽  
Cofactor Regeneration ◽  
Keto Esters

scholarly journals Efficient production of (S)-1-phenyl-1, 2-ethanediol using xylan as co-substrate by a coupled multi-enzyme Escherichia coli system

2020 ◽  
Author(s):  
Junchao Rao ◽  
Rongzhen Zhang ◽  
Guanyu Xu ◽  
Lihong Li ◽  
Yan Xu
Keyword(s):  
Escherichia Coli ◽  
Glucose Dehydrogenase ◽  
Optical Purity ◽  
Carbonyl Reductase ◽  
Cofactor Regeneration ◽  
Efficient Production ◽  
Chiral Synthesis ◽  
E Coli ◽  
Concomitant Reduction ◽  
Different Strains

Abstract Background: ( S )-1-phenyl-1,2-ethanediol is an important chiral intermediate in the synthesis of liquid crystals and chiral biphosphines.(S)-carbonyl reductase II from Candida parapsilosis catalyzes the conversion of 2-hydroxyacetophenone to ( S )-1-phenyl-1,2-ethanediol with NADPH as a cofactor. Glucose dehydrogenase with a Ala258Phe mutation is able to catalyze the oxidation of xylose with concomitant reduction of NADP + to NADPH, while endo-β-1,4-xylanase 2 catalyzes the conversion of xylan to xylose. In the present work, the Ala258Phe glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 were introduced into the ( S )-carbonyl reductase II-mediated chiral pathway to strengthen cofactor regeneration by using xylan as a naturally abundant co-substrate. Results: We constructed several coupled multi-enzyme systems by introducing ( S )-carbonyl reductase II, the A258F glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 into Escherichia coli . Different strains were produced by altering the location of the encoding genes on the plasmid. Only recombinant E. coli /pET-G-S-2 expressed all three enzymes, and this strain produced ( S )-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone as a substrate and xylan as a co-substrate. The optical purity was 100% and the yield was 98.3% (6 g/L 2-HAP) under optimal conditions of 35°C, pH 6.5 and a 2:1 substrate-co-substrate ratio. The introduction of A258F glucose dehydrogenase and endo-β-1,4-xylanase 2 into the ( S )-carbonyl reductase II-mediated chiral pathway caused a 54.6% increase in yield, and simultaneously reduced the reaction time from 48 h to 28 h. Conclusions: This study demonstrates efficient chiral synthesis using a pentose as a co-substrate to enhance cofactor regeneration. This provides a new approach for enantiomeric catalysis through the inclusion of naturally abundant materials.

Characterization and further stabilization of a new anti-prelog specific alcohol dehydrogenase from Thermus thermophilus HB27 for asymmetric reduction of carbonyl compounds

2012 ◽  
Vol 103 (1) ◽  
pp. 343-350 ◽  
Author(s):  
Javier Rocha-Martín ◽  
Daniel Vega ◽  
Juan M. Bolivar ◽  
Aurelio Hidalgo ◽  
José Berenguer ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Carbonyl Compounds ◽  
Asymmetric Reduction ◽  
Thermus Thermophilus

scholarly journals Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus

2008 ◽  
Vol 74 (13) ◽  
pp. 3949-3958 ◽  
Author(s):  
Angela Pennacchio ◽  
Biagio Pucci ◽  
Francesco Secundo ◽  
Francesco La Cara ◽  
Mosè Rossi ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Thermus Thermophilus ◽  
Critical Role ◽  
Enantiomeric Excess ◽  
Short Chain ◽  
Gene Encoding ◽  
Keto Esters ◽  
Adh Gene ◽  
Strict Requirement

ABSTRACT The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh Tt) was heterologously overexpressed in Escherichia coli, and the protein (ADHTt) was purified to homogeneity and characterized. ADHTt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ∼73°C and a 30-min half-inactivation temperature of ∼90°C, as well as good tolerance to common organic solvents. ADHTt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and α-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, α-tetralone, and α-methyl and α-ethyl benzoylformates to (S)-(−)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-α-(trifluoromethyl)benzyl alcohol (93% ee), (S)-α-tetralol (>99% ee), methyl (R)-(−)-mandelate (92% ee), and ethyl (R)-(−)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

scholarly journals Efficient biotransformation of (S)-1-phenyl-1, 2-ethanediol using xylan as co-substrate by a coupled multi-enzyme Escherichia coli system

2020 ◽  
Author(s):  
Junchao Rao ◽  
Rongzhen Zhang ◽  
Guanyu Xu ◽  
Lihong Li ◽  
Yan Xu
Keyword(s):  
Escherichia Coli ◽  
Glucose Dehydrogenase ◽  
Optical Purity ◽  
Carbonyl Reductase ◽  
Cofactor Regeneration ◽  
Chiral Synthesis ◽  
E Coli ◽  
Concomitant Reduction ◽  
Different Strains ◽  
Encoding Genes

Abstract Background: ( S )-1-phenyl-1,2-ethanediol is an important chiral intermediate in the synthesis of liquid crystals and chiral biphosphines. ( S )-carbonyl reductase II from Candida parapsilosis catalyzes the conversion of 2-hydroxyacetophenone to ( S )-1-phenyl-1,2-ethanediol with NADPH as a cofactor. Glucose dehydrogenase with a Ala258Phe mutation is able to catalyze the oxidation of xylose with concomitant reduction of NADP + to NADPH, while endo-β-1,4-xylanase 2 catalyzes the conversion of xylan to xylose. In the present work, the Ala258Phe glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 were introduced into the ( S )-carbonyl reductase II-mediated chiral pathway to strengthen cofactor regeneration by using xylan as a naturally abundant co-substrate. Results: We constructed several coupled multi-enzyme systems by introducing ( S )-carbonyl reductase II, the A258F glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 into Escherichia coli . Different strains were produced by altering the location of the encoding genes on the plasmid. Only recombinant E. coli /pET-G-S-2 expressed all three enzymes, and this strain produced ( S )-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone as a substrate and xylan as a co-substrate. The optical purity was 100% and the yield was 98.3% under optimal conditions of 35°C, pH 6.5 and a 2:1 substrate-co-substrate ratio. The introduction of A258F glucose dehydrogenase and endo-β-1,4-xylanase 2 into the ( S )-carbonyl reductase II-mediated chiral pathway caused a 54.6% increase in yield, and simultaneously reduced the reaction time from 48 h to 28 h. Conclusions: This study demonstrates efficient chiral synthesis using a pentose as a co-substrate to enhance cofactor regeneration. This provides a new approach for enantiomeric catalysis through the inclusion of naturally abundant materials.

scholarly journals A Redox-Neutral, Two-Enzyme Cascade for the Production of Malate and Gluconate from Pyruvate and Glucose

2021 ◽  
Vol 11 (11) ◽  
pp. 4877
Author(s):  
Ravneet Mandair ◽  
Pinar Karagoz ◽  
Roslyn M. Bill
Keyword(s):  
Malate Dehydrogenase ◽  
Closed System ◽  
Glucose Dehydrogenase ◽  
Sulfolobus Solfataricus ◽  
Cofactor Regeneration ◽  
Triple Mutant ◽  
Thermococcus Kodakarensis ◽  
Platform Chemicals ◽  
Enzyme Cascade

A triple mutant of NADP(H)-dependent malate dehydrogenase from thermotolerant Thermococcus kodakarensis has an altered cofactor preference for NAD+, as well as improved malate production compared to wildtype malate dehydrogenase. By combining mutant malate dehydrogenase with glucose dehydrogenase from Sulfolobus solfataricus and NAD+/NADH in a closed reaction environment, gluconate and malate could be produced from pyruvate and glucose. After 3 h, the yield of malate was 15.96 mM. These data demonstrate the feasibility of a closed system capable of cofactor regeneration in the production of platform chemicals.

scholarly journals Cascading Old Yellow Enzyme, Alcohol Dehydrogenase and Glucose Dehydrogenase for Selective Reduction of (E/Z)-Citral to (S)-Citronellol

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 931
Author(s):  
Yunpeng Jia ◽  
Qizhou Wang ◽  
Jingjing Qiao ◽  
Binbin Feng ◽  
Xueting Zhou ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Glucose Dehydrogenase ◽  
Selective Reduction ◽  
Coli Strain ◽  
Severe Reaction ◽  
High Catalytic Activity ◽  
Old Yellow Enzyme ◽  
One Pot ◽  
Nadph Regeneration ◽  
By Products

Citronellol is a kind of unsaturated alcohol with rose-like smell and its (S)-enantiomer serves as an important intermediate for organic synthesis of (-)-cis-rose oxide. Chemical methods are commonly used for the synthesis of citronellol and its (S)-enantiomer, which suffers from severe reaction conditions and poor selectivity. Here, the first one-pot double reduction of (E/Z)-citral to (S)-citronellol was achieved in a multi-enzymatic cascade system: N-ethylmaleimide reductase from Providencia stuartii (NemR-PS) was selected to catalyze the selective reduction of (E/Z)-citral to (S)-citronellal, alcohol dehydrogenase from Yokenella sp. WZY002 (YsADH) performed the further reduction of (S)-citronellal to (S)-citronellol, meanwhile a variant of glucose dehydrogenase from Bacillus megaterium (BmGDHM6), together with glucose, drove efficient NADPH regeneration. The Escherichia coli strain co-expressing NemR-PS, YsADH, and BmGDHM6 was successfully constructed and used as the whole-cell catalyst. Various factors were investigated for achieving high conversion and reducing the accumulation of the intermediate (S)-citronellal and by-products. 0.4 mM NADP+ was essential for maintaining high catalytic activity, while the feeding of the cells expressing BmGDHM6 effectively eliminated the intermediate and by-products and shortened the reaction time. Under optimized conditions, the bio-transformation of 400 mM citral caused nearly complete conversion (>99.5%) to enantio-pure (S)-citronellol within 36 h, demonstrating promise for industrial application.

scholarly journals Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Langxing Liao ◽  
Yonghui Zhang ◽  
Yali Wang ◽  
Yousi Fu ◽  
Aihui Zhang ◽  
...  
Keyword(s):  
Glucose Dehydrogenase ◽  
Catalytic Efficiency ◽  
Space Time ◽  
Cofactor Regeneration ◽  
Enzyme Complex ◽  
Regeneration System ◽  
Regeneration Rate ◽  
Leucine Dehydrogenase ◽  
Fusion Enzyme ◽  
Space Time Yield

Abstract Background Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of l-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. Results In this work, a novel fusion enzyme (GDH–R3–LeuDH) for the efficient biosynthesis of l-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH–R3–LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). Conclusions It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize l-tle and reach the highest space–time yield up to now. These results demonstrated the great potential of the GDH–R3–LeuDH fusion enzyme for the efficient biosynthesis of l-tle.

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