scholarly journals Two Biotechnological Approaches to the Preparative Synthesis of Natural Dihydrocoumarin

Catalysts ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 28
Author(s):  
Stefano Serra ◽  
Stefano Marzorati ◽  
Mattia Valentino
Keyword(s):  
Glucose Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Catalytic Amount ◽  
Microbial Reduction ◽  
Preparative Synthesis ◽  
Enzymatic System ◽  
Recombinant Enzymes ◽  
Old Yellow Enzyme ◽  
Preparative Scale ◽  
Natural Coumarin

In this work, we describe two different biotechnological processes that provide the natural flavour dihydrocoumarin in preparative scale. Both the presented approaches are based on the enzyme-mediated reduction of natural coumarin. The first one is a whole-cell process exploiting the reductive activity of the yeast Kluyveromyces marxianus, a Generally Recognized As Safe (GRAS) microorganism that possesses high resistance to the substrate toxicity. Differently, the second is based on the reduction of natural coumarin by nicotinamide adenine dinucleotide phosphate (NADPH) and using the Old Yellow Enzyme reductase OYE2 as catalyst. NADPH is used in catalytic amount since the co-factor regeneration is warranted employing an enzymatic system based on glucose oxidation, in turn catalysed by a further enzyme, namely glucose dehydrogenase (GDH). Both processes compare favourably over the previously reported industrial method as they work with higher coumarin concentration (up to 3 g/L for the enzymatic process) yet allowing the complete conversion of the substrate. Furthermore, the two approaches have significant differences. The microbial reduction is experimentally simple but the isolated dihydrocoumarin yield does not exceed 60%. On the contrary, the enzymatic approach requires the use of two specially prepared recombinant enzymes, however, it is more efficient, affording the product in 90% of isolated yield.

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 New Insights into the Potential of Endogenous Redox Systems in Wheat Bread Dough

Antioxidants ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 190 ◽  
Author(s):  
Nicolas Navrot ◽  
Rikke Buhl Holstborg ◽  
Per Hägglund ◽  
Inge Povlsen ◽  
Birte Svensson
Keyword(s):  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Thioredoxin Reductase ◽  
Heat Stability ◽  
Wheat Bread ◽  
Enzymatic System ◽  
Redox Systems ◽  
Bread Dough ◽  
Seed Formation ◽  
Protein Substrates ◽  
Thioredoxin System

Various redox compounds are known to influence the structure of the gluten network in bread dough, and hence its strength. The cereal thioredoxin system (NTS), composed of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) and thioredoxin (Trx), is a major reducing enzymatic system that is involved in seed formation and germination. NTS is a particularly interesting tool for food processing due to its heat stability and its broad range of protein substrates. We show here that barley NTS is capable of remodeling the gluten network and weakening bread dough. Furthermore, functional wheat Trx that is present in the dough can be recruited by the addition of recombinant barley NTR, resulting in dough weakening. These results confirm the potential of NTS, especially NTR, as a useful tool in baking for weakening strong doughs, or in flat product baking.

The mechanism of cyclic phosphorylation by illuminated chloroplasts

1963 ◽  
Vol 157 (968) ◽  
pp. 332-345 ◽  
Keyword(s):  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reducing Power ◽  
Catalytic Amount ◽  
Adenosine Diphosphate ◽  
Hill Reaction ◽  
Hydrogen Transport ◽  
Hydrogen Acceptor ◽  
Light Reaction ◽  
The Hill ◽  
Evolution Of Oxygen

In the reaction discovered by Hill (1937, 1939), chloroplasts isolated from the cell were shown to be capable, upon illumination, of reducing an artificial hydrogen acceptor with the concurrent evolution of oxygen. The ‘Hill reaction’ was regarded as a partial model of the light reaction in photosynthesis where limited reducing power and molecular oxygen arose from the photolysis of water. Attempts to relate this reaction to the photochemical events preceding the dark reduction of carbon dioxide in photosynthesis received their first direct support from the finding of San Pietro & Lang (1958) that nicotinamide adenine dinucleotide phosphate ( NADP )can serve as an effective acceptor of hydrogen in the photochemical reaction when the system is supplemented with a catalytic amount of a soluble protein extracted from leaves. Added significance was given to this finding by the further observation of Amon, Whatley & Allen (1959), that hydrogen transport in the reaction could be coupled to the phosphorylation of adenosine diphosphate { ADP ) to yield adenosine triphosphate { ATP ) concurrently with the reduction of NADP and the production of oxygen in the stoicheiometric proportions: l NADP LL 2 jl ATP /^0 2 . They had previously demonstrated a similar coupling of phosphorylation to hydrogen transport when the artificial reagent, ferricyanide, served as hydrogen acceptor (Arnon, Whatley & Allen 1958). In this work, Amon et al. made the further important observation that hydrogen transport in the ferricyanide reaction is strongly stimulated when phosphorylation occurs concurrently.

Oxidative decarboxylation of isocitric acid in the presence of montmorillonite

Clay Minerals ◽  
1990 ◽  
Vol 25 (1) ◽  
pp. 27-37 ◽  
Author(s):  
A. Naidja ◽  
B. Siffert
Keyword(s):  
Catalytic Activity ◽  
Reaction Mechanism ◽  
Nicotinamide Adenine Dinucleotide ◽  
Reaction Rate ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Oxidative Decarboxylation ◽  
Enzymatic System ◽  
Exchangeable Cation ◽  
Isocitric Acid ◽  
Dehydrogenase Enzyme

AbstractIsocitric acid oxidative decarboxylation was realized in the absence and in the presence of homoionic Na+-, Mn2+-, and Cu2+-montmorillonite. The catalytic activity of the clay depends upon the nature of the interlayer exchangeable cation. Isocitric acid is transformed into α-ketoglutaric acid under the action of the clay mineral saturated with Na+ cations which do not form a complex with the isocitrate anion. Nevertheless, the reaction rate is very much lower than in the presence of the enzymatic system (isocitrate dehydrogenase enzyme and nicotinamide adenine dinucleotide phosphate coenzyme). The reaction mechanism in the presence of clay is given showing the different steps of the transformation.

scholarly journals From Cell-Free Protein Synthesis to Whole-Cell Biotransformation: Screening and Identification of Novel α‑Ketoglutarate-Dependent Dioxygenases for Preparative-Scale Synthesis of Hydroxy-l-Lysine

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1038
Author(s):  
Jascha Rolf ◽  
Philipp Nerke ◽  
Annette Britner ◽  
Sebastian Krick ◽  
Stephan Lütz ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Specific Activity ◽  
Value Added ◽  
Small Scale ◽  
Preparative Synthesis ◽  
Preparative Scale ◽  
Whole Cell ◽  
Free Protein ◽  
Starting Point ◽  
Cell Free Protein Synthesis

The selective hydroxylation of non-activated C-H bonds is still a challenging reaction in chemistry. Non-heme Fe2+/α-ketoglutarate-dependent dioxygenases are remarkable biocatalysts for the activation of C-H-bonds, catalyzing mainly hydroxylations. The discovery of new Fe2+/α-ketoglutarate-dependent dioxygenases with suitable reactivity for biotechnological applications is therefore highly relevant to expand the limited range of enzymes described so far. In this study, we performed a protein BLAST to identify homologous enzymes to already described lysine dioxygenases (KDOs). Six novel and yet uncharacterized proteins were selected and synthesized by cell-free protein synthesis (CFPS). The subsequent in vitro screening of the selected homologs revealed activity towards the hydroxylation of l-lysine (Lys) into hydroxy-l-lysine (Hyl), which is a versatile chiral building block. With respect to biotechnological application, Escherichia coli whole-cell biocatalysts were developed and characterized in small-scale biotransformations. As the whole-cell biocatalyst expressing the gene coding for the KDO from Photorhabdus luminescens showed the highest specific activity of 8.6 ± 0.6 U gCDW−1, it was selected for the preparative synthesis of Hyl. Multi-gram scale product concentrations were achieved providing a good starting point for further bioprocess development for Hyl production. A systematic approach was established to screen and identify novel Fe2+/α-ketoglutarate-dependent dioxygenases, covering the entire pathway from gene to product, which contributes to accelerating the development of bioprocesses for the production of value-added chemicals.

scholarly journals Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Catalysts ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 531
Author(s):  
Svenja Kochius ◽  
Jacqueline van Marwijk ◽  
Ana Ebrecht ◽  
Diederik Opperman ◽  
Martha Smit
Keyword(s):  
Glucose Dehydrogenase ◽  
Reaction Medium ◽  
Cofactor Regeneration ◽  
Alkane Hydroxylase ◽  
P450 Monooxygenase ◽  
Preparative Scale ◽  
Alkane Hydroxylation ◽  
Shake Flasks ◽  
Total Turnover

Some of the most promising results for bacterial alkane hydroxylation to alcohols have been obtained with the cytochrome P450 monooxygenase CYP153A6. CYP153A6 belongs to the class I CYPs and is generally expressed from an operon that also encodes the ferredoxin (Fdx) and ferredoxin reductase (FdR) which transfer electrons to CYP153A6. In this study, purified enzymes (CYP, Fdx, FdR and dehydrogenases for cofactor regeneration) were used to deconstruct the CYP153A6 system into its separate components, to investigate the factors limiting octane hydroxylation in vitro. Proteins in the cytoplasm (cell-free extract) were found to better enhance and stabilize hydroxylase activity compared to bovine serum albumin (BSA) and catalase. Optimization of the CYP:Fdx:FdR ratio also significantly improved both turnover frequencies (TFs) and total turnover numbers (TTNs) with the ratio of 1:1:60 giving the highest values of 3872 h−1 and 45,828 moloctanol molCYP−1, respectively. Choice and concentration of dehydrogenase for cofactor regeneration also significantly influenced the reaction. Glucose dehydrogenase concentrations had to be as low as possible to avoid fast acidification of the reaction medium, which in the extreme caused precipitation of the CYP and other proteins. Cofactor regeneration based on glycerol failed, likely due to accumulation of dihydroxyacetone. Scaling the reactions up from 1 mL in vials to 60 mL in shake flasks and 120 mL in bioreactors showed that mixing and shear forces will be important obstacles to overcome in preparative scale reactions.

scholarly journals The proton-translocating nicotinamide–adenine dinucleotide (phosphate) transhydrogenase of rat liver mitochondria

1973 ◽  
Vol 132 (3) ◽  
pp. 571-585 ◽  
Author(s):  
Jennifer Moyle ◽  
Peter Mitchell
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Isocitrate Dehydrogenase ◽  
Proton Translocation ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Catalytic Amount ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Ph Change ◽  
Carbonyl Cyanide

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an →H+/2e− quotient of 1.94±0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K+-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of −450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.

scholarly journals Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 5040
Author(s):  
Tairan Wang ◽  
Ran Wei ◽  
Yingting Feng ◽  
Lijun Jin ◽  
Yunpeng Jia ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rational Design ◽  
Asymmetric Reduction ◽  
Glucose Dehydrogenase ◽  
Direct Synthesis ◽  
Cascade Reaction ◽  
Old Yellow Enzyme ◽  
Complete Reduction ◽  
Novel Approach ◽  
Double Substitution

The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.

scholarly journals One-pot Synthesis of (R)- and (S)-phenylglycinol From Bio-based L-phenylalanine by an Artificial Biocatalytic Cascade

2021 ◽  
Author(s):  
Jiandong Zhang ◽  
Ning Qi ◽  
Lili Gao ◽  
Jing Li ◽  
Chaofeng Zhang ◽  
...  
Keyword(s):  
Glucose Dehydrogenase ◽  
Value Added ◽  
Microbial Consortia ◽  
Acid Decarboxylase ◽  
Grand Challenge ◽  
Preparative Scale ◽  
E Coli ◽  
Ferulic Acid Decarboxylase ◽  
Cascade Biocatalysis ◽  
Cell Module

Abstract Chiral phenylglycinol is a very important chemical in the pharmaceutical manufacturing. Current methods for synthesis of chiral phenylglycinol often suffered from unsatisfied selectivity, low product yield and using the non-renewable resourced substrates, then the synthesis of chiral phenylglycinol remain a grand challenge. Design and construction of synthetic microbial consortia is a promising strategy to convert bio-based materials to high value-added chiral compounds. In this study, we reported a six-step artificial cascade biocatalysis system for conversion of biobased L-phenylalanine to yield chiral phenylglycinol. The cascade biocatalysis system was conducted by a microbial consortium composed of two engineered recombinant Escherichia coli cells modules, one recombinant E. coli cell module co-expression of six different enzymes (phenylalanine ammonia lyase/ferulic acid decarboxylase/phenylacrylic acid decarboxylase/styrene monooxygenase/epoxide hydrolase/alcohol dehydrogenase) for efficient conversion of L-phenylalanine into 2-hydroxyacetophenone. The second recombinant E. coli cell module expression of an (R)-ω-transaminase or co-expression of the (S)-ω-transaminase, alanine dehydrogenase and glucose dehydrogenase for conversion of 2-hydroxyacetophenone to (S)- or (R)-phenylglycinol, respectively. Combining the two engineered E. coli cell modules, after the optimization of bioconversion conditions (including pH, temperature, glucose concentration, amine donor concentration and cell ratio), L-phenylalanine could be easily converted to (R)-phenylglycinol and (S)-phenylglycinol with up to 99% conversion and >99% ee. Preparative scale biotransformation was also conducted on 100 mL scale, (S)-phenylglycinol and (R)-phenylglycinol were obtained in 71.0% and 80.5% yield, >99% ee, and 5.19 g/L.d and 4.42 g/L.d productivity, respectively. The salient features of this biocatalytic cascade system are good yields, excellent ee, mild reaction conditions and no need for additional cofactor (NADH/NAD+), provide a practical biocatalytic method for sustainable synthesis of (S)-phenylglycinol and (R)-phenylglycinol from biobased L-phenylalanine.

scholarly journals Confining enzyme clusters in supramolecular nanoreactors enhances cofactor-dependent cascade for chiral alcohol synthesis

2020 ◽  
Author(s):  
Yan-Qing Zhang ◽  
Yufei Cao ◽  
Xiao-Yan Zhang ◽  
Tao Wang ◽  
Mario Roque Huanca Nina ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Catalytic Efficiency ◽  
Chiral Alcohols ◽  
Local Concentration ◽  
Preparative Synthesis ◽  
Confined Space ◽  
Living Organisms ◽  
Supramolecular Ensemble

Abstract Enzymes in living organisms work efficiently in confined environments through spatial organization. Constituting a bio-cascade reaction in nano-confined space in vitro for the efficient synthesis of high-value chiral chemicals is challenging. Herein, we confined a cofactor-dependent cascade in bacteriophage P22 nanoparticles for the synthesis of chiral alcohols. Compared to free enzymes, this supramolecular ensemble, P22-SP-BmGDH-SsCR, exhibited enhanced catalytic efficiency up to 14.5-fold towards various ketones and improved stereoselectivity up to > 99% ee towards 8 substrates, and 10 chiral alcohols with > 96% ee were synthesized. The recycling efficiency of nicotinamide adenine dinucleotide phosphate (NADPH) was increased by 7.5-fold. We demonstrated that the enhancement in cofactor recycling originates from the higher local concentration of NADPH in the nanoparticles due to the proximity effect of enzymes and confinement of nanoparticles. The preparative synthesis of chiral alcohols showed that the consumption of NADPH can be reduced by one magnitude compared with the conventional free enzyme system.

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