Multi‐enzymatic one‐pot reduction of dehydrocholic acid to 12‐keto‐ursodeoxycholic acid with whole‐cell biocatalysts

Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non‐surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven‐step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo‐enzymatic preparation of UDCA—the two‐step reduction of dehydrocholic acid (DHCA) to 12‐keto‐ursodeoxycholic acid using a mutant of 7β‐hydroxysteroid dehydrogenase (7β‐HSDH) from Collinsella aerofaciens and 3α‐hydroxysteroid dehydrogenase (3α‐HSDH) from Comamonas testosteroni. Three different one‐pot reaction approaches were investigated using whole‐cell biocatalysts in simple batch processes. We applied one‐biocatalyst systems, where 3α‐HSDH, 7β‐HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two‐biocatalyst systems, where 3α‐HSDH and 7β‐HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one‐pot reaction. The best result was achieved by the one‐biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100 mM DHCA to >99.5 mM 12‐keto‐UDCA within 4.5 h in a simple batch process on a liter scale. Biotechnol. Bioeng. 2013; 110: 68–77. © 2012 Wiley Periodicals, Inc.     

Mechanistic model for prediction of formate dehydrogenase kinetics under industrially relevant conditions

Formate dehydrogenase (FDH) from Candida boidinii is an important biocatalyst for the regeneration of the cofactor NADH in industrial enzyme‐catalyzed reductions. The mathematical model that is currently applied to predict progress curves during (semi‐)batch reactions has been derived from initial rate studies. Here, it is demonstrated that such extrapolation from initial reaction rates to performance during a complete batch leads to considerable prediction errors. This observation can be attributed to an invalid simplification during the development of the literature model. A novel mechanistic model that describes the course and performance of FDH‐catalyzed NADH regeneration under industrially relevant process conditions is introduced and evaluated. Based on progress curve instead of initial reaction rate measurements, it was discriminated from a comprehensive set of mechanistic model candidates. For the prediction of reaction courses on long time horizons (>1 h), decomposition of NADH has to be considered. The model accurately describes the regeneration reaction under all conditions, even at high concentrations of the substrate formate and thus is clearly superior to the existing model. As a result, for the first time, course and performance of NADH regeneration in industrial enzyme‐catalyzed reductions can be accurately predicted and used to optimize the cost efficiency of the respective processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol

The efficiency of biocatalysis is often affected by an insufficient supply and regeneration of cofactors and redox equivalents. To alleviate this shortcoming, a cofactor self-sufficient system was developed for enhanced production of 2-phenylethanol (2-PE) in E. coli. A “bridge” between the amino acid and its corresponding alcohol was designed in the system using glutamate dehydrogenase. By coupling glutamate dehydrogenase with transaminase and alcohol dehydrogenase, the cosubstrate (2-oxoglutarate) and redox equivalents (NAD(P)H) were regenerated simultaneously, so that no external cofactor or redox source was required. Thus, a cofactor self-sufficient system was developed, which improved the biocatalyst efficiency 3.8-fold. The ammonium generated in this process was removed using zeolite, which further improved the biosynthetic efficiency and resulted in a cleaner system. To the best of our knowledge, this system yielded the highest titer of 2-PE ever obtained in E. coli. Additionally, the wider applicability of this self-sufficient strategy was demonstrated in the production of D-phenyllactic acid. This study thus offers a new method to resolve the cofactor/redox imbalance problem and demonstrates the feasibility of the cofactor self-sufficient strategy for enhanced production of diverse chemicals.     

Enantioselectivity of alcohol dehydrogenase-catalyzed oxidation of 1,2-diols and aminoalcohols

The alcohol dehydrogenase from horse liver is able to catalyze the oxidation of a number of 1,2-diols and α-aminoalcohols enantioselectively to l-α-hydroxyaldehydes and l-α-amino aldehydes. A decrease of enantioselectivity was found in reactions with 1,3-diols and substrates with hydrophobic substituent at position 3. α-Aminoalcohols are not substrates for yeast alcohol dehydrogenase, but the enzyme can catalyze the oxidation of most of the diols to l-hydroxyaldehydes. New methods for determination of the optical purity of α-hydroxy-and α-aminoaldehydes via converting them in situ to the corresponding acids, catalyzed by the aldehyde dehydrogenase from yeast, have been developed. The coupled alcohol dehydrogenase/aldehyde dehydrogenase has been extended to preparatory scale synthesis of optically pure l-α-hydroxyacids in the presence of a cofactor regeneration system. The active-site cubic-space section model has been shown not to be applicable to all substrates.     

Whole-cell biocatalysis: Evaluation of new hydrophobic ionic liquids for efficient asymmetric reduction of prochiral ketones

A biphasic process design is often applied in whole-cell biocatalysis if substrate and product have low water solubility, are unstable in water or toxic for the biocatalyst. Some water immiscible ionic liquids (ILs) with adequate distribution coefficients have already been applied successfully as second liquid phase, which acts as a substrate reservoir and in situ extractant for the product. In this work, 12 new ILs were evaluated with respect to their applicability in biphasic asymmetric reductions of prochiral ketones in comparison to 9 already published ILs. The ILs under study are composed of seven different cations and three different anions. Recombinant Escherichia coli was used as whole-cell biocatalyst overexpressing the genes of a Lactobacillus brevis alcohol dehydrogenase (LB-ADH) and a Candida boidinii formate dehydrogenase (CB-FDH) for cofactor regeneration. Best results were achieved if ionic liquids with [PF6]- and [NTF]-anions were applied, whereas [FAP]-ILs showed minor qualification, e.g., the use of [HMPL][NTF] as second liquid phase for asymmetric synthesis of (R)-2-octanol resulted in a space–time-yield of 180 g L⁻¹ d⁻¹, a chemical yield of 95% and an enantiomeric excess of 99.7% in a simple batch process.     

Construction of an engineered biocatalyst system for the production of medium-chain α,ω-dicarboxylic acids from medium-chain ω-hydroxycarboxylic acids

Medium-chain α,ω-dicarboxylic acids produced from renewable long-chain fatty acids are valuable as precursors in the chemical industry. However, they are difficult to produce biologically at high concentrations. Although improved biocatalyst systems consisting of engineering of Baeyer–Villiger monooxygenases are used in the production of ω-hydroxycarboxylic acids from long-chain fatty acids, the engineering of biocatalysts involved in the production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids has been rarely attempted. Here, we used highly active bacterial enzymes, Micrococcus luteus alcohol dehydrogenase and Archangium violaceum aldehyde dehydrogenase, for the efficient production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids and constructed a biocatalyst with cofactor regeneration system by introducing NAD(P)H flavin oxidoreductase as the NAD(P)H oxidase. The inhibition of the biocatalyst by hydrophobic substrates was attenuated by engineering a biocatalyst system with an adsorbent resin, which allowed us to obtain 196 mM decanedioic, 145 mM undecanedioic, and 114 mM dodecanedioic acid from 200 mM of C10, C11, and C12 hydroxyl saturated carboxylic acids, respectively, and 141 mM undecanedioic acid from 150 mM C11 unsaturated carboxylic acids, with molar conversions of 98%, 97%, 95%, and 94%, respectively. The concentration of undecanedioic acid obtained was approximately 40-fold higher than that in the previously highest results. Our results from this study can be applied for the industrial production of medium-chain α,ω-dicarboxylic acids from renewable long-chain fatty acids.     

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.     

Biocatalytic process optimization based on mechanistic modeling of cholic acid oxidation with cofactor regeneration

Reduction and oxidation of steroids in the human gut are catalyzed by hydroxysteroid dehydrogenases of microorganisms. For the production of 12-ketochenodeoxycholic acid (12-Keto-CDCA) from cholic acid the biocatalytic application of the 12α-hydroxysteroid dehydrogenase of Clostridium group P, strain C 48-50 (HSDH) is an alternative to chemical synthesis. However, due to the intensive costs the necessary cofactor (NADP(+) ) has to be regenerated. The alcohol dehydrogenase of Thermoanaerobacter ethanolicus (ADH-TE) was applied to catalyze the reduction of acetone while regenerating NADP(+) . A mechanistic kinetic model was developed for the process development of cholic acid oxidation using HSDH and ADH-TE. The process model was derived by identifying the parameters for both enzymatic models separately using progress curve measurements of batch processes over a broad range of concentrations and considering the underlying ordered bi-bi mechanism. Both independently derived kinetic models were coupled via mass balances to predict the production of 12-Keto-CDCA with HSDH and integrated cofactor regeneration with ADH-TE and acetone as co-substrate. The prediction of the derived model was suitable to describe the dynamics of the preparative 12-Keto-CDCA batch production with different initial reactant and enzyme concentrations. These datasets were used again for parameter identification. This led to a combined model which excellently described the reaction dynamics of biocatalytic batch processes over broad concentration ranges. Based on the identified process model batch process optimization was successfully performed in silico to minimize enzyme costs. By using 0.1 mM NADP(+) the HSDH concentration can be reduced to 3-4 μM and the ADH concentration to 0.4-0.6 μM to reach the maximal possible conversion of 100 mM cholic acid within 48 h. In conclusion, the identified mechanistic model offers a powerful tool for a cost-efficient process design.     

TADH,the thermostable alcohol dehydrogenase from <Emphasis Type="Italic">Thermus</Emphasis> sp. ATN1: a versatile new biocatalyst for organic synthesis

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD⁺/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.     

Redox self-sufficient whole cell biotransformation for amination of alcohols

Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD⁺ was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best.     

Purification and characterization of a novel alcohol dehydrogenase from Leifsonia sp. strain S749: a promising biocatalyst for an asymmetric hydrogen transfer bioreduction

To find microorganisms that could reduce phenyl trifluoromethyl ketone (PTK) to (S)-1-phenyltrifluoroethanol [(S)-PTE], styrene-assimilating bacteria (ca. 900 strains) isolated from soil samples were screened. We found that Leifsonia sp. strain S749 was the most suitable strain for the conversion of PTK to (S)-PTE in the presence of 2-propanol as a hydrogen donor. The enzyme corresponding to the reaction was purified homogeneity, characterized and designated Leifsonia alcohol dehydrogenase (LSADH). The purified enzyme had a molecular weight of 110,000 and was composed of four identical subunits (molecular weight, 26,000). LSADH required NADH as a cofactor, showed little activity with NADPH, and reduced a wide variety of aldehydes and ketones. LSADH catalyzed the enantioselective reduction of some ketones with high enantiomeric excesses (e.e.): PTK to (S)-PTE (>99% e.e.), acetophenone to (R)-1-phenylethanol (99% e.e.), and 2-heptanone to (R)-2-heptanol (>99% e.e.) in the presence of 2-propanol without an additional NADH regeneration system. Therefore, it would be a useful biocatalyst.     

Influence of water-miscible organic solvents on kinetics and enantioselectivity of the (R)-specific alcohol dehydrogenase from Lactobacillus brevis

Using the organic solvents acetonitrile and 1,4-dioxane as water-miscible additives for the alcohol dehydrogenase (ADH)-catalyzed reduction of butan-2-one, we investigated the influence of the solvents on enzyme reaction behavior and enantioselectivity. The NADP(+)-dependent (R)-selective ADH from Lactobacillus brevis (ADH-LB) was chosen as biocatalyst. For cofactor regeneration, the substrate-coupled approach using propan-2-ol as co-substrate was applied. Acetonitrile and 1,4-dioxane were tested from mole fraction 0.015 up to 0.1. Initial rate experiments revealed a complex kinetic behavior with enzyme activation caused by the substrate butan-2-one, and increasing K(M) values with increasing solvent concentration. Furthermore, these experiments showed an enhancement of the enantioselectivity for (R)-butan-2-ol from 37% enantiomeric excess (ee) in pure phosphate buffer up to 43% ee in the presence of 0.1 mol fraction acetonitrile. Finally, the influence of the co-solvents on water activity of the reaction mixture and on enzyme stability was investigated.     

Production of a doubly chiral compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone,by two-step enzymatic asymmetric reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD(+), and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol.

ADH: alcohol dehydrogenase; LDH: Lactose dehydrogenase; GDH: Glucose dehydrogenase; FDH: Formate dehydrogenase; LB-ADH: alcohol dehydrogenase from Lactobacillus brevis; HL-ADH: alcohol dehydrogenase from horse liver; TB-ADH: alcohol dehydrogenase from Thermoanaerobicum brockii; PS-GDH: Glucose dehydrogenase from Pseudomonas species; [BMIM][PF₆]: Butyl-methyl-imidazoliumhexafluorophosphate     

Cofactor regeneration by a soluble pyridine nucleotide transhydrogenase for biological production of hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP(+)-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.     

Design of a cytochrome P450BM3 reaction system linked by two‐step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenase

A cytochrome P450BM3‐catalyzed reaction system linked by a two‐step cofactor regeneration was investigated in a cell‐free system. The two‐step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD⁺‐dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD⁺) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP⁺ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3‐catalyzed reaction linked by the two‐step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10‐fold under initial reaction conditions. In contrast, a 10‐fold increase in STH units resulted in about a 9‐fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate‐determining step. In the system lacking the two‐step cofactor regeneration, 34% conversion of 50 μM of a model substrate (p‐nitrophenoxydecanoic acid) was attained using 50 μM NADPH. In contrast, with the two‐step cofactor regeneration, the same amount of substrate was completely converted using 5 μM of oxidized cofactors (NAD⁺ and NADP⁺) within 1 h. Furthermore, a 10‐fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD⁺ and NADP⁺. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Displacement of equilibrium in electroenzymatic reactor for acetaldehyde production using yeast alcohol dehydrogenase

Electrochemical regeneration of NAD was performed in a bench scale reactor in which yeast alcohol dehydrogenase catalyzed the oxidation of ethanol. By recycling one of the products of the reaction, it was possible to displace the equilibrium and favor the production of acetaldehyde. The flow-through electrode was made of graphite felt and had a specific area of 275 cm(-1). A mathematical model taking into account the enzymatic and electrochemical reaction rates as well as the mass transfer to the electrode was used to analyze the results. The limiting steps in the reactor are the electrochemical reaction for low potentials and the cofactor mass transfer for high potentials.     

Cofactor Regeneration by a Soluble Pyridine Nucleotide Transhydrogenase for Biological Production of Hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP⁺-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.     

Cofactor regeneration for sustainable enzymatic biosynthesis

Oxidoreductases are attractive catalysts for biosynthesis of chiral compounds and polymers, construction of biosensors, and degradation of environmental pollutants. Their practical applications, however, can be quite challenging since they often require cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These cofactors are generally expensive. Efficient regeneration of cofactors is therefore critical to the economic viability of industrial-scale biotransformations using oxidoreductases. The chemistry of cofactor regeneration is well known nowadays. The challenge is mostly regarding how to achieve the regeneration with immobilized enzyme systems which are preferred for industrial processes to facilitate the recovery and continuous use of the catalysts. This has become a great hurdle for the industrialization of many promising enzymatic processes, and as a result, most of the biotransformations involving cofactors have been traditionally performed with living cells in industry. Accompanying the rapidly growing interest in industrial biotechnology, immobilized enzyme biocatalyst systems with cofactor regeneration have been the focus for many studies reported since the late 1990s. The current paper reviews the methods of cofactor retention for development of sustainable and regenerative biocatalysts as revealed in these recent studies, with the intent to complement other reviewing articles that are mostly regeneration chemistry-oriented. We classify in this paper the methods of sustainable cofactor regeneration into two categories, namely membrane entrapment and solid-attachment of cofactors.