Exploitation of the alcohol dehydrogenase‐acetone NADP‐regeneration system for the enzymatic preparative‐scale production of 12‐ketochenodeoxycholic acid

Fossati, Elena; Polentini, Fausto; Carrea, Giacomo; Riva, Sergio

doi:10.1002/bit.20753

Cited by 32 publications

(15 citation statements)

References 16 publications

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“…In the first general strategy, NADPH is regenerated using a rare NADP-dependent dehydrogenase, ADH 15,16 and glucose dehydrogenase, 17,18 or an engineered FDH that can accept NADP þ instead of its inherent substrate NAD þ . 19,20 The second intriguing approach involves the use of the soluble pyridine nucleotide transhydrogenase (STH), which catalyzes the transfer of reducing equivalents between NAD(P) þ and NAD(P)H (Scheme 1).…”

Section: Introductionmentioning

confidence: 99%

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

Mouri

Shimizu

Kamiya

et al. 2009

Biotechnology Progress

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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 microM of a model substrate (p-nitrophenoxydecanoic acid) was attained using 50 microM NADPH. In contrast, with the two-step cofactor regeneration, the same amount of substrate was completely converted using 5 microM 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(+).

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Section: Introductionmentioning

confidence: 99%

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

Mouri

Shimizu

Kamiya

et al. 2009

Biotechnology Progress

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“…Differences can be caused by using progress curves for analysis in the presence of cholic acid. Fossati et al (2006) compared high concentrations of acetone up to 25% (v/v) for the enzymatic oxidation of cholic acid in order to switch the equilibrium in favor of 12-Keto-CDCA. Therefore, initial conditions of substrate concentrations for progress curve measurements were chosen on a very high level as well as the addition of 100 mM cholic acid to cover typical concentrations of industrial applications to simulate process conditions.…”

Section: Resultsmentioning

confidence: 99%

“…The reaction mixtures were prepared based on published data of preparative-scale oxidation of cholic acid (Fossati et al, 2006). A total of 50 mM of potassium phosphate buffer (pH 8.0) was supplemented with all reactants as indicated in the figure legends.…”

Section: Enzymatic Oxidation Of Cholic Acid To 12-keto-cdca With Intementioning

confidence: 99%

“…In spite of this, some applications are investigated and currently applied (Kroutil et al, 2004). In the regiospecific enzymatic oxidation of cholic acid to 12-Keto-CDCA, the successful use of different alcohol dehydrogenases (ADH) for NADP þ regeneration could be shown (Fossati et al, 2006). In Figure 1, the reaction scheme is depicted for this biocatalytic system.…”

Section: Introductionmentioning

confidence: 98%

“…ADH can also be used for regeneration of the cofactor NADPH (Eckstein et al, 2004) or NADP þ (Fossati et al, 2006). In our laboratory, we expressed a NADPH-dependent ADH from Thermoanaerobacter ethanolicus (ADH-TE).…”

Section: Introductionmentioning

confidence: 99%

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

Braun¹,

Link²,

Liu³

et al. 2011

Biotech & Bioengineering

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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.

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Biocatalysis

Torrelo

Hanefeld

Hollmann

2015

Kirk-Othmer Encyclopedia of Chemical Technology

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Biocatalysis, the use of enzymes—either as isolated enzymes or in whole cells—to accelerate chemical transformations, is one of the approaches available to the organic chemist. Key advantages of biocatalysis are its high selectivity and the high rate of chemical transformations at relatively mild reaction conditions, and as a result, the simplified downstream processing and more cost‐effective synthesis. Therefore, it is not surprising that interest in industrial biocatalysis has been on the rise. The aim of this chapter is to provide the process chemist with a general overview of the available enzymes and the chemistries they enable and to discuss concepts in biocatalysis highlighted by selected examples.

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Exploitation of the alcohol dehydrogenase‐acetone NADP‐regeneration system for the enzymatic preparative‐scale production of 12‐ketochenodeoxycholic acid

Cited by 32 publications

References 16 publications

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

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

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

Biocatalysis

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