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. 2024 Jun 29;108(1):399.
doi: 10.1007/s00253-024-13221-3.

Sources and control of impurity during one-pot enzymatic production of dehydroepiandrosterone

Affiliations

Sources and control of impurity during one-pot enzymatic production of dehydroepiandrosterone

Jiawei Dai et al. Appl Microbiol Biotechnol. .

Abstract

Dehydroepiandrosterone (DHEA) has a promising market due to its capacity to regulate human hormone levels as well as preventing and treating various diseases. We have established a chemical esterification coupled biocatalytic-based scheme by lipase-catalyzed 4-androstene-3,17-dione (4-AD) hydrolysis to obtain the intermediate product 5-androstene-3,17-dione (5-AD), which was then asymmetrically reduced by a ketoreductase from Sphingomonas wittichii (SwiKR). Co-enzyme required for KR is regenerated by a glucose dehydrogenase (GDH) from Bacillus subtilis. This scheme is more environmentally friendly and more efficient than the current DHEA synthesis pathway. However, a significant amount of 4-AD as by-product was detected during the catalytic process. Focused on the control of by-products, we investigated the source of 4-AD and identified that it is mainly derived from the isomerization activity of SwiKR and GDH. Increasing the proportion of glucose in the catalytic system as well as optimizing the catalytic conditions drastically reduced 4-AD from 24.7 to 6.5% of total substrate amount, and the final yield of DHEA achieved 40.1 g/L. Furthermore, this is the first time that both SwiKR and GDH have been proved to be promiscuous enzymes with dehydrogenase and ketosteroid isomerase (KSI) activities, expanding knowledge of the substrate diversity of the short-chain dehydrogenase family enzymes. KEY POINTS: • A strategy of coupling lipase, ketoreductase, and glucose dehydrogenase in producing DHEA from 4-AD • Both SwiKR and GDH are identified with ketosteroid isomerase activity. • Development of catalytic strategy to control by-product and achieve highly selective DHEA production.

Keywords: DHEA; Impurity control strategy; Ketosteroid isomerase activity; Promiscuous enzymes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Scheme 1
Scheme 1
Chemical and biosynthesis preparation of DHEA
Scheme 2
Scheme 2
Chemical esterification coupled biocatalytic preparation of DHEA by 4-AD
Fig. 1
Fig. 1
Screening of lipases with the capacity to hydrolyze 3-ethoxy-androsta-3,5-dien-17-one. a The TLC profile of the in vitro enzyme assay illustrates the hydrolytic activity of lipase. Lines 1 and 2 represent lipase samples without or with hydrolytic target substrate activity, respectively. CK (control) indicates standard compounds. Solvent system: ethyl acetate:n-hexane (1:2, v/v), chromogenic agent: 0.8 M potassium permanganate. b Comparison of substrate conversion rates of different lipases in second screening. The catalytic generation of 5-AD was assayed at pH 6, 30 °C for 8 h
Fig. 2
Fig. 2
Optimization of reaction conditions of the hydrolysis reaction: a the reaction pH; b the reaction temperature. The buffer used in the assay mixture with the following: acetate (circles) (pH 4.0–6.0), phosphate (square) (pH 6.0–8.0), and Tris–HCl (triangles) (pH 8.0–10.0). The black line represents the relative reaction rate of the target product 5-AD, and the red line represents the yield of the by-product 4-AD. The maximum generation rate of 5-AD was set to 100%
Fig. 3
Fig. 3
Organic solvent influence on the Lipase PS Amano SD hydrolysis activity. a Effect of different organic solvents on lipase activity. b Effect of logP value of organic solvent on lipase activity. c Process monitoring of Lipase PS Amano SD hydrolysis of 3-ethoxy-androsta-3,5-dien-17-one was assayed under optimal conditions for 8 h. The hydrolysis activity for the generation of 5-AD in the pure aqueous phase was set to 100%
Fig. 4
Fig. 4
Results of a screening for the catalytic generation of DHEA by dual enzyme coupling system and sources of 4-AD generation. a The HPLC chromatography of reaction mixture composition of SwiKR and GDH and standard compounds was shown. The reaction was assayed at pH 6.3, 25 g/L 5-AD, 25 g/L glucose, 250 mg/L NAD+ and NADP+, at 35 °C. b Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) analysis of SwiKR and GDH expression and purification. Lane M, marker; lane 1, purified GDH; lane 2, purified SwiKR; and loading volume, 5 μL. c The source of by-product 4-AD produced by each part of the catalytic system. SwiKR or GDH pure enzyme were used in the activity assay. The 4-AD content produced by the enzyme-free system was set as a control, and the amount of 4-AD produced by the remaining three parts was removed from the control
Fig. 5
Fig. 5
Factors influencing the isomerization activity of GDH enzyme and their catalytic reactions: a Effects of glucose and NAD(P)+ on the isomerization activity of GDH. b Primary/side catalytic reactions of GDH in dual enzyme coupling system. The control was the pure enzyme system without the addition of any other substrates. The reaction was assayed at pH 6.0, 10 g/L 5-AD, 50 g/L glucose, or 500 mg/L NAD+ and NADP+, at 30 °C
Fig. 6
Fig. 6
Effect of glucose and NAD(P)+ inhibition on GDH isomerization products: a The isomerization products of GDH pure enzyme were compared in the presence of 25 g/L glucose, 0–2.0 g/L NAD(P)+; b the isomerization products of GDH pure enzyme were compared in the presence of 500 mg/L NAD(P)+, 0–400 g/L glucose
Fig. 7
Fig. 7
Factors influencing the isomerization activity of SwiKR enzyme and their catalytic reactions: a Effects of oxidation or reduction form cofactors on the isomerization activity of SwiKR; b primary/side catalytic reactions of SwiKR in dual enzyme coupling system. The control was the pure enzyme system without the addition of any other substrates. The reaction was assayed at pH 6.0, 10 g/L 5-AD, 50 g/L glucose, or 500 mg/L NAD+ and NADP+, at 30 °C
Fig. 8
Fig. 8
Effect of glucose on catalytic results: a Time course analysis of the production of 4-AD in the dual enzyme coupling system after optimization of glucose and cofactors. The catalytic process was monitored with or without 50 g/L glucose and 500 mg/L of NAD(P)+; b comparison of by-products containing products in the catalytic solution after the reaction

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