Abstract12α‐Hydroxysteroid dehydrogenase (12α‐HSDH) has the potential to convert cheap and readily available cholic acid (CA) to 12‐oxochenodeoxycholic acid (12‐oxo‐CDCA), a key precursor for chemoenzymatic synthesis of the therapeutic bile acid ursodeoxycholic acid (UDCA). In this work, a native nicotinamide adenine dinucleotide (NAD+)‐dependent 12α‐hydroxysteroid dehydrogenase (Rr12α‐HSDH) from Rhodococcus ruber was identified using a structure‐guided genome mining (SSGM) approach, which is based on the structure of cofactor pocket and the conserved nicotinamide cofactor binding motif alignment. Rr12α‐HSDH was heterologously overexpressed in Escherichia coli BL21 (DE3), purified and characterized. The purified Rr12α‐HSDH showed a high oxidative activity of 290 U mg−1protein toward CA, with a catalytic efficiency (kcat/KM) of 5.10×103 mM−1 s−1. In a preparative biotransformation (100 mL), CA (200 mM, 80 g L−1) was efficiently converted to 12‐oxo‐CDCA in 1 h, with a 85% isolated yield and a space‐time yield (STY) of up to 1632 g L−1 d−1. Furthermore, Rr12α‐HSDH was shown to be able to catalyze the oxidation of other 12α‐hydroxysteroids at high substrate loads (up to 200 mM), giving the corresponding 12‐oxo‐hydroxysteroids in 71%–85% yields, indicating the great potential of Rr12α‐HSDH as a promising biocatalyst for the synthesis of various therapeutic bile acids.magnified image
Background: Carbonic anhydrase (CA, EC 4.2.1.1), an ancient enzyme and the fastest among many enzymes, is a useful biocatalyst for carbon capture use and storage (CCUS). The use of alkaline buffers and high temperatures are favorable for biomineralization. Hence, the stability of CA under such harsh conditions is extremely important for its practical application.
Methods and results:Herein, we report a new thermostable and alkaline-tolerant α-CA (designated as LdCA), with only 26 % identity to bovine CA (BCA), which was identified by genome mining from Lactobacillus delbrueckii CGMCC 8137. It was overexpressed in Escherichia coli in a soluble form and purified to electrophoretic homogeneity by HisTrap affinity chromatography. The dimer protein had a subunit molecular weight of 23.8 kDa and showed extremely high stability at pH 6.0-11.0 and 30-60 °C. Its activity was maintained even after incubation at 90 °C for 15 min. The half-lives of the enzyme measured at 30, 40, and 50 °C were 630, 370, and 177 h, respectively. At pH 9.0, 10.0, and 11.0, its half-lives were 105, 65, and 41 min, respectively. LdCA was applied at 50 °C to accelerate the formation of calcium carbonate in a vaterite phase.
Conclusions:In summary, a new CA with high thermal and alkaline stability was identified from a general bacterium, demonstrating an effective strategy for discovering new and useful biocatalysts.
In the last 50 years the number of known natural organo-halogen compounds has grown from a dozen to more than 4000 today. 1 The most studied group of halogenase biocatalysts are haloperoxidases, which are able to accelerate a wide range of halogenation reactions following the mode of action represented in Scheme 11.1.More recently new halogenases have been identified such as 2-oxoglutarate irondependent halogenase, nucleophilic halogenases that can introduce fluorine and FADH 2 dependant halogenases. The FADH 2 cofactor enzymes can halogenate substrates with excellent regioselectivity. A good example of this reaction is the synthesis of isolated 7chlorotriptophan by Walsh et al. 2 a target synthesis that would involve many steps using traditional organic synthesis methods (Scheme 11.2).Hence this group of enzymes offers the chemical industry significant improvements in synthetic methodologies as low regio-selectivities are usually obtained in traditional halogenation reagents and these reagents are limited to reaction at specific activated sites.Halogenases are now known that regioselectively halogenate all four positions of the indole benzene ring which is not possible with chemical reagents. By replacing chloride salts by bromide salts in the reaction media bromination is possible in many cases.Dehalogenases have been known to efficiently dehalogenate activated halogen compounds and are applied on the large scale for the synthesis of (S)-chloropropionic acid as Practical Methods for Biocatalysis and Biotransformations 2, First Edition. Edited by John Whittall and Peter W. Sutton.
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