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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Characterization of a Novel Thermostable 7α-Hydroxysteroid Dehydrogenase

Author(s): Deshuai Lou, Yangyang Cao, Hongtao Duan, Jun Tan*, Binyan Li, Yuanjun Zhou and Dong Wang*

Volume 31, Issue 2, 2024

Published on: 26 January, 2024

Page: [153 - 160] Pages: 8

DOI: 10.2174/0109298665279004231229100320

Price: $65

Abstract

Background: 7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays a pivotal role in vivo in the biotransformation of secondary bile acids and has great potential in industrial biosynthesis due to its broad substrate specificity. In this study, we expressed and characterized a novel thermostable 7α-HSDH (named Sa 7α-HSDH).

Methods: The DNA sequence was derived from the black bear gut microbiome metagenomic sequencing data, and the coding sequence of Sa 7α-HSDH was chemically synthesized. The heterologous expression of the enzyme was carried out using the pGEX-6p-1 vector. Subsequently, the activity of the purified enzyme was studied by measuring the absorbance change at 340 nm. Finally, the three-dimensional structure was predicted with AlphaFold2.

Results: Coenzyme screening results confirmed it to be NAD(H) dependent. Substrate specificity test revealed that Sa 7α-HSDH could catalyze taurochenodeoxycholic acid (TCDCA) with catalytic efficiency (kcat/Km) 3.81 S-1 mM-1. The optimum temperature of Sa 7α-HSDH was measured to be 75°C, confirming that it belongs to thermophilic enzymes. Additionally, its thermostability was assessed using an accelerated stability test over 32 hours. The catalytic activity of Sa 7α-HSDH remained largely unchanged for the first 24 hours and retained over 90% of its functionality after 32 hours at 50°C. Sa 7α-HSDH exhibited maximal activity at pH 10. The effect of metal ions-K+, Na+, Mg2+ and Cu2+-on the enzymatic activity of Sa 7α-HSDH was investigated. Only Mg2+ was observed to enhance the enzyme’s activity by 27% at a concentration of 300 mM. Neither K+ nor Na+ had a significant influence on activity. Only Cu2+ was found to reduce enzyme activity.

Conclusion: We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.

Keywords: 7α-Hydroxysteroid dehydrogenase, thermostability, catalytic activity, short-chain dehydrogenase/reductase, metagenomics, bioconversion.

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