A High-specific-activity 7α-hydroxysteroid Dehydrogenase from Metagenome： Structural and Functional Characterization

doi:10.13560/j.cnki.biotech.bull.1985.2025-1466

Abstract

Abstract:

Objective To explore the 7α-hydroxysteroid dehydrogenase (7α-HSDH) gene from the gut microbiota of Nomascus concolor, heterologously express and purify it, and study its enzymatic properties, so as to provide an enzyme for the biosynthesis of ursodeoxycholic acid. Method Through bioinformatics analysis, the 7α-HSDH gene was screened from the fecal microbial metagenome of Nomascus concolor. After PCR amplification, it was cloned into the pEASY-E2 vector and heterologously expressed in E. coli BL21(DE3). The recombinant enzyme was purified and characterized. The yield of 7-ketolithocholic acid (7K-LCA), the precursor of UDCA, was determined by high-performance liquid chromatography. Result A previously uncharacterized 7α-hydroxysteroid dehydrogenase, designated NC3-2, was obtained. The enzyme is a tetramer with a molecular weight of 27.03 kD and is NAD(H)-dependent. NC3-2 exhibited the highest specific activity (485.53 U/mg) among 7α-HSDHs from other microorganisms. The optimum pH was 10.0, and the optimum temperature was 50 ℃. NC3-2 showed good pH stability, retaining over 60% residual activity after 12 h of incubation at pH 9.0-11.0. The enzyme also exhibited good thermal stability, with more than 50% residual activity after 12 h at 25, 37, 50, and 60 ℃. Ag⁺ completely inactivated NC3-2, while EDTA increased its activity to 128%. Triton X-100 and Tween-80 had strong inhibitory effects, and SDS completely inactivated the enzyme. The concentration of 7K-LCA reached 0.55 mg/mL after 8 h of catalysis by NC3-2. Conclusion A novel 7α-hydroxysteroid dehydrogenase gene, NC3-2, was obtained from the fecal microbial metagenome of Nomascus concolor and successfully heterologously expressed. The recombinant enzyme NC3-2 exhibits high specific activity and good alkali resistance. It can catalyze 20 mmol/L chenodeoxycholic acid (CDCA) to produce 7K-LCA, with an optimal reaction time of 8 h, demonstrating potential for application in the biosynthesis of ursodeoxycholic acid.

Key words: metagenome, ursodeoxycholic acid, 7α-hydroxysteroid dehydrogenase, heterologous expression, enzymatic properties

LIU Yu-ting, XIAO En-le, GONG Yu-xia, TANG Xiang-hua, HUANG Zun-xi, XU Bo. A High-specific-activity 7α-hydroxysteroid Dehydrogenase from Metagenome： Structural and Functional Characterization[J]. Biotechnology Bulletin, 2026, 42(11): 274-282.

Figures/Tables 7

Fig. 1 PCR cloning of NC3-2 (A), SDS-PAGE analysis (B), and Native-PAGE analysis (C)A: M: DNA marker; 1: NC3-2; B: M: Protein molecular weight standard; 1: Escherichia coli cell lysate containing empty plasmid pEASY-E2; 2: Unpurified 7α- HSDH NC3-2; 3: purified 7α-HSDH NC3-2; C: M: Protein molecular weight standard; 1: purified NC3-2

Table 1 Effects of metal ions and chemical reagents on 7α- HSDH NC3-2

Metal ions and chemical reagents		Relative activity （%）
Metal ions （1.25 mmol/L）	Control	100 ± 4.97
	Co²⁺	108.58 ± 7.13
	Cu²⁺	105.59 ± 13.03
	Ag⁺	0.00
	Mn²⁺	84.62 ± 8.01
	K⁺	95.03 ± 9.54
	Al³⁺	82.66 ± 9.96
	Ni⁺	97.43 ± 4.86
	Zn²⁺	92.38 ± 4.70
	Li⁺	91.49 ± 5.36
	Mg²⁺	94.46 ± 2.18
	Na⁺	97.08 ± 3.10
	Ca²⁺	86.64 ± 7.02
Chemical reagents （1%， v/v）	Acetone	90.26 ± 9.35
	EDTA	128.72 ± 10.49
	Tween-80	44.93 ± 4.54
	SDS	0.00
	DTT	94.72 ± 4.71
	TritonX-100	7.11 ±1.83

Fig. 2 Phylogenetic tree of NC3-2 and other 7α-hydroxysteroid dehydrogenases from different microorganisms

Fig. 3 Three-dimensional structure model of homotetramer of NC3-2 and visualization of binding interactionsA: The overall structure of the complex; B: binding pocket interaction (3D); C: 2D binding interaction diagram, where light green is van der Waals force, dark green is hydrogen bond, red is donor, and pink indicates alkyl interactions

Fig. 4 Optimal temperature (A) and temperature stability (B) of 7α-HSDH NC3-2

Fig. 5 Optimal pH (A) and pH stability (B) of 7α-HSDH NC3-2

Fig. 6 Yield of 7K-LCA

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