宏基因组来源高比活7α-羟基类固醇脱氢酶结构和功能表征

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

摘要/Abstract

摘要：

目的挖掘肠道微生物7α羟基类固醇脱氢酶（7α-HSDH）基因，异源表达、纯化并研究其酶学性质，为熊去氧胆酸（UDCA）的生物合成提供酶原。方法通过生物信息学分析，从西黑冠长臂猿粪便微生物宏基因组中筛选7α-HSDH基因，经PCR扩增后克隆至pEASY-E2载体，在大肠杆菌BL21(DE3)中进行异源表达。重组酶经纯化和酶学性质表征，利用高效液相色谱法测定该酶催化鹅去氧胆酸（CDCA）生成UDCA的前体7-酮石胆酸（7K-LCA）的产量。结果获得未被表征的7α-HSDH NC3-2，该酶为四聚体，分子量为27.03 kD，辅酶与NAD(H)相关。NC3-2较其他微生物来源的7α-HSDH 具有最高比活485.53 U/mg，最适作用条件为pH 10.0、50 ℃。该酶pH稳定性较好，pH 9.0-11.0条件下孵育12 h，保持60%以上剩余酶活；热稳定性有待提升，25、37、50、60 ℃下分别孵育12 h，剩余酶活保持在50%以上。EDTA可将其酶活性提高至128%；Triton X-100和Tween-80抑制作用较大，Ag⁺与SDS使其完全失活。NC3-2催化CDCA反应8 h，7K-LCA浓度即可达到0.55 mg/mL。结论从西黑冠长臂猿粪便微生物宏基因组中获得7α-HSDH基因NC3-2并成功实现异源表达。重组酶NC3-2具有较高的比活力与良好的耐碱性，在pH 10.0、30 ℃条件下可催化20 mmol/L CDCA生成7K-LCA，反应8 h时产物浓度趋于饱和。该酶在熊去氧胆酸的生物合成中具有应用潜力。

关键词: 宏基因组, 熊去氧胆酸, 7α-羟基类固醇脱氢酶, 异源表达, 酶学性质

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. Methods 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. Results 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 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

刘雨婷, 肖恩乐, 龚雨霞, 唐湘华, 黄遵锡, 许波. 宏基因组来源高比活7α-羟基类固醇脱氢酶结构和功能表征[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1466.

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

图/表 7

图1 NC3-2的PCR克隆（A）、SDS-PAGE分析（B）与Native-PAGE分析（C）A：M：DNA marker；1：NC3-2；B：M：蛋白分子量标准；1：含空载质粒pEASY-E2的大肠杆菌细胞裂解液；2：未纯化7α-HSDH NC3-2；3：7α-HSDH NC3-2纯酶；C：M：蛋白分子量标准；1：NC3-2纯酶

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

表1 金属离子和化学试剂对7α-羟基类固醇脱氢酶NC3-2的影响

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

图2 NC3-2与不同微生物来源的7α-羟基类固醇脱氢酶的系统进化树

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

图3 NC3-2的同源四聚体三维结构模型及结合作用力可视化A：复合物整体结构；B：结合口袋相互作用（3D）；C：结合作用力图（2D），其中：浅绿色为范德华力，深绿色为氢键，红色为供体，粉色为烷基

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

图4 7α-羟基类固醇脱氢酶NC3-2的最适温度（A）和温度稳定性（B）

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

图5 7α-羟基类固醇脱氢酶NC3-2的最适pH（A）和pH稳定性（B）

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

图6 7K-LCA产量

Fig. 6 Yield of 7K-LCA

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