一种新型恒温 7α- 羟类固醇脱氢酶的特性。

IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein and Peptide Letters Pub Date : 2024-01-01 DOI:10.2174/0109298665279004231229100320
Deshuai Lou, Yangyang Cao, Hongtao Duan, Jun Tan, Binyan Li, Yuanjun Zhou, Dong Wang
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引用次数: 0

摘要

背景:7α-羟类固醇脱氢酶(7α-HSDH)在体内仲胆汁酸的生物转化中发挥着关键作用,由于其广泛的底物特异性,在工业生物合成中具有巨大潜力。在本研究中,我们表达并鉴定了一种新型恒温 7α-HSDH (命名为 Sa 7α-HSDH):方法:DNA序列来自黑熊肠道微生物组元基因组测序数据,Sa 7α-HSDH的编码序列由化学合成。利用 pGEX-6p-1 载体对该酶进行了异源表达。随后,通过测量 340 纳米波长处的吸光度变化研究了纯化酶的活性。最后,利用 AlphaFold2.Results 预测了酶的三维结构:结果:辅酶筛选结果表明该酶依赖于 NAD(H)。底物特异性测试表明,Sa 7α-HSDH 可催化牛磺鹅去氧胆酸(TCDCA),催化效率(kcat/Km)为 3.81 S-1 mM-1。测得Sa 7α-HSDH的最适温度为75℃,证实它属于嗜热型酶。此外,还通过 32 小时的加速稳定性测试评估了它的热稳定性。在最初的 24 小时内,Sa 7α-HSDH 的催化活性基本保持不变,在 50°C 温度下 32 小时后,其功能保持了 90% 以上。Sa 7α-HSDH 在 pH 值为 10 时表现出最大活性。研究了金属离子-K+、Na+、Mg2+ 和 Cu2+ 对 Sa 7α-HSDH 酶活性的影响。在浓度为 300 mM 时,只有 Mg2+ 能使酶的活性提高 27%。K+ 和 Na+ 对酶活性都没有显著影响。只有 Cu2+ 会降低酶的活性:我们鉴定了恒温 7α-HSDH 的特性,它为类固醇在高温反应条件下的生物转化提供了一种前景广阔的生物催化剂。
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Characterization of a Novel Thermostable 7α-Hydroxysteroid Dehydrogenase.

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.

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来源期刊
Protein and Peptide Letters
Protein and Peptide Letters 生物-生化与分子生物学
CiteScore
2.90
自引率
0.00%
发文量
98
审稿时长
2 months
期刊介绍: Protein & Peptide Letters publishes letters, original research papers, mini-reviews and guest edited issues in all important aspects of protein and peptide research, including structural studies, advances in recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, and drug design. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallization and preliminary structure determination of biologically important proteins are considered only if they include significant new approaches or deal with proteins of immediate importance, and preliminary structure determinations of biologically important proteins. Purely theoretical/review papers should provide new insight into the principles of protein/peptide structure and function. Manuscripts describing computational work should include some experimental data to provide confirmation of the results of calculations. Protein & Peptide Letters focuses on: Structure Studies Advances in Recombinant Expression Drug Design Chemical Synthesis Function Pharmacology Enzymology Conformational Analysis Immunology Biotechnology Protein Engineering Protein Folding Sequencing Molecular Recognition Purification and Analysis
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