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.
CONCLUSIONS: We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.
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.
CONCLUSIONS: We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.
摘要：
背景：7α-羟基类固醇脱氢酶（7α-HSDH）在体内次级胆汁酸的生物转化中起着关键作用，并且由于其广泛的底物特异性，在工业生物合成中具有巨大的潜力。在这项研究中,我们表达并表征了一种新型的热稳定性7α-HSDH（命名为Sa7α-HSDH）。
方法：DNA序列来源于黑熊肠道微生物组宏基因组测序数据，化学合成了Sa7α-HSDH的编码序列。使用pGEX-6p-1载体进行酶的异源表达。随后，通过测量340nm处的吸光度变化来研究纯化酶的活性。最后,用AlphaFold2预测三维结构。
结果：辅酶筛选结果证实它是NAD（H）依赖性的。底物特异性测试表明，Sa7α-HSDH可以催化牛磺鹅去氧胆酸（TCDCA），催化效率（kcat/Km）为3.81S-1mM-1。Sa7α-HSDH的最佳温度为75°C，确认它属于嗜热酶。此外,在32小时内使用加速稳定性测试评估其热稳定性。Sa7α-HSDH的催化活性在最初24小时内基本保持不变，在50°C下32小时后保留了90%以上的功能。Sa7α-HSDH在pH10时表现出最大活性。金属离子-K+的作用,Na+,研究了Mg2+和Cu2+-对Sa7α-HSDH酶活性的影响。在300mM的浓度下,仅观察到Mg2+使酶活性增强27%。K+和Na+对活性均无显著影响。仅发现Cu2+降低酶活性。
结论：我们表征了热稳定的7α-HSDH，它为类固醇在高反应温度下的生物转化提供了有前途的生物催化剂。
方法：DNA序列来源于黑熊肠道微生物组宏基因组测序数据，化学合成了Sa7α-HSDH的编码序列。使用pGEX-6p-1载体进行酶的异源表达。随后，通过测量340nm处的吸光度变化来研究纯化酶的活性。最后,用AlphaFold2预测三维结构。
结果：辅酶筛选结果证实它是NAD（H）依赖性的。底物特异性测试表明，Sa7α-HSDH可以催化牛磺鹅去氧胆酸（TCDCA），催化效率（kcat/Km）为3.81S-1mM-1。Sa7α-HSDH的最佳温度为75°C，确认它属于嗜热酶。此外,在32小时内使用加速稳定性测试评估其热稳定性。Sa7α-HSDH的催化活性在最初24小时内基本保持不变，在50°C下32小时后保留了90%以上的功能。Sa7α-HSDH在pH10时表现出最大活性。金属离子-K+的作用,Na+,研究了Mg2+和Cu2+-对Sa7α-HSDH酶活性的影响。在300mM的浓度下,仅观察到Mg2+使酶活性增强27%。K+和Na+对活性均无显著影响。仅发现Cu2+降低酶活性。
结论：我们表征了热稳定的7α-HSDH，它为类固醇在高反应温度下的生物转化提供了有前途的生物催化剂。
