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.
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。