Liu Yahui, Wang Xuming, Ma Shuo, Liu Keyu, Li Wei, Zhang Lulu, DU Jie, Zhang Honglei
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, College of Chemistry and Materials Sciences, Hebei University, Baoding 071002, Hebei, China.
Hebei MuQun Biotechnology Co., Ltd., Baoding 071001, Hebei, China.
Sheng Wu Gong Cheng Xue Bao. 2025 Jan 25;41(1):321-332. doi: 10.13345/j.cjb.240238.
Screening carbonyl reductases with the ability to catalyze the reduction of complex carbonyl compounds is of great significance for the biosynthesis of -tolvaptan(-TVP). In this study, the target carbonyl reductase in the crude enzyme extract of rabbit liver was separated, purified, and identified by ammonium sulfate precipitation, gel-filtration chromatography, ion exchange chromatography, affinity chromatography, and protein mass spectrometry. With the rabbit liver genome as the template, the gene encoding the carbonyl reductase was amplified by PCR and the recombinant strain was successfully constructed. After RLSR5 was purified by affinity chromatography, its enzymatic properties were characterized. The results indicated that the gene sequence of was 972 bp, encoding a protein with a molecular weight of 40 kDa. RLSR5 was a dimeric protein, and each monomer was composed of a (α/β)-barrel structure. RLSR5 could asymmetrically reduce 7-chloro-1-[2-methyl-4-[(2- methylbenzoyl)amino]benzoyl]-5-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine (prochiral ketone, PK) to synthesize -TVP. The specific activity of the enzyme was 36.64 U/mg, and the optical purity of the product was 99%. This enzyme showcased the optimal performance at pH 6.0 and 30 °C. It was independent of metal ions, with the activity enhanced by Mn. This study lays a foundation for the biosynthesis of tolvaptan of optical grade.
筛选具有催化复杂羰基化合物还原能力的羰基还原酶对于托伐普坦（-TVP）的生物合成具有重要意义。在本研究中，通过硫酸铵沉淀、凝胶过滤色谱、离子交换色谱、亲和色谱和蛋白质质谱对兔肝脏粗酶提取物中的目标羰基还原酶进行了分离、纯化和鉴定。以兔肝脏基因组为模板，通过PCR扩增编码羰基还原酶的基因，并成功构建了重组菌株。通过亲和色谱对RLSR5进行纯化后，对其酶学性质进行了表征。结果表明，的基因序列为972 bp，编码一种分子量为40 kDa的蛋白质。RLSR5是一种二聚体蛋白，每个单体由一个（α/β）-桶状结构组成。RLSR5可以不对称还原7-氯-1-[2-甲基-4-[(2-甲基苯甲酰基)氨基]苯甲酰基]-5-氧代-2,3,4,5-四氢-1H-1-苯并氮杂卓（前手性酮，PK）以合成-TVP。该酶的比活性为36.64 U/mg，产物的光学纯度为99%。该酶在pH 6.0和30°C时表现出最佳性能。它不依赖于金属离子，Mn可增强其活性。本研究为光学级托伐普坦的生物合成奠定了基础。
