催化合成R型托伐普坦的新型羰基还原酶

A novel carbonyl reductase for the synthesis of (R)-tolvaptan

摘要:

筛选具有催化还原复杂羰基化合物能力的羰基还原酶对于R型托伐普坦(R-tolvaptan,R-TVP)的生物合成具有重要意义。本研究分别采用硫酸铵盐析、葡聚糖分子排阻、离子交换层析、亲和层析以及蛋白质质谱的方法对兔肝脏粗酶中的目标羰基还原酶进行分离纯化和鉴定。以兔肝脏基因组为模板，PCR扩增得到羰基还原酶rlsr5的基因片段，并成功构建重组表达菌株。对诱导表达的RLSR5进行亲和层析纯化后，表征其酶学性质。研究结果表明，rlsr5基因序列为972 bp，编码蛋白的分子量为40 kDa，属于二聚体蛋白，每个单体由(α/β)₈-桶结构组成。RLSR5可以不对称还原前手性酮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-苯氮䓬(prochiral ketone,PK)合成R-TVP。该酶的比活为36.64 U/mg，产物的光学纯度e.e.值为99%。该酶反应最适pH值为6.0，最适温度为30℃。该酶不是金属离子依赖性的酶，Mn²⁺对酶活力有促进作用。本研究为光学纯托伐普坦的生物催化合成奠定了基础。

关键词:兔肝脏;蛋白纯化;羰基还原酶;不对称还原;托伐普坦

Abstract:

Screening carbonyl reductases with the ability to catalyze the reduction of complex carbonyl compounds is of great significance for the biosynthesis of R-tolvaptan(R-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 rlsr5 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 rlsr5 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)₈-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 R-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 ℃. It was independent of metal ions, with the activity enhanced by Mn²⁺. This study lays a foundation for the biosynthesis of tolvaptan of optical grade.

Key words:rabbit liver;protein purification;carbonyl reductase;asymmetric reduction;tolvaptan