A novel carbonyl reductase for the synthesis of (<i>R</i>)-tolvaptan.

Yahui LIU; Xuming WANG; Shuo MA; Keyu LIU; Wei LI; Lulu ZHANG; Jie DU; Honglei ZHANG

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

Author: Yahui LIU ¹ ; Xuming WANG ¹ ; Shuo MA ¹ ; Keyu LIU ¹ ; Wei LI ¹ ; Lulu ZHANG ² ; Jie DU ¹ ; Honglei ZHANG ¹
Author Information

1. 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.
2. Hebei MuQun Biotechnology Co., Ltd., Baoding 071001, Hebei, China.
Publication Type:Journal Article
Keywords: asymmetric reduction; carbonyl reductase; protein purification; rabbit liver; tolvaptan
MeSH: Animals; Rabbits; Alcohol Oxidoreductases/biosynthesis*; Recombinant Proteins/metabolism*; Escherichia coli/metabolism*; Liver/enzymology*
From: Chinese Journal of Biotechnology 2025;41(1):321-332
CountryChina
Language:Chinese
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 °C. 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.