Application of 2,4-dinitrophenylhydrazine (DNPH) in high-throughput screening for microorganism mutants accumulating 9a-Hydroxyandrost-4-ene-3,17-dione (9a-OH-AD)

Abstract

To develop a quick method for the preliminarily screening of mutant strains that can accumulate 9a-hydroxyandrost-4-ene-3,17-dione (9a-OH-AD), a high-throughput screening method was presented by applying the principle that 2,4-dinitrophenylhydrazine (DNPH) can react with ketones to produce precipitation. The optimal color assay conditions were the substrate androst-4-ene-3,17-dione (AD) concentration at 2.0 g/L, the ratio of AD to DNPH solution at 1:4, and the sulfuric acid and ethanol solution percentages in DNPH solution at 2% and 35%, respectively. This method was used to preliminarily screen the mutants of Rhodococcus rhodochrous DSM43269, from which the three ones obtained could produce more 9a-OH-AD. This DNPH color assay method not only broadens screening methods and increases screening efficiency in microbial mutation breeding but also establishes a good foundation for obtaining strains for industrial application.