Abstract
To develop a quick method for the preliminarily screening of mutant strains that can accumulate 9α-hydroxyandrost-4-ene-3,17-dione (9α-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 9α-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.
Highlights
9α-Hydroxysteroids, which contain a hydroxyl group in the C-9α position of their steroid polyheterocyclic molecules, can be used as starting compounds for the synthesis of 9α-halo-11βhydroxysteroids, such as 9α-fluorohydrocortisone, triamcinolone, dexamethasone and synaflan that possess high antiallergic, antishock and anti-inflammatory activities [1, 2]
In addition to 0.5 g/L of AD, we inoculated the wild type R. rhodochrous DSM43269 in mineral medium with different AD concentration that ranged from 0 g/L to 3.0 g/L
Regardless of how much is the steroid concentration, the strain cannot normally grow in any steroid mineral medium that displays no significant difference from the control medium
Summary
9α-Hydroxysteroids, which contain a hydroxyl group in the C-9α position of their steroid polyheterocyclic molecules, can be used as starting compounds for the synthesis of 9α-halo-11βhydroxysteroids, such as 9α-fluorohydrocortisone, triamcinolone, dexamethasone and synaflan that possess high antiallergic, antishock and anti-inflammatory activities [1, 2]. Chemical processes and microbial transformations can be employed to produce 9α-hydroxysteroids. It is difficult to introduce hydroxyl groups into the C-9α position of the steroid molecules by organic synthesis. Microbial transformations, which are enzymatic reactions catalyzed by microorganisms, can exert reactions of stereo- and region- selectivity, and have many other advantages over chemical processes, e.g., mild reaction conditions and lower costs
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