Over-expression of Mycobacterium neoaurum 3-ketosteroid-∆ 1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3, 17-dione to androst-1, 4-diene-3, 17-dione

Background: 3-Ketosteroid-∆¹-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen asa new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksdd n) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 µM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADDfromADefficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production.

protective effects of demethoxyviridine and 1-alpha-hydroxy-demethoxyviridine in the livers of male rats treated with diethylnitrosamine and 2-acetylaminflourene

To determine the protective effects of a fungal metabolite of demethoxyviridine [DMV] and its derivative, 1-alpha-hydroxy-DMV in the livers of 2-month-old male Spraque-Dawley rats treated with diethylnitrosamine [DEN] and 2-acetylaminflourene [2-AAF]. This study was performed in the Department of Medical Biology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey from May 2006. Animals were divided into 10 groups. Those were the control, olive oil, dimethyl sulfoxide [DMSO], DMV, 1-alpha-hydroxy-DMV, DEN, 2-AAF, DEN+2-AAF, DEN+2-AAF+DMV, and DEN+2-AAF+1-alpha-hydroxy-DMV-treated animal groups. The liver microsomes were prepared from rats and the levels of expression of cytochrome P450 1A2 [CYP1A2] enzymes were determined with western blot technique. The liver tissue slides were evaluated histopathologically with hematoxylin and eosin staining and immunohistochemically for Harvey-retrovirus associated DNA sequences [Ha-Ras], glutathione S- transferase [GST-p], and connexion-32 [Cx32] proteins. Notably, there were no appreciable differences in CYP1A2 level among control, olive oil, and DMSO-treated animals. The CYP1A2 level was significantly decreased in 2-AAF, DEN+2-AAF, DEN, DEN+2-AAF+DMV, DEN+2-AAF+1-alpha-hydroxy-DMV, 1-alpha-hydroxy-DMV, and DMV-treated animals as compared to the control. Most prenoplastic focus was found in DEN+2-AAF treated group. Demethoxyviridine and 1-alpha-hidroksi-DMV had protective effect in the livers of DEN, 2-AAF and DEN+2-AAF induced rats

Transformation of sitosterol to androsta-1, 4-diene-3, 17-dione by immobilized Mycobacterium cells.

Transformation of sitosterol to androsta-1, 4-diene-3, 17-dione was studied with Mycobacterium cells entrapped in various polymeric matrices. Of the three supports viz. alginate, carageenan, agarose and polyacrylamide, studied, the polyacrylamide immobilized cells showed optimum catalytic stability and reusability.