Enhancing the sustainability of KsdD as a biocatalyst for steroid transformation by immobilization on epoxy support

The A1-dehydrogenation of 3-ketosteroid substrates is a crucial reaction in the production of steroids. Although 3-ketosteroid A1-dehydrogenase (KsdD) catalyzes this reaction with high efficiency and selectivity, the low stability and high cost of the purified enzyme catalyst have limited its industrial application. In this study, an epoxy support was used to evaluate the covalent immobilization of KsdD from Pimelobacter simplex, and the best androsta-1,4-diene-317-dione (ADD) production was achieved after optimized immobilization of KsdD enzyme in 1.5 M NaH2PO4- Na2HPO4 buffer (pH 6.5) for 12 h at 25 ?C. The immobilized KsdD exhibited higher tolerance toward 20 % methanol. The dehydrogenation reaction reached a conversion efficiency of up to 90.0 % in 2 h when using 0.6 mg/mL of 4-androstene-317-dione (AD). The W299A and W299 G mutants of KsdD were also immobilized, and both showed the better catalytic performance with higher kcat/KM values compared with the wild type (WT). The immobilized W299A, W299 G and WT KsdD respectively maintained 70.5, 65.7 and 38.7 % of their initial activity at the end of 15 reaction cycles. Furthermore, the W299A retained 66.3 % of the initial activity after 30 days of incubation at 4 ?C, and was more stable than free KsdD, Thus, the immobilized W299A is a promising biocatalyst for steroid dehydrogenation. In this study, we investigated the application of immobilized enzymes for the dehydrogenation of steroids, which will be of great importance for improving the development of green technology and sustainable use of biocatalysts in the steroid manufacturing industry.

Automated summary

The study successfully immobilized KsdD enzyme on an epoxy support, achieving high ADD production and demonstrating high tolerance of immobilized KsdD to 20% methanol. The W299A mutant showed higher stability and catalytic performance, with greater potential for application. The use of immobilized enzymes for steroid dehydrogenation in this study is important for advancing green technology and sustainable biocatalyst use in the steroid manufacturing industry.