Abstract
Dehydrogenases from fungi are attracting attention as industrial biocatalysts due to their high activity and chiral selectivity. However, these enzymes form insoluble aggregates when overexpressed in E. coli, limiting their industrial application. In the present study, we report the systematic development of a refolding process for selected, industrially relevant fungal dehydrogenases, viz., formate dehydrogenase from Candida boidinii (CbFDH) and formate and alcohol dehydrogenases from Geotrichum candium (GcFDH and GcADH, respectively). We first employed a screen to evaluate the effects of different variables on refolding including the buffer system, additives, and rate of dilution. The extent of refolding was determined by enzyme assays, circular dichroism, and tryptophan fluorescence. Our results showed that glycerol and reducing environment are essential for refolding of these dehydrogenases. Further, slow dilution of solubilized protein over 16 h dramatically improved the recovery of refolded enzymes compared to rapid dilution. The importance of slow dilution was further confirmed in a 10-fold scaled-up refolding trial. Overall, we demonstrate a robust method for refolding of fungal dehydrogenases, thus improving their availability for various biocatalytic applications.
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