Covalent immobilization of Candida methylica formate dehydrogenase on short spacer arm aldehyde group containing supports

• The molecular weight of monomer of CmFDH was found to be 42408.0 Da.
• Immobilized FDHs used as effective biocatalysts for in situ regeneration of NADH was prepared.
• Aldehyde-functionalized Immobead 150 is better support for the immobilization of CmFDH.
• CmFDH showed 1.32 times higher activity when it was immobilized on modified Immobead 150.
• 3.6 times more stabilization was obtained for CmFDH by immobilizing on modified Immobead 150.

Formate dehydrogenases are promising enzymes for in situ regeneration of reduced form of nicotinamide adenine dinucleotide. However, the low operational stability of free form of formate dehydrogenases restricts their industrial uses. In this study, three different aldehyde group containing supports, such as glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 were evaluated for the covalent immobilization of formate dehydrogenase from Candida methylica. The immobilization – activity yields were determined as 75–68%, 60–84%, and 90–132%, respectively for glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 supports for formate oxidation. The formate production activity of Candida methylica formate dehydrohenase was tested for different initial bicarbonate concentrations; however, no product was determined. The optimum pHs of free FDH and immobilized FDH on aldehyde-functionalized Immobead 150 were both 7.0, whereas the optimum pHs of the immobilized FDHs on glyoxyl silica and glyoxyl agarose were both 8.0. The optimum temperatures of all the FDH preparations were determined as 35 °C. KM values of free FDH and immobilized FDH on glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 supports were determined as 4.18 ± 0.22, 3.06 ± 0.14, 3.22 ± 0.18, and 2.79 ± 0.15 mM, respectively for formate and 0.51 ± 0.05, 0.49 ± 0.02, 0.59 ± 0.05, and 0.15 ± 0.04 mM, respectively for NAD+. The half-life time values of immobilized FDH on glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 supports were 2.8, 3.1 and 3.6 times higher than that of the free FDH, respectively at 35 °C. The immobilized FDH on glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 supports were remained 60, 56 and 51% of their initial activities, respectively at the end of 10 reuses. These results show that the immobilized FDHs may be used for in situ regeneration of NADH along with oxidoreductases.

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