Affinity interaction of hydroxypyruvate reductase from Methylophilus spp. with Cibacron blue F3GA-derived poly(HEMA EGDMA) microspheres: partial purification and characterization

Date

1999-06-01

Author

Arica, MY
Halicigil, C
Alaeddinoglu, G
Denizli, A

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A methylotrophic hydroxypyruvate reductase was partially purified and characterized from Methylophilus spp. using the biomimetic dye, Cibacron Blue F3FA attached to poly(HEMA-EGDMA) microspheres. The absorption capacities of the dye-affinity microspheres were determined by changing pH and the concentration of the proteins in the adsorption medium. Hydroxypyruvate reductase was desorbed from the dye-affinity support specifically with 2 mM NADH solution. The enzyme was purified 10.4-fold with 47% yield. The molecular mass and subunit molecular mass of the enzyme was estimated to be 75 kDa and 37 kDa on the basis of its mobility in polyacrylamide and SDS-polyacrylamide gels, respectively. This suggested a homogeneous dimer structure. The optimal pH was between 5.0 and 7.0, and the maximum enzyme activity was obtained at 50 degrees C. The K-m values of hydroxpyruvate reductase were 0.222 mM for hydroxpyruvate and 0.067 mM for NADH.

Subject Keywords

Hydroxypyruvate reductase, NADH dependent enzyme, Methylophilus spp., Affinity adsorption, Poly(HEMA-EGDMA) microspheres, Cibacron Blue F3GA

URI

https://hdl.handle.net/11511/67723

Journal

PROCESS BIOCHEMISTRY

DOI

https://doi.org/10.1016/s0032-9592(98)00104-6

Collections

Department of Biology, Article

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Citation Formats

M. Arica, C. Halicigil, G. Alaeddinoglu, and A. Denizli, “Affinity interaction of hydroxypyruvate reductase from Methylophilus spp. with Cibacron blue F3GA-derived poly(HEMA EGDMA) microspheres: partial purification and characterization,” PROCESS BIOCHEMISTRY, pp. 375–381, 1999, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/67723.