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

Arica M., Halicigil C., Alaeddinoglu G., Denizli A.

PROCESS BIOCHEMISTRY, vol.34, no.4, pp.375-381, 1999 (Peer-Reviewed Journal) identifier identifier

  • Publication Type: Article / Article
  • Volume: 34 Issue: 4
  • Publication Date: 1999
  • Doi Number: 10.1016/s0032-9592(98)00104-6
  • Journal Indexes: Science Citation Index Expanded, Scopus
  • Page Numbers: pp.375-381


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. (C) 1999 Elsevier Science Ltd. All rights reserved.