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A. A. Pometun, K. M. Boyko, S. A. Zubanova, A. Yu. Nikolaeva, D. L. Atroshenko, S. S. Savin, V. I. Tishkov

Preparation of recombinant formate dehydrogenase from thermotolerant yeast Ogataea Parapolymorpha and crystallization of apo- and holo- forms of the enzyme


NAD+-dependent formate dehydrogenase from thermotolerant yeast Ogataea parapolymorpha DL-1 (OpaFDH, EC with additional Gly residue at N-terminus and it double mutant OpaFDH_AD were over expressed in E. colicells with yield 6000 and 6200 U per liter of cultivation medium, respectively. Purified enzymes were obtained as homogeneous preparations with activity yield 62%. Purification procedure included ultrasonic cell disruption, heat treatment of cell free extract at 55 oC for 15 min and hydrophobic chromatography on Phenyl Sepharose Fast Flow. Crystallization experiments with wild-type OpaFDH resulted in preparation of crystals of apo- but not holo- form. Crystals of holo-form were obtained with mutant OpaFDH_AD only in the presence of 7 mM NAD+ and 10 mM sodium azide. Size and quality of crystals were enough for collection of X-ray diffraction data.
Key words: formate dehydrogenase, Ogataea parapolymorpha DL-1,expression, purification, crystallization, apo- and holo- forms.
Moscow University Chemistry Bulletin.
2021, Vol. 62, No. 2, P. 130

Copyright (C) Chemistry Dept., Moscow State University, 2002
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