Research Article: Influence of Ion Strength and pH on Thermal Stability of Yeast Formate Dehydrogenase

Date Published: July , 2010

Publisher: A.I. Gordeyev

Author(s): V.I. Tishkov, S.V. Uglanova, V.V. Fedorchuk, S.S. Savin.



The kinetics of the thermal inactivation of recombinant wild–type formate dehydrogenase
from Candida boidinii yeast was studied in the temperature range of
53–61oC and pH 6.0, 7.0, and 8.0. It was shown that the loss of the
enzyme’s activity proceeds via a monomolecular mechanism. Activation parameters
∆Н­ and ∆S­ were calculated based on the temperature
relations dependence of inactivation rate constants according to the transition state theory.
Both parameters are in a range that corresponds to globular protein denaturation processes.
Optimal conditions for the stability of the enzyme were high concentrations of the phosphate
buffer or of the enzyme substrate sodium formate at pH = 7.0.

Partial Text

NAD+–dependent formate dehydrogenase (EC, FDH)
belongs to the superfamily of D–specific dehydrogenases of 2–hydroxyacids [1]. Because of the simplicity of the catalyzed reaction, which
is a simple transfer of the hydride ion in the active site between the formate and the C4 atom
of the nicotinamide ring with no acidic–basic catalysis involved, FDH is
used as a model system for studying the enzyme catalytic mechanism of the whole superfamily.

For this work we used a preparation of recombinant formate dehydrogenase originating from
wild–type Candida boidinii yeast. Cultivation of E. coli
(BL21(DE3)/pCboFDH) cells expressing the C. boidinii FDH was performed at 25°C in 250 ml or 1l shaker flasks with baffles using 50 or 250 ml
of medium, respectively. The medium consisted of 16 g/l tryptone, 1 0 g/ l of yeast extract, 1
g/l of sodium chloride, 1.5 g/l of H2NaPO4, 1 g/l of HK2PO4, 100 micrograms/ml of ampicillin,
and 25 micrograms/ml of chloramphenicol. The volume of the bacterial inoculate was equal to
10–15% of the medium volume. Lactose was used for the unduction of FDH
biosynthesis and an inducer was added to a final concentration of 20 mg/ml when absorbance of
the cell suspension at 600 nm ( А 600 ) reached the value
0.5 – 0. 7 . The cells were then grown in maximum aeration overnight. Then, the cells
were spun down on a Beckman J–21 (United States) centrifuge at 8000 rpm for 20 minutes at
4°С. Recombinant CboFDH was then purified according to the standard
protocol developed for Pseudomonas sp.101 FDH [10]. The enzyme purification procedure involved the
destruction of the 10% w/v cell suspension in a 0.1 М potassium–phosphate buffer,
0.02 M EDTA, and рН 8.0 using a Braun Sonic ultrasound disintegrator (Germany) at
0°С, the precipitation of some of the ballast proteins with ammonium sulfate (35% of
saturation), hydrophobic chromatography on a Fast Protein Liquid Chromatography (FPLC)
apparatus (Pharmacia Biotech, Sweden) using a column with Phenyl Sepharose Fast Flow from the
same company, and gel filtration on a Sephacryl S200 column. The obtained preparations were at
least 95% pure as assayed by an analytical gel electrophoresis in a 12% polyacrylamide gel in
denturating conditions.

Effect of pH on the Thermal Inactivation Rate of Recombinant C.
boidinii FDH.

Formate dehydrogenase is widely used in dehydrogenase catalyzed synthesis of chiral compounds
as a coenzyme regenerating catalyst, and high concentrations (up to 2–3 M) of
formate–ion, substrate of FDH, are used to achive high turnover of
coenzyme. This is why we decided to examine CboFDH thermal inactivation
kinetics upon varying sodium formate concentrations at two pH values: 7.0 and 8.0 (Fig. 6) since these are the values which are most often used for
enzymatic synthesis processes involving dehydrogenases. As can be seen in Fig. 6, the dramatic stabilization of the enzyme is observed at high
concentrations of sodium formate. This effect is especially notable at concentrations of sodium
formate reaching 1.5 М. As in the case of the relation between the inactivation rate
constant and the concentration of the phosphate ion, the stabilization effect is more
pronounced at рН 8.0 than at р Н 7. 0 (Fig. 6).