Nitrate reductase from yeasts, the preparation and use thereof

A process for the preparation of an NAD(P)H-dependent nitrate reductase comprising:

• cultivating yeast cells, wherein the yeast cells are of the genera Candida or Hansenula, in a synthetic medium comprising nitrate as the sole nitrogen source;
• disrupting said cells in a phosphate buffer having a pH of about 6.5 to about 8.5, in the presence of a complexing agent, mercaptoethanol, FAD, potassium hexacyanoferrate, and a protease inhibitor, to form a cell extract;
• removing cell debris from said cell extract by centrifugation, the debris produced by said disrupting;
• chromatographing said cell extract on an anion exchange material to obtain fractions comprising nitrate reductase;
• mixing said fractions with about 10% to about 50% protein;
• concentrating said fractions by ultrafiltration;
• and
• drying said concentrated fractions by fluidized bed granulation.

A process for the preparation of an NAD(P)H-dependent nitrate reductase comprising:

• cultivating yeast cells in a synthetic medium comprising nitrate as the sole nitrogen source wherein the yeast cells are of the genera Candida or Hansenula;
• disrupting said cells in a phosphate buffer having a pH of about 6.5 to about 8.5, in the presence of a complexing agent, mercaptoethanol, FAD, potassium hexacyanoferrate, and a protease inhibitor, to form a cell extract;
• removing cell debris from said cell extract by centrifugation, the debris produced by said disrupting;
• chromatographing said cell extract, obtained directly from said centrifugation, on an anion exchange material to obtain fractions comprising nitrate reductase;
• mixing said fractions obtained directly from said chromatography with about 10% to about 50% protein;
• concentrating by ultrafiltration said fractions obtained directly after mixing;
• and
• drying said concentrated fractions by fluidized bed granulation.