Stabilized, NAD(P)H-dependent, soluble nitrate reductase, a process for the preparation thereof and a reagent containing it

US5169758A · kind A · utility

Assignee

• Boehringer Mannheim GmbH · DE

Inventors

• Stephan Fischer · Weilheim, DE
• Barbel Wurst · Pöcking, DE
• Hans-Otto Beutler · Tutzing, DE
• Georg-Burkhard Kresse · Penzberg, DE
• Herwig Brunner · Stuttgart, DE

Key dates

• Filing date: Aug 1, 1990
• Grant date: Dec 8, 1992
• Expiry date: Aug 1, 2010

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Y107/01002
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

The present invention provides a stabilized, NAD(P)H-dependent, soluble nitrate reductase of the assimilatory type, characterized by a molecular weight of about 90,000 D in the case of electrophoresis in the presence of sodium dodecyl sulphate and a residual activity after 3 weeks at 35.degree. C. of more than 60%, obtainable by preparing a suspension of the comminuted starting material in tris/tartaric acid buffer (pH 7-8.5), adding soluble polyethyleneimine thereto in such an amount that 10 to 20% of the nitrate reductase activity passes into the precipitation obtained, separating off the precipitate and working up the supernatant according to conventional biochemical methods of fractionation in the above-mentioned digestion buffer, dialyzing the enzyme solution obtained and lyophilizing in a zwitterionic buffer.