Assay for D-allose using a NAD cofactor coupled D-allose dehydrogenase

• US 5,567,605 A
• Filed: 11/21/1994
• Issued: 10/22/1996
• Est. Priority Date: 11/21/1994
• Status: Expired due to Term

• Current Assignee: UOP Llc (Honeywell International Inc.)
• Original Assignee: UOP Llc (Honeywell International Inc.)
• Inventors: St. Martin, Edward J.
• Primary Examiner(s): Kight, John
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US08/342,502
• Time in Patent Office: 701 Days
• Field of Search: 435/26, 435/25, 435/4, 435/23, 435/14, 435/24, 435/94, 436/34, 436/63, 436/74, 436/501, 536/1.11
• US Class Current: 435/26
• CPC Class Codes: C12Q 1/32 involving dehydrogenase

Abstract

A D-allose specific dehydrogenase has been isolated which can be used with NAD as a cofactor in a sensitive, specific, quantitative assay for D-allose in aqueous media. A qualitative, color-based test for the presence of D-allose results when an election-accepting dye is coupled to NAD.

Claims

1. A method of determining the amount of only D-allose present in an aqueous medium, said aqueous medium optionally containing at least one other D-aldohexose, comprising selectively oxidizing said D-allose with a D-allose-specific dehydrogenase elaborated by a strain of Exophiala pisciphila in the presence of nicotinamide adenine dinucleotide at a pH between about 8.5 and about 9.5 and at a temperature from about 25°

to about 75°

C., measuring the formation of the reduced form of nicotinamide adeninc dinucleotide (NADH₂), and relating the rate of NADH₂ formation to D-allose concentration using a standard curve of NADH₂ formation rate versus D-allose concentration.

2. The method of claim 1 where the pH is between about 8.8 and about 9.2.

3. The method of claim 1 where the formation of NADH₂ is measured in the ultraviolet spectrum at a wavelength of about 340 nm.

4. A qualitative method of determining the presence of D-allose in an aqueous medium comprising oxidizing said D-allose with a D-allose dehydrogenase in the presence of an electron accepting dye at a pH between about 7.5 and about 8.5 and in the temperature range of between about 25°

and about 35°

C., and determining the color change accompanying reduction of said dye.

5. The method of claim 4 where the dye is a tetrazolium salt.

6. The method of claim 5 where the dye is nitro blue tetrazolium.