Preparation of a novel NADP linked alcohol-aldehyde/ketone oxidoreductase from thermophilic anaerobic bacteria for analytical and commercial use

• US 4,352,885 A
• Filed: 05/09/1980
• Issued: 10/05/1982
• Est. Priority Date: 05/09/1980
• Status: Expired due to Term

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Zeikus, Joseph G., Lamed, Raphael J.
• Primary Examiner(s): Shapiro, Lionel M.
• Application Number:
  US06/148,243
• Time in Patent Office: 879 Days
• Field of Search: 435/189, 435/190, 435/822, 435/842, 435/221, 435/222, 435/25, 435/26, 435/814
• US Class Current: 435/189
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 19/36 Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
• CPC Class Codes: C12P 7/02 containing a hydroxy group
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12R 2001/145 Clostridium
• CPC Class Codes: Y10S 435/814 Enzyme separation or purification
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales
• CPC Class Codes: Y10S 435/842 Clostridium

Abstract

A partially purified NADP specific thermostable alcohol, aldehyde/ketone oxidoreductase is prepared which can react with a wide range of alcohols, ketones and aldehydes. The enzyme has a unique preference for secondary over primary alcohols. The thermostability, broad range of operating temperatures and lack of sensitivity to metal ions and complexing agents, in addition to the absolute specifity for the coenzyme, increase the utility of the enzyme in asymmetric organic synthesis and NADPH regeneration.

Claims

1. A method for producing thermostable enzymes, said method comprising the steps of:

(a) inoculating a culture medium with a strain of thermophilic bacteria selected from the group consisting of Thermoanaerobium brockii and Clostridium thermohydrosulfuricum;

(b) incubating said bacteria at a temperature between 50°

C. and 75°

C.;

(c) purifying the bacteria from spent culture supernatant fluids by heating to a temperature in the range of 85°

-98°

C.; and

(d) isolating from the bacteria thermostable NADP linked alcohol, aldehyde/ketone oxidoreductases that are formed.

2. A method as claimed in claim 1 wherein the thermostable enzymes are isolated by centrifugation.

3. A method in accordance with claim 1 wherein said thermostable enzymes are isolated by applying said incubated bacteria to a chromatographic adsorbant selected from the group consisting of diethylaminoethyl cellulose, blue dextran-sephadex and sephadex.

4. A method as claimed in claim 1 in which the purification step is carried out by heating to a temperature within the range of 80°

-98°

C. for a time ranging inversely from 1-10 minutes.

5. A method as claimed in claim 4 in which the heat treatment is for a time of about 5 minutes at 90°

C. to 10 minutes at 85°

C.

6. A thermostable enzyme as claimed in claim 1 comprising an NADP linked alcohol dehydrogenase derived from a strain of thermophilic bacteria selected from the group consisting of Thermoanaerobium brockii and Clostridium thermohydrosulfuricum said dehydrogenase being stable after heating for 80 minutes at 80°

C.