Enzyme-based system and sensor for measuring acetone

• US 7,794,994 B2
• Filed: 11/08/2002
• Issued: 09/14/2010
• Est. Priority Date: 11/09/2001
• Status: Active Grant

• Current Assignee: Invoy Holdings, Inc.
• Original Assignee: Kemeta, LLC
• Inventors: Sun, Larry, Strickland, Alan D., McIntyre, James A., Rosner, Bettina M., Miller, Theodore E. Jr., Allen, Jeffrey R., Subramanian, Venkiteswaran, Danowski, Kristine L., Cranley, Paul E.
• Primary Examiner(s): Pak; Yong D
• Application Number:
  US10/494,923
• Publication Number:
  US 20050084921A1
• Time in Patent Office: 2,867 Days
• Field of Search: None
• US Class Current: 435/190
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12P 7/28 Acetone-containing products
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence

Abstract

Described are enzyme systems specific for acetone and methods of using these enzyme systems to detect acetone in biological or environmental samples. Biosensors containing these enzyme systems are disclosed, in which detection of acetone may be achieved by linking electrochemical, photometric, or other detection means to one or more acetone-specific enzyme reactions or pathways. Methods of using such acetone-specific biosensors include subject management of weight loss, disease detection, and bioavailability monitoring of therapeutics.

Claims

1. An acetone-specific enzyme system comprising a secondary alcohol dehydrogenase isolated from Xanthobacter autotrophicus Py2 (ATCC®

PTA-4779) that selectively catalyzes the reduction of acetone to isopropanol in the presence of NAD(P)H, coupled to a detectable signal mediator, wherein the secondary alcohol dehydrogenase comprises a polypeptide having a tetradecameric N-terminal amino acid sequence of SEQ ID NO;

7, said polypeptide being capable of being degraded to form one or more fragments each having the amino acid sequence selected from SEQ ID NO;

8 to SEQ ID NO;

19.

2. The acetone-specific enzyme system according to claim 1, wherein the acetone-specific enzyme system is coupled to a signal mediator detected by electrochemical or photometric means.

3. The acetone-specific enzyme system according to claim 2, wherein the acetone-specific enzyme system is secondary alcohol dehydrogenase (S-ADH) with concomitant NAD(P)H oxidation, or S-ADH catalyzed NAD(P)⁺ formation coupled to H₂O₂ production.

4. The acetone-specific enzyme system according claim 2, wherein the signal mediator is a member of any one of organic cofactors, inorganic cofactors, indicators, electron transfer mediators, photometric mediators, enzyme reaction by-products, or combinations thereof.

5. The acetone-specific enzyme system according to claim 4, wherein a signal from the electrochemically detected signal mediator is linearly or exponentially amplified by recycling enzyme substrates to magnify electrochemical signal output.

6. The enzyme-system of claim 1 wherein the secondary alcohol dehydrogenase isolated from Xanthobacter autotrophicus Py2 (ATCC®

PTA-4779) comprises a protein having NAD⁺-dependent secondary alcohol dehydrogenase activity, having the ability to reduce acetone to isopropanol, and having specific activity for ketones and secondary alcohols;

(A) said protein (1) having, for the oxidation of isopropanol to acetone, a pH optimum of approximately 7.8, and (2) having, for the oxidation of alcohols, an average specific activity ratio for secondary-to-primary alcohols of at least 50;

1 when tested at pH 7.8 under equivalent conditions individually with C3-C5 straight chain secondary alcohols and with C2-C5 straight chain primary alcohols;

(B) said protein having, for the reduction of acetone to isopropanol, (1) a pH optimum of approximately 6.2, (2) an apparent K_m of approximately 144+/−

18 μ

M, (3) an apparent V_max of approximately 43.4+/−

1.2 μ

mmol acetone reduced·

min<sup>−

1</sup>·

mg<sup>−

1</sup> protein, (4) an apparent k_cat of approximately 30.4 sec<sup>−

1</sup>, (5) an apparent k_cat/K_m of approximately 2.1×

10⁵, and (6) a K_m for NADH of approximately 5.1+/−

0.4 μ

M; and

(C) said protein comprising at least one polypeptide molecule, (1) said polypeptide molecule having (a) a molecular mass of approximately 37.1 kDa as determined by mass spectrometry, (b) a pI of approximately 7.4 as determined by isofocusing electrophoresis, and (c) a tetradecameric N-terminal amino acid sequence of SEQ ID NO;

7, and (2) said polypeptide molecule being capable of being degraded to form fragments having the amino acid sequences of SEQ ID NO;

8 to SEQ ID NO;

19.

7. The enzyme-system of claim 6 wherein the secondary alcohol dehydrogenase isolated from Xanthobacter autotrophicus Py2 (ATCC®

PTA-4779) comprises a polypeptide molecule purified from the protein according to claim 6, (1) said polypeptide molecule having (a) a molecular mass of approximately 37.1 kDa as determined by mass spectrometry, (b) a pI of approximately 7.4 as determined by isofocusing electrophoresis, and (c) a tetradecameric N-terminal amino acid sequence of SEQ ID NO;

7, and (2) said polypeptide molecule being capable of being degraded to form fragments having the amino acid sequences of SEQ ID NO;

8 to SEQ ID NO;

19.