NOVEL GLUCOSE DEHYDROGENASE

• US 20130183716A1
• Filed: 09/24/2008
• Published: 07/18/2013
• Est. Priority Date: 09/26/2007
• Status: Active Grant

• Current Assignee: Kaneka Corporation
• Original Assignee: Kaneka Corporation
• Inventors: Yasohara, Yoshihiko, Kawano, Shigeru
• Granted Patent: US 9,303,280 B2
• Time in Patent Office: Days
• US Class Current: 435/90
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: C12Y 101/01047 Glucose 1-dehydrogenase (1.1.1.47)

Abstract

The invention relates to a glucose dehydrogenase showing an NADP/NAD activity ratio, namely the value obtained by dividing the enzyme activity value obtained by using NADP as a coenzyme by the enzyme activity value obtained by using NAD as a coenzyme, of not lower than 300, to a gene coding therefor, and to a transformant harboring that gene. The enzyme of the invention is very high in NADP specificity and can be suitably used, for example, for NADPH production, for coenzyme regeneration in enzymatic reduction reactions, and in biosensors for glucose concentration measurements.

Claims

1. A NADP-specific glucose dehydrogenase showing an NADP/NAD activity ratio of not lower than 300.

2. The glucose dehydrogenase according to claim 1 which shows an NADP/NAD activity ratio of not lower than 10000.

3. The glucose dehydrogenase according to claim 1, which is derived from among lactic acid bacteria.

4. The glucose dehydrogenase according to claim 3, wherein the lactic acid bacteria are bacteria belonging to the genus Lactobacillus.

5. The glucose dehydrogenase according to claim 4, wherein the bacteria belonging to the genus Lactobacillus are bacteria belonging to the species Lactobacillus plantarum or Lactobacillus pentosus.

6. The glucose dehydrogenase according to claim 3, wherein the lactic acid bacteria are bacteria belonging to the genus Pediococcus.

7. The glucose dehydrogenase according to claim 6, wherein the bacteria belonging to the genus Pediococcus are bacteria belonging to the species Pediococcus parvulus.

8. The glucose dehydrogenase according to claim 2, which is a polypeptide selected from the group consisting of the following (a1), (a2), (a3), (b1), (b2), (b3), (c1), (c2) and (c3):

(a1) a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

1;

(a2) polypeptides having an amino acid sequence derived from the amino acid sequence described in the sequence listing under SEQ ID NO;

1 by substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues and capable of acting on glucose and NADP to form gluconolactone and NADPH;

(a3) polypeptides having a sequence homology of at least 90% with the amino acid sequence described in the sequence listing under SEQ ID NO;

1 and capable of acting on glucose and NADP to form gluconolactone and NADPH;

(b1) a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

3;

(b2) polypeptides having an amino acid sequence derived from the amino acid sequence described in the sequence listing under SEQ ID NO;

3 by substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues and capable of acting on glucose and NADP to form gluconolactone and NADPH;

(b3) polypeptides having a sequence homology of at least 85% with the amino acid sequence described in the sequence listing under SEQ ID NO;

3 and capable of acting on glucose and NADP to form gluconolactone and NADPH;

(c1) a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

1 1;

(c2) polypeptides having an amino acid sequence derived from the amino acid sequence described in the sequence listing under SEQ ID NO;

11 by substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues and capable of acting on glucose and NADP to form gluconolactone and NADPH;

(c3) polypeptides having a sequence homology of at least 85% with the amino acid sequence described in the sequence listing under SEQ ID NO;

11 and capable of acting on glucose and NADP to form gluconolactone and NADPH.

9. The glucose dehydrogenase according to claim 8 which is a polypeptide selected from the group consisting of the following (a1), (a2) and (a3):

(a1) a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

1;

(a2) polypeptides having an amino acid sequence derived from the amino acid sequence described in the sequence listing under SEQ ID NO;

1 by substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues and capable of acting on glucose and NADP to form gluconolactone and NADPH;

(a3) polypeptides having a sequence homology of at least 90% with the amino acid sequence described in the sequence listing under SEQ ID NO;

1 and capable of acting on glucose and NADP to form gluconolactone and NADPH.

10. The glucose dehydrogenase according to claim 8 which is a polypeptide selected from the group consisting of the following (b1), (b2) and (b3):

(b1) a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

3;

(b2) polypeptides having an amino acid sequence derived from the amino acid sequence described in the sequence listing under SEQ ID NO;

3 by substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues and capable of acting on glucose and NADP to form gluconolactone and NADPH;

(b3) polypeptides having a sequence homology of at least 85% with the amino acid sequence described in the sequence listing under SEQ ID NO;

3 and capable of acting on glucose and NADP to form gluconolactone and NADPH.

11. The glucose dehydrogenase according to claim 8 which is a polypeptide selected from the group consisting of the following (c1), (c2) and (c3):

(c1) a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

11;

(c2) polypeptides having an amino acid sequence derived from the amino acid sequence described in the sequence listing under SEQ ID NO;

11 by substitution, deletion, insertion and/or addition of one or a plurality of amino acid residues and capable of acting on glucose and NADP to form gluconolactone and NADPH;

(c3) polypeptides having a sequence homology of at least 85% with the amino acid sequence described in the sequence listing under SEQ ID NO;

11 and capable of acting on glucose and NADP to form gluconolactone and NADPH.

12. A DNA coding for the glucose dehydrogenase according to claim 1.

13. The DNA according to claim 12 which is any of the following (A1) to (A4):

(A1) a DNA having the base sequence shown in the sequence listing under SEQ ID NO;

2;

(A2) a DNA coding for a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

1;

(A3) a DNA hybridizing with a DNA having a base sequence complementary to the base sequence shown in the sequence listing under SEQ ID NO;

2 under stringent conditions and coding for a polypeptide acting on glucose and NADP to form gluconolactone and NADPH;

(A4) a DNA having a sequence homology of at least 90% with the base sequence shown in the sequence listing under SEQ ID NO;

2 and coding for a polypeptide acting on glucose and NADP to form gluconolactone and NADPH.

14. The DNA according to claim 12 which is any of the following (B1) to (B4):

(B1) a DNA having the base sequence shown in the sequence listing under SEQ ID NO;

4;

(B2) a DNA coding for a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

3;

(B3) a DNA hybridizing with a DNA having a base sequence complementary to the base sequence shown in the sequence listing under SEQ ID NO;

4 under stringent conditions and coding for a polypeptide acting on glucose and NADP to form gluconolactone and NADPH;

(B4) a DNA having a sequence homology of at least 85% with the base sequence shown in the sequence listing under SEQ ID NO;

4 and coding for a polypeptide acting on glucose and NADP to form gluconolactone and NADPH.

15. The DNA according to claim 12 which is any of the following (Cl) to (C4):

(C1) a DNA having the base sequence shown in the sequence listing under SEQ ID NO;

12;

(C2) a DNA coding for a polypeptide having the amino acid sequence described in the sequence listing under SEQ ID NO;

11;

(C3) a DNA hybridizing with a DNA having a base sequence complementary to the base sequence shown in the sequence listing under SEQ ID NO;

12 under stringent conditions and coding for a polypeptide acting on glucose and NADP to form gluconolactone and NADPH;

(C4) a DNA having a sequence homology of at least 85% with the base sequence shown in the sequence listing under SEQ ID NO;

12 and coding for a polypeptide acting on glucose and NADP to form gluconolactone and NADPH.

16. A recombinant vector containing the DNA according to claim 12.

17. A transformant obtained by transforming a host cell with the vector according to claim 16.

18. A method of producing NADPH which comprises using a NADP-specific glucose dehydrogenase showing an NADP/NAD activity ratio of not lower than 300 or the transformant according to claim 17 for the formation of NADPH from NADP and glucose.

19. A reduction reaction system for an enzymatic reduction reaction using a reductase source requiring NADPH as a coenzyme, wherein NADPH is regenerated using a NADP-specific glucose dehydrogenase showing an NADP/NAD activit ratio of not lower than 300 or the transformant according to claim 17.

20. The reduction reaction system according to claim 19, which comprises a transformant harboring both a DNA coding for a NADP-specific glucose dehydrogenase showing an NADP/NAD activity ratio of not lower than 300 and a DNA coding for a reductase requiring NADPH as a coenzyme as introduced therein and thus having the activities of both the enzymes.

21. A biosensor wherein a NADP-specific glucose dehydrogenase showing an NADP/NAD activity ratio of not lower than 300 or the transformant according to claim 17 is used.

22. The biosensor according to claim 21 which is used for detecting NADPH and/or NADP and/or measuring the concentration thereof.

23. The biosensor according to claim 21 which is used for detecting D-glucose or 2-deoxy-D-glucose and/or measuring the concentration thereof.