D-glucaric acid producing bacterium, and method for manufacturing D-glucaric acid
The present invention provides a D-glucaric acid-producing bacterium and a method for producing D-glucaric acid. The present invention is characterized in that D-glucaric acid or a salt thereof is produced from one or more saccharides selected from the group consisting of D-glucose, D-gluconic acid and D-glucuronic acid with catalytic action of a specific alcohol dehydrogenase PQQ-ADH (1) and a specific aldehyde dehydrogenase PQQ-ALDH (2), and that D-glucaric acid or a salt thereof is produced by using a microorganism having the PQQ-ADH (1) and the PQQ-ALDH (2) or a processed product thereof in the presence of the one or more saccharides. The present invention can provide a microorganism having improved productivity of D-glucaric acid to be used for production of D-glucaric acid and a method for efficiently producing D-glucaric acid.
1. A method for producing D-glucaric acid or a salt thereof, which comprises mixing
(i) an isolated alcohol dehydrogenase ADH (1); and
(ii) an isolated aldehyde dehydrogenase ALDH (2)
in an aqueous solution of one or more saccharides selected from the group consisting of D-glucose, D-gluconic acid, and L-guluronic acid, whereby Cl aldehyde residue of D-glucose is specifically oxidized, and then C6 hydroxymethyl residue of D-gluconic acid is specifically oxidized to form D-glucaric acid without generating D-glucaraldehyde or D-glucuronic acid, to progress the following enzymatic reactions to generate the D-glucaric acid or the salt thereof,
D-glucose→D-gluconic acid→L-guluronic acid→D-glucaric acid
(Intermediate B)
wherein said isolated alcohol dehydrogenase ADH (1) comprises a pyrroloquinoline quinone-dependent enzyme PQQ-ADH (1) having a molecular weight of 64,000±5,000 as measured by SDS-PAGE and a molecular weight of 120,000±10,000 as measured by gel filtration chromatography and comprises the amino acid sequence represented by SEQ ID NO: 1, and
wherein said isolated aldehyde dehydrogenase ALDH (2) comprises a pyrroloquinoline quinone-dependent enzyme PQQ-ALDH (2) having a molecular weight of 61,000±5,000 as measured by SDS-PAGE and a molecular weight of 180,000±10,000 as measured by gel filtration chromatography and comprises the amino acid sequence represented by SEQ ID NO: 2, and
recovering the D-glucaric acid or the salt thereof resulting therefrom.
2. The method according to claim 1 , wherein the isolated alcohol dehydrogenase ADH (1) is encoded by a nucleic acid molecule comprising SEQ ID NO: 7.
3. The method according to claim 1 , wherein the isolated aldehyde dehydrogenase ALDH (2) is encoded by a nucleic acid molecule comprising SEQ ID NO: 8.
4. The method according to claim 1 , wherein L-guluronic acid (intermediate B) is produced in the presence of D-glucose, D-gluconic acid, or both.
5. The method according to claim 1 , which further comprises adjusting the pH of the reaction mixture to be in the range of 5 to 8.
6. The method according to claim 1 , which further comprises adjusting the concentration of D-glucose in the reaction mixture to 1 to 2% (w/v).
7. The method according to claim 1 , wherein the isolated alcohol dehydrogenase ADH (1) is obtained from the strain of Pseudogluconobacter saccharoketogenes Rh47-3 (FERM BP-10820).
8. The method according to claim 1 , wherein the isolated aldehyde dehydrogenase ALDH (2) is obtained from the strain of Pseudogluconobacter saccharoketogenes Rh47-3 (FERM BP-10820).