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Novel alcohol/aldehyde dehydrogenases

a technology of alcohol/aldehyde dehydrogenase and recombinant enzymes, which is applied in the field of recombinant enzymes, can solve the problem of no reports of cloning of such genes so far

Inactive Publication Date: 2005-04-28
ROCHE VITAMINS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0024] Before describing the present invention in more detail a short explanation of the attached figures is given.

Problems solved by technology

However, there have been no reports so far of the cloning of such genes.

Method used

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  • Novel alcohol/aldehyde dehydrogenases
  • Novel alcohol/aldehyde dehydrogenases
  • Novel alcohol/aldehyde dehydrogenases

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of AADH Genes

(1) Construction of a Genomic Library of G. oxydans DSM No. 4025

[0104] Chromosomal DNA was prepared as follows. G. oxydans DSM No. 4025 was cultivated on an agar plate containing 20 ml of NS2 medium consisting of 5.0% D-mannitol, 0.25% MgSO4.7H2O, 1.75% corn steep liquor, 5.0% baker's yeast (Oriental Yeast Co., Osaka, Japan), 0.5% CaCO3, 0.5% urea (sterilized separately) and 2.0% agar (pH 7.0 before sterilization) at 27° C. for 3 days. The cells were collected from the agar plate, washed with 10 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA and resuspended in 5 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 20 mM EDTA. The cell suspension was treated with lysozyme (Sigma Chemicals Co., St. Louis, Mo., USA) at a final concentration of 400 μg / ml at 37° C. for 30 minutes, then with pronase (400 units) at 37° C. 30 minutes and with 1% SDS at 37° C. for 1 hour. Chromosomal DNA was treated with phenol and RNase A (Boheringer Mannheim, GmbH, Mannheim, G...

example 2

Nucleotide Sequencing

[0112] Nucleotide sequences of the genes for Enzymes A, A′, A″ and B were determined with the plasmids, p24D4, p1E2, p26C3, and pSS31, respectively, by the dideoxynucleotide chain termination method using M13mp18 and M13mp19 (Boehringer Mannheim). One open reading frame (ORF) for each gene was found; the nucleotide sequences of the four genes are shown in the sequence list SEQ ID NOS. 1 to 4 and the amino acid sequences deduced from the nucleotide sequences were shown in the sequence list SEQ ID NOS. 5 to 8. The ORFs for Enzymes A, A′, A″ and B genes are 1737, 1737, 1734, and 1737-bp long and encode 579, 579, 578 and 579 amino acid residues all including 23 amino acid of signal sequences.

[0113] The homologies between Enzymes A, A′, A″ and B are shown in Table 7.

TABLE 7Homologies of amino acid sequences among AADHs.(%)Enzyme AEnzyme A′Enzyme A″Enzyme BEnzyme A100———Enzyme A′89100——Enzyme A″8586100—Enzyme B838281100

FIG. 5 shows the amino acid sequences of matu...

example 3

Subcloning of AADH Genes

[0114] Enzyme A gene was originally cloned as a cosmid clone of p24D4 which has about 25 kb insert in EcoRI site of pVK100. Then, it was further subcloned to use as an Enzyme A gene cassette. The 2.7 kb EcoRV fragment which includes ORF of Enzyme A gene with about 500 bp of non-coding regions at the both ends was excised from 3.4 kb NruI fragment, which was isolated from p24D4 in M13 mp18, and was ligated to HindIII site of pUC18 with HindIII linker (CAAGCTTG). The resulting plasmid was designated pSSA202. Enzyme A gene cassette (2.7 kb HindIII fragment) was then inserted at HindIII site of pVK102 to produce pSSA102R. The plasmid pSSA102R was introduced into nalidixic acid resistant P. putida [ATCC 21812] by a conjugal mating method as described in Example 1-(1). The transconjugant of P. putida carrying pSSA102R was selected on MB agar medium containing 50 μg / ml nalidixic acid and 10 μg / ml tetracycline (MNT agar medium) and subjected to a mini-resting cell r...

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Abstract

The present invention is directed to a recombinant enzymes having alcohol and aldehyde dehydrogenase activity which comprises one or more recombinant polypeptides selected from the group consisting of polypeptides which are identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and chimeric recombinant polypeptides that are a chimeric combination of at least two of the following amino acid sequences identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and functional derivatives of the polypeptides identified above which contain addition, insertion, deletion and / or substitution of one or more amino acid residues, wherein said enzymatic polypeptides have said alcohol and aldehyde dehydrogenase activity. DNA molecules encoding the recombinant polypeptides, vectors comprising such DNA molecules, host cells transformed by such vectors and processes for the production of such recombinant enzymes are provided. Furthermore, the recombinant enzymes having alcohol and aldehyde dehydrogenase activity are used for obtaining aldehydes, ketones or carboxylic acids, and specifically, 2-keto-L-gulonic acid an intermediate for the production of L-ascorbic acid (vitamin C).

Description

FIELD OF THE INVENTION [0001] The present invention relates to recombinant enzymes, particularly, novel recombinant alcohol / aldehyde dehydrogenases (hereinafter referred to as AADH or AADHs) having alcohol and aldehyde dehydrogenase activity. The present invention also relates to novel recombinant DNA molecules encoding AADHs, recombinant expression vectors containing said DNAs, and recombinant organisms containing said recombinant DNA molecules and / or said recombinant expression vectors. Furthermore, the present invention relates to a process for producing recombinant AADHs and a process for producing aldehydes, carboxylic acids and ketones, especially, 2-keto-L-gulonic acid (herein after referred to as 2KGA) by utilizing said recombinant enzymes, and a process for producing aldehydes, carboxylic acids and ketones, especially, 2KGA by utilizing said recombinant organisms. BACKGROUND OF THE INVENTION [0002] 2-KGA is an important intermediate for the production of L-ascorbic acid (vi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/04C12P7/24C12P7/26C12P7/40C12P7/60C12P17/00
CPCC12N9/0006C12P7/24C12P17/00C12P7/40C12P7/60C12P7/26
Inventor ASAKURA, AKIRAHOSHINO, TATSUOOJIMA, SETSUKOSHINJOH, MASAKOTOMIYAMA, NORIBUMI
Owner ROCHE VITAMINS INC
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