Hoffmann-la Roche Inc.

Dsm IP Assets B.v.

Roche Vitamins Inc.

Teruhide Sugisawa

Tatsuo Hoshino

Taro Miyazaki

Akiko Fujiwara

Setsuko Ojima

Masako Shinjoh

Yutaka Setoguchi

Peter Karl Matzinger

Setuko Ojima

Setsuko Nomura

The present invention provides a process for the production of vitamin C from a substrate, such as for instance L-sorbosone using a microorganism belonging to the genus

Microbial production of vitamin C

The present invention relates to a process for the production of vitamin C from L sorbosone using an aldehyde dehydrogenase which is isolated fromDSM 4025 (FERM BP-3812), said enzyme having the following physicochemical properties: (a) molecular weight of 150,000&#xb1;6,000 Da or 230,000&#xb1;9,000 Da (consisting of 2 or 3 homologous subunits, each subunit having a molecular weight of 75,000&#xb1;3,000 Da); (b) substrate specificity as active on aldehyde compounds; (c) Cofactors are pyrroloquinoline quinone and heme c; (d) Optimum pH between 6.4 and 8.2 for vitamin C production from L-sorbosone; and (e) as inhibitors Co, Cu, Fe, Ni, Zn, monoiodoacetate and ethylendiamine tetraacetic acid. The process is performed in the presence of a suitable electron acceptor and the vitamin C isolated from the reaction mixture.

Vitamin C from sorbosone

The present invention provides a process for the production of vitamin C from different substrates like D-sorbitol, L-sorbose, L-sorbosone or L-gulose using a microorganism selected from the group consisting ofDMS 4025 (FERM BP-3812), a microorganism belonging to the genusand having identifying characteristics ofDSM 4025 (FERM BP-3812) and mutants thereof.

The present invention relates to a novel DNA which encodes aldehyde dehydrogenase (SNDH), an expression vector containing the DNA and recombinant organisms containing said DNA. Furthermore, the present invention concerns a process for producing recombinant aldehyde dehydrogenase protein and a process for producing L-ascorbic acid (vitamin C) and/or 2-keto-L-gulonic acid (2-KGA) from L-sorbosone by using the recombinant aldehyde dehydrogenase protein or recombinant organisms containing the expression vector. Also provided is a process for the production of 2KGA with a microorganism in which the gene encoding said aldehyde dehydrogenase is disrupted.

Aldehyde dehydrogenase gene

A new aldehyde dehydrogenase having the physico-chemical properties: molecular weight:150,000&#xb1;6,000 or 230,000&#xb1;9,000; substrate specificity active on aldehyde compounds; cofactors:pyrroloquinoline quinone and heme c; optimum pH: 7.0-8.5; and inhibitors: Co, Cu, Fe, Ni, Zn, monoiodoacetate and EDTA, is derived from a microorganism belonging to the genus. Said aldehyde dehydrogenase can be produced by cultivating a microorganism of the genuswhich is capable of producing an aldehyde dehydrogenase having the above properties, in an aqueous nutrient medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the aldehyde dehydrogenase from the cell-free extract of the disrupted cells of the microorganism. 2-Keto-L-gulonic acid (2-KGA) can be produced from L-sorbosone by contacting L-sorbosone with (i) the aldehyde dehydrogenase in the presence of an electron acceptor, (ii) amicroorganism capable of producing the aldehyde dehydrogenase in an aqueous medium under aerobic conditions or (iii) a cell-free extract of said microorganism, and in each case isolating the resulting 2-KGA from the reaction mixture.

Method of making an aldehyde dehydrogenase with gluconobacter

A new aldehyde dehydrogenase having the physico-chemical properties: molecular weight: 150,000&plusmn;6,000 or 230,000&plusmn;9,000; substrate specificity:active on aldehyde compounds; cofactors:pyrroloquinoline quinone and heme c; optimum pH: 7.0-8.5; and inhibitors: Co,, Cu,, Fe,, Ni,, Zn,, monoiodoacetate and EDTA, is derived from a microorganism belonging to the genus Gluconobacter. Said aldehyde dehydrogenase can be produced by cultivating a microorganism of the genus Gluconobacter which is capable of producing an aldehyde dehydrogenase having the above properties, in an aqueous nutrient medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the aldehyde dehydrogenase from the cell-free extract of the disrupted cells of the microorganism. 2-Keto-L-gulonic acid (2-KGA) can be produced from L-sorbosone by contacting L-sorbosone with (i) the aldehyde dehydrogenase in the presence of an electron acceptor, (ii) a Gluconobacter microorganism capable of producing the aldehyde dehydrogenase in an aqueous medium under aerobic conditions or (iii) a cell-free extract of said microorganism, and in each case isolating the resulting 2-KGA from the reaction mixture.

Aldehyde dehydrogenase

An aldehyde dehydrogenase having the physico-chemical properties: molecular weight: 91,000.+-.5,000; substrate specificity:active on aldehyde compounds; inhibition: by Cu.sup.2+, Zn.sup.2+, Ni.sup.2 + and ethylenediamine tetraacetic acid; optimum pH: 6.0-8.5; optimum temperature: 20.degree.-40.degree. C.; and stimulator: Ca.sup.2+ and pyrroloquinoline quinone, is derived from a microorganism belonging to the genus Gluconobacter. Said aldehyde dehydrogenase can be produced by cultivating a microorganism of the genus Gluconobacter which is capable of producing an aldehyde dehydrogenase having the above properties, in an aqueous nutrient medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the aldehyde dehydrogenase from the cell-free extract of the disrupted cells of the microorganism. The aldehyde dehydrogenase is useful for the preparation of 2-keto-L-gulonic acid (2-KGA) which can be produced from L-sorbosone by contacting L-sorbosone with (i) the aldehyde dehydrogenase in the presence of an electron acceptor, (ii) a Gluconobacter microorganism capable of producing the aldehyde dehydrogenase in an aqueous medium under aerobic conditions or (iii) a cell-free extract of said microorganism, and in each case isolating the resulting 2-keto-L-gulonic acid from the reaction mixture.

Aldehyde dehydrogenase enzyme

A D-sorbitol dehydrogenase in homogenous form, which catalyzes the oxidation of D-sorbitol to L-sorbose, has a molecular structure consisting of homologous subunits of molecular weight 79,000.+-.5,000 each, a substrate specificity for polyols and an optimum pH of 6.0-7.0. Said D-sorbitol dehydrogenase is producible by a process comprising cultivating a microorganism belonging to the genus Gluconobacter or Acetobacter, or a mutant or variant thereof, which is capable of producing the D-sorbitol dehydrogenase in the cells and isolating it from the cells, e.g. by disrupting the cells and isolation from cell-free extract of the disrupted cells. The so-isolated D-sorbitol dehydrogenase is useful for catalyzing the oxidation of D-sorbitol to L-sorbose, the latter being an important intermediate for the production of vitamin C.

D-sorbitol dehydrogenase

A process for producing 2-keto-L-gulonic acid which comprises converting L-sorbose and/or D-sorbitol into 2-keto-L-gulonic acid with the aid of a microorganism or its cell free extract, said microorganism belonging to the species Gluconobacter oxydans capable of producing 2-keto-L-gulonic acid and having L-sorbose dehydrogenase activity. Also disclosed are specific microorganisms useful in such process.

Gluconobacter oxydans strains

The present invention relates to a process for producing 2-keto-L-gulonic acid by fermentative conversion of D-sorbitol in high yield. 2-Keto-L-gulonic acid is an important intermediate for the production of L-ascorbic acid into which it can be converted.

Process for producing 2-keto-L-gulonic acid

The present invention concerns a novel process for the microbiological manufacture of L-ascorbic acid from L-gulono-gamma-lactone, a process for preparing the enzyme being responsible for the microbiological oxidation of L-gulono-gamma-lactone, and said enzyme as a homogeneous protein, which is a L-gulono-gamma-lactone dehydrogenase.

L-gulono-gamma-lactone-dehydrogenase for producing vitamin C

A process of producing 2-keto-L-gulonic acid by fermentative conversion of L-sorbose utilizing a fermentation system composed of component produced from a microorganism having the identifying characteristics of strain DSM No. 4025 and a yeast component.

Fermentation process to produce 2-keto-L-gluonic acid

The present invention relates to novel coenyme independent L-sorbose dehydrogenase and a process for producing the same. The enzyme provided by the present invention catalyzes the oxidation of L-sorbose to L-sorbosone, the precursor of 2-keto-L-gulonic acid which is an important intermediate in the production of vitamin C. Due to the release duties in foreign countries, the original deposits with respect to the following strains have been transferred to the deposit under the Budapest Treaty at Agency of Industrial Science and Technology, Fermentation Research Institute, Japan on Jan. 29, 1987. And the following accession numbers have been allotted to each strain.

Enzyme and process for producing the same

The present invention relates to novel enzyme having L-sorbosone dehydrogenase activity and a process for producing the same. This L-Sorbosone dehydrogenase enzyme provided by the present invention catalyzes the oxidation of L-sorbosone to 2-keto-L-gulonic acid in the presence of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a coenzyme. 2-KGA is an important precursor for the production of vitamin C.

Growing community of inventors

Yokohama, Japan

Teruhide Sugisawa