Europaisches Patentamt (19) European Patent Office Office europeenpeen des brevets EP 0 486 289 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) intci.6: C12P7/42, C12P 13/02, of the grant of the patent: C12P 41/00 09.07.1997 Bulletin 1997/28 (21) Application number: 91310490.7 (22) Date of filing: 13.11.1991 (54) Biological process for producing alpha-hydroxyamide or alpha-hydroxy acid Biologisches Verfahren zur Herstellung von Alpha-Hydroxyamid und Alpha-Hydroxysaure Procede biologique pour la production d'un alpha-hydroxyamide ou d'un acide alpha hydroxyle (84) Designated Contracting States: (74) Representative: Pearce, Anthony Richmond DE FR GB MARKS & CLERK, Alpha Tower, (30) Priority: 14.11.1990 JP 308269/90 Suffolk Street Queensway Birmingham B1 1TT (GB) (43) Date of publication of application: 20.05.1992 Bulletin 1992/21 (56) References cited: EP-A- 0 348 901 FR-A- 2 245 585 (73) Proprietor: NITTO CHEMICAL INDUSTRY CO., FR-A- 2 294 999 LTD. Tokyo (JP) • CHEMICAL ABSTRACTS, vol. 105, no. 7, 18 August 1986, Columbus, Ohio, US; abstract no. (72) Inventors: 59481x, KAWAKAMI, KYOSHI ET AL. 'Microbial • Endo, Takakazu, c/o Nitto Chemical lnd.Co.,Ltd. production of alpha hydroxy acid and its salts.' Yokohama-shi, Kanagawa (JP) page 518 ; • Yamagami, Tomohide Neyagawa-shi, Osaka (JP) Remarks: • Tamura, Koji, c/o Nitto Chemical lnd.Co.,Ltd. The file contains technical information submitted Yokohama-shi, Kanagawa (JP) after the application was filed and not included in this specification DO O) 00 CM CO 00 ^- Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice the Patent Office of the Notice of shall be filed in o to European opposition to European patent granted. opposition a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. a. 99(1) European Patent Convention). LU Printed by Jouve, 75001 PARIS (FR) EP 0 486 289 B1 Description FIELD OF THE INVENTION 5 The invention relates to a biological process for producing an a-hydroxyamide or an a-hydroxy acid. BACKGROUND OF THE INVENTION Known biological processes for producing an a-hydroxyamide include a process comprising reacting lactonitrile, 10 hydroxyacetonitrile, a-hydroxymethylthiobutyronitrile etc. with a microorganism belonging to the genus Bacillus, Bac- teridium, Micrococcus or Brevibacterium to obtain a corresponding amide as disclosed in JP-B-62-21 51 9 (correspond- ing to U.S. Patent 4,001 ,081 ) (the term "JP-B" as used herein means an "examined published Japanese patent appli- cation"). It is also reported in Grant, D.J.W., Antonie van Leeuwenhoek; J. Microbiol. Serol. Vol. 39, p. 273 (1 973) that hydrolysis of lactonitrile by the action of a microorganism belonging to the genus Corynebacterium to produce lactic is acid involves accumulation of lactamide as an intermediate. Known biological processes for producing an a-hydroxy acid include a process for obtaining glycolic acid, lactic acid, a-hydroxyisobutyric acid etc. from the corresponding a-hydroxynitrile by the action of a microorganism of the genus Corynebacterium (see JP-A-61 -56086, the term "JP-A" as used herein means an "unexamined published Jap- anese patent application"), a process for obtaining lactic acid, glycolic acid etc. from the corresponding a-hydroxynitrile 20 by using a microorganism of the genus Bacillus, Bacteridium, Micrococcus or Brevibacterium (see JP-B-58-15120, corresponding to U.S. Patent 3,940,316), a process for obtaining lactic acid, a-hydroxyisobutyric acid, mandelic acid, a-hydroxybutyric acid, a-hydroxyvaleric acid, a-hydroxy-a-phenylpropionic acid, a-hydroxy-a-(p-isobutylphenyl)pro- pionic acid etc. from the corresponding a-hydroxynitrile by using a microorganism of the genus Pseudomonas, Arthro- bacter, Aspergillus, Penicillium, Cochliobolus or Fusarium (see JP-A-63-222696) and a process for obtaining a-hy- 25 droxy-p.p-dimethyl-y-butyrolactone from the corresponding a-hydroxynitrile by using a microorganism of the genus Arthrobacter, Aspergillus, Bacillus, Bacteridium, Brevibacterium, Cochliobolus, Corynebacterium, Micrococcus, No- cardia, Penicillium, Pseudomonas or Fusarium (see JP-A-64-1 0996). It is known, however, that an a-hydroxynitrile is, more or less, partially dissociated into the corresponding aldehyde and hydrogen cyanide in a polar solvent as taught in V. Okano et al., J. Am. Chem. Soc, Vol. 98, p. 4201 (1976). Since 30 an aldehyde generally is bound to proteins to deactivate enzymes as described in G.E. Means et al. (ed.), Chemical Modification of Proteins, p. 1 25, Holden-Day (1 971 ), enzymatic hydration or hydrolysis of an a-hydroxynitrile is accom- panied with the problem that the enzyme is deactivated in a short time. It therefore has been difficult to obtain an a- hydroxyamide or an a-hydroxy acid in high concentration with high productivity. 35 SUMMARY OF THE INVENTION To overcome the above-described problem associated with conventional biological processes for producing an a- hydroxyamide or an a-hydroxy acid, the inventors conducted extensive investigations and, as a result, found that the presence of a sulfite ion, a disulfite ion or a dithionite ion in the reaction system markedly improves activity and stability 40 of enzymes and solved the above-described problem. The present invention provides a biological process for producing an a-hydroxyamide or an a-hydroxy acid rep- resented by formula (III): 45 OH R - C - X (III 50 H wherein R represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a sub- stituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted 55 aryl group, a substituted or unsubstituted aryloxy group or a substituted or unsubstituted and saturated or unsaturated heterocyclic group; and X represents an amido group or a carboxyl group, comprising reacting an a-hydroxynitrile represented by formula (I): 2 EP 0 486 289 B1 OH R - C - CN (I) 5 | H wherein R is as defined above, 10 or a mixture of an aldehyde represented by formula (II): R-CHO (II) 15 wherein R is as defined above, and hydrogen cyanide with a microorganism capable of producing such an amide or acid from the corresponding a- hydroxynitrile, in which the reaction system contains a sulfite ion, a disulfite ion or a dithionite ion. DETAILED DESCRIPTION OF THE INVENTION 20 A sulfite ion, a disulfite ion or a dithionite ion added to the reaction system is supplied as, for example, sodium sulfite, sodium bisulfite, sodium dithionite, potassium sulfite, potassium bisulfite, potassium dithionite, ammonium sulfite, ammonium bisulfite or ammonium dithionite. The following mechanism is suggested to account for the action of those ions. A sulfite ion, a disulfite ion or a 25 dithionite ion has properties of forming a complex with an aldehyde. That is, those ions rapidly react with an aldehyde released by dissociation of an a-hydroxynitrile in a polar solvent to form a complex, thus serving to maintain a low free aldehyde concentration in the reaction system. The complex formed undergoes a nucleophilic reaction with a proton or hydrogen cyanide to release reversibly an aldehyde or an a-hydroxynitrile, respectively. According to the present invention, hydration or hydrolysis of a nitrile can be carried on while maintaining a low 30 aldehyde concentration in the reaction system by a combination of those actions so that the enzyme inhibitory action of the aldehyde is minimized and the reaction can last for an extended period of time, in a stable manner, without causing drastic deactivation of the enzyme making it possible to accumulate the produced amide or acid in high con- centrations. It is still obscure, however, whether the enzyme stabilization mechanism by a sulfite ion, a disulfite ion or a dithionite ion is attributable only to the reduction in free aldehyde concentration in the reaction system. 35 Considering the above-stated fact that an aldehyde generally has properties of binding to proteins to deactivate an enzyme, it appears that the inhibitory effect on enzyme activity by a sulfite ion, a disulfite ion or a dithionite ion applies to all biological reactions which involve an aldehyde. In other words, the microorganism which can be used in the production of an amide or an acid from an a-hydroxynitrile according to the present invention is not particularly limited as long as the microorganism is capable of producing such an amide or acid from the corresponding a-hydrox- 40 ynitrile. Likewise, the a-hydroxynitrile substrate is not particularly limited as long as it reaches a dissociation equilibrium with an aldehyde in the reaction system. Microorganisms capable of hydrolyzing an a-hydroxynitrile to convert it to the corresponding acid include those belonging to the genus Pseudomonas, Alcaligenes, Acinetobacter, Caseobacter, Corynebacterium, Brevibacterium, Nocardia, Rhodococcus, Arthrobacter, Bacillus, Aureobacterium, Enterobacter, Escherichia, Micrococcus, Streptomy- 45 ces, Flavobacterium, Aeromonas, Mycoplana, Cellulomonas, Erwinia, Candida, Bacteridium, Aspergillus, Penicillium, Cochliobolus, Fusarium or Rhodopseudomonas. Specific species of those microorganisms are Pseudomonas sp. BC13-2 (FERM BP-3319), Pseudomonas sp. BC1 5-2 (FERM BP-3320), Pseudomonas sp. SK1 3 (FERM BP-3325), Pseudomonas sp. SK31 (FERM P-1 1 31 0), Pseu- domonas sp. SK87 (FERM P-1 1311), Pseudomonas synxanta IAM 12356, Alcaligenes sp. BC12-2 (FERM P-1 1263), 50 Alcaligenes sp. BC20 (FERM P-1 1264), Alcaligenes sp. BC35-2 (FERN BP-3318), Acinetobacter sp. BC9-2 (FERM BP-331 7), Caseobacter sp. BC4 (FERM BP-331 6), Caseobacter BC23 (FERM P-1 1 261 ), Corynebacterium nitrilophilus ATCC 21419, Brevibacterium acetylicum IAM 1 790, Brevibacterium helvolum ATCC 1 1 822, Nocardia sp. N-775 (FERM BP-961), Nocardia asteroides IFO 3384, Nocardia calcarea KCC A0191 , Nocardia polychromogenes IFM 19, Rhodo- coccus sp. SK70 (FERM P-1 1304), Rhodococcus sp.