TW521088B - A production method of Α-hydroxy acid by using a microorganism - Google Patents

Abstract

This invention provides a production method of Α-hydroxy acids [II]: RCH(OH)COOH (wherein, R represents hydrogen atom, substituted C1-C6 alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkoxyl, substituted aryl, substituted aryloxy or substituted heterocyclic groups), which uses a microorganism to hydrolyze Α-hydroxynitrile [I]: RCH(OH)CN (wherein, R is as defined as before), so as to convert said compounds into Α-hydroxy acids [II]. Preferably, it is related to a production method of Α-hydroxy acids [II], which makes Α-hydroxy acid [II] be generated and accumulated in an aqueous solvent in the presence of cyanide by using a microorganism having concentration-resistance against Α-hydroxynitrile [I] and/or Α-hydroxy acid [II] as well as durable activity. More specifically, this invention enables to accumulate Α-hydroxy acid [II] in a high concentration by using a microorganism, which has concentration-resistance against Α-hydroxynitrile [I] and/or Α-hydroxy acid [II] as well as long-time-lasted durable activity, and using said microorganism repeatedly, so as to produce Α-hydroxy acid [II] efficiently. Moreover, addition of cyanide into the reaction system makes the production of Α-hydroxy acid [II] more efficient.

Description

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the Invention (1) Technical Field: The present invention relates to a method for producing α-hydroxyacid by hydrolyzing α-hydroxynitrile with microorganisms. Lactic acid can be used for food, mining and industrial purposes, and 2-hydroxy-4_butyric acid is a useful animal feed additive. Technological background: The method of producing α-hydroxy acid [II] from α-hydroxynitrile [I] by microorganisms is known, for example: (1) from Bacillus, Bacterium ， Cruciol® 靥 （Micrococcus) and short rod @ Brevibacteriuin production method of manufacturing lactic acid, glycolic acid, etc. [Kokusho 5 8 -15120], (2) by rod _ genus (C 〇rynebacteri ϋ m ) Production of lactic acid, glycolic acid, and 2-hydroxyisobutyric acid by 擞 擞 Bio [Japanese Patent Application Laid-Open No. 6B 5 6 0 8 6], (3) Pseudomonas (Pseud om nas), joints Arthrobater. Arthrobater: Aspergillus, Penicillium (Penic 丨 Π iu in), Spirulina (Cocch 1 丨 〇b 〇bjs), Fusarium A method for the production of lactic acid, 2-hydroxyisobutyric acid, 2-hydroxy-2-hydroxyphenylpropionic acid and mandelic acid by microorganisms of Fusari U m [Japanese Patent Application Laid-Open No. 5 2 -2 2 2 6 9 6], (4) By Arthrobacillus, Aspergillus, Bacillus, Aspergillus, Brevibacterium, Sporocystis, Corynebacterium, Micrococcus, Soil Mycelia (Nocardia >, a method for producing 2-hydroxy-3,3-dimethyl-4-butyllactone by microorganisms of Penicillium, Pseudomonas, and Fusarium spp. [To be opened 6 4-1 0 9 9 6],-3-This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

Printed by the Employees' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the invention (2) (5) Rhodococcus (Rh 〇d 〇c 〇ccus), Pseudomonas _ _, Arthrosporium spp., And the production method of 2-hydroxyisobutyric acid by microorganisms of Brevibacterium [To be Kaiping 4-4 0 8 9 7], (6) Casease (bacteria), Pseudomonas, alkali-producing Method for producing α-hydroxy-4 -methylthiobutyric acid by microorganisms of genus AU a 丨 U enes, Corynebacterium, Brevibacterium, Agrobacterium, Rhodococcus, and Arthrobacillus To be Kaiping 4-40898], (7) Production method of 4-methylthiobutyric acid produced by microorganisms of the genus Alkali, Rhodococcus and Gordona [WO 96/09403], ( 8) Pantoea, a method for producing α-hydroxy-4-methylthiobutyric acid by microorganisms of the genus Micrococcus and non-bacillus [Japanese Patent Laid-Open No. 8-1 7 3 1 7 5] and the like. However, the production of these 〇 (-hydroxy acids) does not necessarily meet the purpose of generating accumulation substances at high concentrations. For example, lactic acid can be confirmed to be from 9.8% by weight of microorganisms belonging to the genus Corynebacterium. [Japanese Patent Application Publication No. 6 1 -56086], only 10% by weight of pupae organisms derived from Pseudomonas spp. [To be opened 6 3-2 2 2 6 9 6], microorganisms derived from Arthrobacillus pupae were only 0.1% 5% by weight [Japanese Patent Laid-Open No. 6 3-2 2 2 6 9 6]. Also, the amount of α-hydroxyisobutyric acid that can be confirmed separately is only 0.8% by weight from microorganisms of the genus Pseudomonas [Japanese Patent Laid-Open] Sho 63-222696], cc-hydroxy-4-methylthiobutyric acid can be confirmed that the accumulation amount comes from microorganisms of the genus only 1 8 m Μ (2.8% by weight) [Japanese Patent Laid-Open No. 4-4 0 8 9 8], only 5.5 m MU (0.8% by weight) from the genus Pleurotus spp. [Japanese Patent Laid-Open No. 4-4 0 8 9 8], and only 9 40 m Μ (14% by weight) [W 0 9 6 ./ 0 9 4 0 3]. -4-This paper size applies to China National Standard (CNS) Α4 size (210 × 297 mm) (Please read the note on the back first Matters then fill out this page)

Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 B7 V. Explanation of the invention (3) The reason for the low concentration of its products is, for example, the dissociation of the corresponding aldehydes in the aqueous solution by α-hydroxy groups into ketones and acetic acid [Chemical Overview (C he in ica 1 Reviews) Vol. 42, p. 139, p. 189), acetic acid will inhibit enzyme activity [/ Uricultural Biological Chemistry (vol. 46, p. 1165, 1) 982]. In addition, the possibility of inactivating the enzyme in a short time due to the action of dissociating aldehyde is pointed out. The solution can be proposed by adding an acidic sulfite ion or a disulfite ion [Japanese Patent Laid-Open No. 5 -1 9 2 1 8 9], Method for adding arsenite ion or hypophosphite ion [Japanese Patent Application Laid-Open No. 7-213296]. In addition, the use of these additives 〇 (-hydroxy acid production accumulation concentration is not high. Generally, under conditions where the product accumulation concentration is low, these manufacturers know that the equipment for large-scale manufacturing is complex. At this point The method of industrially producing α-hydroxy acid has a problem of production efficiency. The object of the present invention is to provide a method for producing α-hydroxy acid by using the efficiency of a high-concentration livestock product of α-hydroxy acid. The invention shows that the inventor of the present invention is false To find the general formula [I] = RCH (0 H) C Ν (where R is a hydrogen atom, a C 1 -C s alkyl group having a substituent, a C 2-C s alkenyl group having a substituent, Α-hydroxynitrile [I] represented by C i-C 3 alkylchloro group of substituent, aryl group having substituent, aryl chloride group having substituent, or heterocyclic group having substituent) to form the general formula [II]: RCH (0H) C00H (wherein, R represents the same meaning as above), a complete organism of α-hydroxy acid [II], at a high concentration of α-hydroxynitrile [I] or α-hydroxy acid [ II] The activity is less inhibited, the activity lasts for a long time and has durability, and it has a high accumulation. The paper size is applicable to Chinese National Standard (CNS) Α4 regulations. (210Χ297 mm) (Please read the back of the precautions to fill out this page)

n-~, 1Tn 521088 A7 _B7_ Λ · Ling Ming Description (4) The concentration of α-hydroxy acid [I I] ability is an industrially useful organism. As a result, the target activity of the microorganisms of the genus V ari ovavra X and Arthrosporum spp. Can be targeted, and then in the reaction bar, by adding the general formula [III]: Mm (CN) n (wherein Where M is hydrogen, ammonium or metal ions, and mw is an integer of 1 to 3), which shows that the enzyme activity is improved, and the present invention has been completed. PJI, the present invention uses a microorganism to hydrolyze α-hydroxynitrile [I] and α-hydroxyacid [II] to form α-hydroxyacid [II]. / Or α-hydroxy acid [II] has a concentration tolerance and durability of the organism, generates α-hydroxy acid [II] in an aqueous solvent and accumulates. At the same time, it adopts a-hydroxy acid [characterized by adding an odorant in the reaction.] II]. Hereinafter, the present invention will be described in detail. There is no particular limitation on the microorganisms used in the invention. Anyone who can achieve the purpose of the present invention, has a concentration tolerance to α-hydroxynitrile [I] or α-hydroxy acid, and a durability that has long-term activity. Examples of microorganisms are Variovorax paradoxus ΙΑΜ12374 and Arthrobacillus

Arthrobacter) NSSC104 (CCRC 910077, FERM P-15424). Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) Among these creatures, Vario '/ orax paradoxus IAM 12374 can be obtained by the Tokyo Institute of Molecular Cell Biology (IA Μ) easy to get. In addition, the sample of Bacillus sp. N S S C 104 was isolated from nature by the inventor of the present case and has been deposited. (Taiwan) Deposit Number: CCRC910077-δ-This paper size is applicable to Chinese National Standard (CNS) A4 size (210X297 mm) 521088 Polymorphic bacillus lysine aerobic member of the Central Standards Bureau of the Ministry of Economic Affairs M Consumer Cooperatives printed A7 B7 5 5. Description of the invention (5) Deposit date: April 8, 1997 (Japanese version) Deposit number: FER Μ BP-5 3 2 9 (Transferred from Micro-Tech Research Biology P-1 5 4 2 4) Deposit Date: February 6, 1996, Domestic deposit, February 20, 1997, International depository place: Japan's East 1-Chome, Tsukuhatsu, Ibaraki, Japan The industrial properties of the Institute of Industrial Technology 5 Research and Arthrobacillus NSSC 1 0 4 are as follows. Morphology Cocoon orchid staining r 〇d-occcus Cyclic transcellular lotus Motile cell wall diamine group Chlorine attitude Chlorase perhydrogenase DNA degradation Keratin liquefaction starch degradation casein degradation Vitamin requirements This paper size applies China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page)

Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the Invention (6) Borrowing the Si-based test-the type of ethyl acetate) m pieces of MK-9 (H2) cell wall nuclei constitute galactophore + glucose + above _The nature of learning, based on Bergy's Man an ii a 1 〇 f

The results of Systematic Bacteriology (1986) search revealed that NSSC 104 strain was identified as a strain of Arthrobacillus. Hereinafter, specific embodiments of the present invention will be described. The microorganisms used in the present invention are cultured on a medium that generally contains enzyme-inducing substances, bio-available sulfonates, nitrogen, inorganic ions, and possibly organic nutrient sources. The enzyme-inducing substance is pseudo-isocyanate and cyclic amidine compounds such as ε-caprolactam. The carbon source is sulfonic compounds such as grapevine, alcohols such as ethanol, and other applicable ammonium salts. Inorganic ions are phosphate ions, calcium ions, magnesium ions, sulfate ions, iron ions, and others should be used as necessary. Organic nutrition is vitamins. Amino acids, etc. and corn concentrates containing these substances, yeast extract, protein extract, meat extract, and others as applicable. Cultivation should be carried out under aerobic conditions in a suitable range of P Η 6 to 9 and temperature 25 to 37 ° C. According to the hydrolysis reaction of the microorganism used in the present invention, the culture blue body shown above is pseudo-immobilized, if necessary, the bacterial body is immobilized, the crude enzyme is prepared, and the enzyme-immobilized enzyme-treated product is treated with α-hydroxynitrile in an aqueous solvent [I] Connect with a matching bracelet. In the case of immobilizing bacteria or enzymes, the support binding method is applicable. -3-This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of Invention (7), including common fixed technologies such as law. In the case of preparing fermented enzymes, it is suitable to use ultrasonic and high-pressure homogenizers to break the bacterial cells, and then use ammonium sulfate salting out, decanting and other commonly used enzyme purification techniques. The bacterial cells having a concentration of 0.0 to 10% by weight based on the dry electricity were filtered after the reaction, and then subjected to a repeated hydrolysis reaction by centrifugation or concentration recovery of an out-of-range filter membrane. Examples of the aqueous solvent include water, an aqueous solution containing a salt such as a buffer or an organic solvent, and it is preferable to separate the two phases. The general formula [I] used in the present invention: α-hydroxynitrile "1" represented by RC Η (0 H) CN, where R is a hydrogen atom, a substituent C: 1-C6 alkyl, and a substituent C 2-C s alkenyl, C jl-C 3 alkylchloro with substituents, aryl with substituents, aryl with substituents or heterocyclic with substituents. Specific examples include hydrogen Atoms, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, second butyl, third butyl, pentyl, hexyl, etc. C 1-C β Rhenylmethylthiomethyl , 1-methylthioethyl, 2-methylthioethyl, 1-methylthiopropyl, 2-methylthiopropyl, 3-methylthiopropyl, 1-methylthiobutyl, methyl Thiobutyl, 3-methylthiobutyl, 4-methylthiobutyl, 6-methylthiohexyl, ethylthiomethyl, 1-ethylthioethyl, 2-ethylthioethyl, 1-ethylthiopropyl, 2-ethylthiopropyl, 3-ethylthiopropyl, 1-ethylthiobutyl, 2-ethylthiobutyl, 3-ethylthiobutyl, 4- Ethylthiobutyl, propylthiomethyl, 1-propylthioethyl, 2-propylthiopropyl, 3-propylthiobutyl, 1-methylthioisopropyl, 2-ethylthio Propyl, 1-propylthiobutyl, 2-propylthiobutyl, 3-propylthiobutyl, 4-propylthiobutyl, -9-This paper size applies to China National Standard (CNS) A4 specifications (210X 297mm) (Please read the notes on the back before filling this page)

521088 A7 B7 V. Description of the invention (8-based ,, ethylpropylbutanyl-6, thiothione CR, propylpropyl 1 **, isoiso-C1 group 2 2 dimethyl, isopropylthiopropyl butyl butyl Alkylsulfane

Thiothiopropane isopropyl isopropyl II-11- 4 Ethyl Ethyl Propene Butyl; Thiothiopropane Propyl Isopropyl Sulfuryl Propionyl S 2-Iso-C 3 Propyl, Hydroxy 2 Butyl, Hydroxy 2- Propyl ， Hydroxybutyrate, isopropylethoxyl via 1-2, ethoxybutyryl vial 1-I il, ethoxybutyryl 3-hydroxyl, etc. Ethyl Α δ CRCRi! Ϋ 0 ^ 0 II; I--1 methyl form, CI amine amino group 2-6 fluorenyl ethyl carboxylate, 06 ethyl _ ethyl carboxyl group 1 ~ thioguanidine 2-Isomethylpropyl C.1-Iso1-, propyl, phenyl, propyl, propyl, hydrazino-methyl, propylmethylcarboxyl, methylformyl, methylcarboxamidine, sulfanil 3-guanidine * 1-I Ϊ Alkyl propyl carboxyl propyl carboxyl yl yl ^ alkane ethyl ethyl (Please read the precautions on the back before filling this page)

, Etc. Ethyl ethylpropyl methanomethamine amino propyl mercaptopropanyl propyl mercaptopropanyl guanidine, y5 propyl nitrodimethyl 3-α 3 benzene, ketones, employees of the Central Standards Bureau of the Ministry of Economic Affairs Consumer co-operative printed methylmethyl group a C ί 2 methyl, CN3, phenyl CI, methyl amidyl methyl 6 ^ tolyl methyl C64-based radical Ϊ-5 \, hydroxy withindyl C1 group 4-equivalent indyl-2-ylmethyl, guanidiphenylmethyl, chlorotolylethyl, etc. 2-phenylmethyl, oxazolylethyl 4 \ — / / (radicals 2, indylacetazol Imidyl 2-methylcarbazolyl tolyloxymethyl, methylformate;} phenylphenyl methinyl alkane »5 indylaryl indyl yl methazolium ring heterocyclic substitution and other basic ethane basic paper size applicable Chinese national standards ( CNS) A4 specification (210X297 mm) 521088 / Ψ Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (9) Vinyl, propenyl, isopropenyl, allyl, 1-chloroallyl , 2-aminoallyl, butenyl may have substituted C 2 -C 3 alkenyl; methyl chloride, ethoxy, propoxy, trifluoromethyl C 1-C 6 chlorinyl with substituents; benzyl, 2-chlorophenyl, p-tolyl, 3-nitrophenyl, 4-cyanophenyl, α-phenyl, _yl, etc. Substituted aryl groups; rudolyl, 2-phenylphenylchloro, p-toluylchloro, 3-nitrophenoxy, α-extractoxy, / 3-naphthyloxy, etc. -2-pyridyl, 3-pyrimidinyl, 4-1% pyridyl, 5-chloro-3-Uttyl, 2-carpentyl, 3-pyridyl, 2-pyrrolyl, 3- Hexyl, 2-furanyl, 3-furanyl is a heterocyclic group containing 3 to 7 members of nitrogen, gas, and sulfur atoms as heteroatoms. Specifically, α-hydroxynitrile [I] is, for example, lactonitrile, Almond, 2-hydroxy-4-methylthiobutyrene, etc. This α-hydroxynitrile [I] is used at a concentration of 0.1 to 50% by weight during the reaction, and it can also be added one by one between the reactions if necessary. The pH value of the reaction solution should be kept between 5 ~ 11 depending on the acid and alkali of the buffer. The reaction temperature should be kept at 4 ~ 50 ° C, preferably 20 ~ 4 0 υ. The present invention Examples of the nitride represented by the general formula [III] include hydrogen halide, sodium halide, potassium halide, calcium cyanide, and cyanide. 1. Tritiated tritium, ammonium cyanide, etc. Gases are generally used in the range of 0.4 to 1000 mM, preferably 4 to 500 mM, and can be added one by one between the reactions if necessary. Therefore, it is 6 hours to 120 hours When α-hydroxynitrile [I] is added over time, the 0 (-hydroxyacid [II] ammonium salt is accumulated at a high concentration of 15% by weight or more. Response > 11- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

Printed by the Employees' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the invention (10) The body is used after repeated low hydrolysis, after repeated hydrolysis. The product is separated and purified by the usual methods of concentration and extraction, and if necessary, it is extracted by organic solvent under thermal conditions, and thermal decomposition is used to separate ammonium. Examples of the product of α-hydroxy acid [I I] include lactic acid, mandelic acid, 2-hydroxy-4-methylthiobutanoic acid, and the like. The best form of Fenshi invention: The present invention will be described in detail by the following examples. (Example 1) A test tube of 20 m 1 containing 0.3% gravy, 0.5% protein, and 0.5% common salt was placed in a conical flask with a baffle capacity of 100 m 1 and a culture medium having the following composition After 2 0 m 1, each goes off at 1 2 1 ° C for _ 15 minutes. Vari ○ OV 『a X paradoxus IAM 12374 strain was implanted with a platinum inoculation ring (platinum ears) in a test tube of 2 m 丨, and cultured overnight at 301 under shaking with salt, and then 0.2 m 1 to In a baffled conical flask, the cells were cultured by shaking at 30 ° C for 3 days. The cells obtained by centrifuging the culture solution were washed in physiological saline, and the dry cells were converted into 1.0% by weight of cells. Suspension in 0.1 M acetic acid buffer (p Η 7.5). Secondly add 2-hydroxy-4-methylthiobutyronitrile to a final concentration of 160 m Μ, and slowly shake at 30 ° C Hydrolysis reaction. The same amount of 2-hydroxy-4-methylthiobutane is added 9 times every 12 hours after the addition, and the reaction is performed for a total of 120 hours. After the reaction is completed, the reaction solution is centrifuged to remove the bell body and centrifuged. The concentration of 2-hydroxy-4-methylthiobutanoic acid in the supernatant was determined by high-speed liquid chromatography (column: TS1 (gel 0 DS-8 0 T M, vehicle: acetonitrile / water / triacetic acid = 2 0/8 0/0, Γ) Quantitative, results, and confirmed accumulation of 2 -5% by weight of 2-hydroxy-4 -methylthiobutyric acid-12- This paper size applies Chinese National Standard (CNS) Α4 Specifications (210 × 297 mm) (Please read the precautions on the back before filling this page)

521088 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 0.5% 0,5% 0,1% 0,1% 0.02¾ 0.02¾ 0.5% 7.2 (adjusted with 2 Ν caustic) A7 B7 V. Description of the invention (U ) $ A salt (yield 98%). Yeast Extract Glycerol Dichloropotassium Dipotrate Dipotassium Hydrogen Potassium Salt

Magnesium sulfate heptahydrate ε-caprolactam pH (Example 2) A test tube containing 20% of a medium containing 0.3% gravy, 0.5% peptone and 0.5% common salt, in a capacity of 10 A conical flask with a baffle of 0 m 1 was filled with a medium having the following composition 2 0 m 1, and each was sterilized at 12 1 ° C for 15 minutes. In a 2 m 1 test tube, a platinum inoculation ring with a platinum inoculation ring was implanted into the joint spore rod® NSSC 104 strain (Taiwan deposit number CCRC 9 1 0 0 7 7), and cultured overnight at 30 t with shaking. Then implant 0.2 ml into a conical flask equipped with a baffle plate, and incubate at 30 υ for 5 days. The bacterial cells obtained by centrifuging the culture solution were washed in physiological saline, and 0.6% by weight of the _ cells were suspended in a 0.1 M phosphate buffer solution (ρΗ7.5) in terms of dry weight. Next, add lucid so that the final concentration is 124 m, and slowly shake at 30 ° C for hydrolysis reaction. After the addition, the same amount of lactonitrile is added 20 times every 5 hours to repeat the reaction for a total of 100 hours. After completion of the reaction, the reaction solution was centrifuged to remove the body, and the concentration of lactic acid in the centrifuged supernatant was subjected to high-speed liquid chromatography (column: TSK gel 0 DS-8 0 T Μ, vehicle: acetonitrile / water-13- This paper size applies to Chinese National Standard (CNS) Α4 size (210 X 297 mm) (Please read the precautions on the back before filling this page)

Corn Juice Dipotassium Magnesium Dichloro Potassium Dipotassium Chloride Dipotassium Chloride Salt Magnesium Sulfate 7 Hydrate Iron Sulfate ε -Caprolactam pH Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the Invention (12) / Trigas acetic acid = 5/95 / 0.1) Quantitative, results, and confirm the accumulation of 23% of lactic acid saddle salt (yield 93%. 1, 0% (do not destroy a) 1 · 〇% (do not sterilize) 0.1 % 0.1% 0.02% 0.02% 0. 0 0 1% (Do not sterilize) 0.5% 7, 2 (Adjusted with 2 N caustic soda) (Example 3) Arthrobacter sp. NSSC prepared in the same way as in Example 2 Cells of 1 0 4 M strain (registered number: CCRC 910077) were suspended in an amount of 4% by weight in terms of dry weight in 0,1 M acid buffer (P7.5), followed by addition of 2-hydroxy-4-methylsulfide Butyl hydroxy was used to make the final concentration of 200 μM, and was slowly shaken to hydrolyze at 30 C. After the addition, 2-hydroxy-4-methylthiobutyronitrile was added 7 times every 1 hour, and then every 1. Add 8 times in 5 hours for a total of 19 hours of hiking. After the reaction, the reaction solution was centrifuged to remove bacteria, and the concentration of 2-hydroxy-4_methylthiobutyric acid in the supernatant was the same as in Example 1. According to the results of the scene setting in this method, it was confirmed that 49% by weight of 2-hydroxy-4-methylthiobutyric acid $ an salt was accumulated (yield 96%.) (Example 4) The joint spores prepared by the same method as in Example 2 _ NSSC 1 0 4 strain (14- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 × 297 mm) (Please read the precautions on the back before filling in this page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the Invention (l3) Deposit Number: CCRC 910077) The bacterial cells were converted to 3.2 weight by dry weight and suspended in distilled water. Next, when the dry weight of the bacteria is 1 g, 2-hydroxy-4-methylthiobutyrene is continuously added at a rate of 0.46 g / hr, and the pH value is adjusted to pH 7, 4 ~ with 0.5M ammonia water. 7.6, hydrolyze at 30 ^ for 20 hours. After the reaction has been completed, the reaction solution is centrifuged to recover the bell body. The recovered bacteria are washed three times with 40 times the weight of distilled water, and then resuspended in the same amount of distilled water in the first _ [for the second reaction. In the same manner as the first round, _-body recovery and bacterial cell washing were performed. This reaction was repeated 10 times, and the concentration of 2-hydroxypyrene-4-methylthiobutyric acid in each centrifugal supernatant was quantified in the same manner as in Example 1. The results are shown below. Repeated reaction (times) 2-hydroxy-4-methylthiobutyric acid (% by weight) Yield (%) 1 36.1 96 3 36.3 97 5 36.4 97 7 38.4 97 10 36.0 96 (Example 5) Example Arthrobacillus HSSC 1 0 4 strain produced by 2 (Reservation number: CCRC 9 1 0 0 7 7) The bacterial cells are converted to 5% by weight based on dry weight. -15- This paper size applies to China National Standard (CNS) Α4 specifications (210 × 297 mm) (Please read the notes on the back before filling in this page)

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 A7 B7 V. Description of the invention (14) The most suspended in 0.1 M sodium nitride aqueous solution. Secondly, 2-hydroxy-4-methylthiobutane was continuously added, and the ρ 7 value was adjusted by the ρ Η controller to 7, 4 ~ 7.6, and hydrolyzed at 30 I for 10 hours. After the end of the reverse mushroom, the reaction solution was centrifuged to remove the blue body. The concentration of lactic acid in the centrifuged supernatant was the same as that in Example 2. The comparison of the reaction results when sodium sulfide is not added is shown below. Production amount of ammonium lactate (% by weight) without adding 20.5 0. 1 M NaCN 40.0 (Example 6) The P. acnes NSSC 1 0 4 fungus prepared in Example 2 (registered number: CCRC 9 1 0 0 7 7) The noodle is suspended in a 0.1 M potassium cyanide aqueous solution in terms of dry weight and converted to 5% by weight, followed by the continuous addition of 2-hydroxy-4-methylthiobutyronitrile, and the value of ρ is adjusted by the ρ Η regulator. ρ Η 7.4-7.6, hydrolyzed at 30 for 10 hours. After the reaction was completed, the reaction solution was centrifuged to remove bacteria, and the concentration of 2-hydroxy-4-methylthiobutyric acid in the centrifuged supernatant was quantified in the same manner as in Example 1. Compared with the case where potassium cyanide was not added The results are shown below. -16.- This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the invention (15) Production amount of ammonium salt of 2-hydroxy-4-methylthiobutyric acid (% by weight) No addition 26.6 0,1M KCN 41.7 (Example 7) The bacterial cells of Arthrobacillus sp. HSSC 104 strain (registration number: CCRC 910077) prepared in Fen Example 2 were suspended in a 40 m H hydrogen cyanide aqueous solution in an amount of 5% by weight in terms of dry weight. Then, 2-hydroxy-4-methylthiobutyronitrile was continuously added, and the p Η value was adjusted to Η 7.4-7 · 6 by the p Η regulator, and the hydrolysis was performed at 30 1C for 10 hours. After the end of the reaction, the reaction solution was centrifuged to remove the blue body, and the concentration of 2-hydroxy-4-methylthiobutyric acid in the centrifuged supernatant was quantified in the same manner as in Example 丨. The reaction without the addition of tritiated hydrogen was compared. The results are shown below. The amount of 2-hydroxy-4-methylthiobutyric acid ammonium salt produced (% by weight) without the addition of 27.2 40mM HCN 44. 5-17- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (Please read the notes on the back before filling this page)

Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the invention () (Reference example) 蕻 Due to the cultivation method and hydrolysis method described in JP-A-4 0 8 98, the joint spore culture NSSC 1 The 0.4 strain was subjected to a catalyst reaction. (i) Culture medium (unit: W / V) Glycerol 2% Yeast extract 1 • 3% potassium dihydrogen acid 0 ♦ 68% m dipotassium hydrogen acid 0.71% sodium 0 .28¾ chloride m 0,04¾ aerated calcium 0. 0 0 4% sulphur manganese 4 X 10-4¾ m 6 X 10-5% zinc sulfate 0 X 10-5% agar 1. 8% a-acetonitrile 0.5% pH 7. 5 (2) Cultivation conditions # Take 1 platinum inoculation fungus from slant culture medium, spread on steam agar plate culture medium, and incubate at 30 ° C for 48 hours under aerobic conditions. (3) Hydrolysis reaction: The bacterial cells were taken from the agar plate culture medium, and the bacterial cells were separated by centrifugation and washed three times in a 0.05 M acetic acid buffer solution (p Η 7.5). The precipitated bacteria were resuspended in 1.5 m L of the same buffer when the value of 0 0630 was 25, and the final concentration was added to 1000 m Μ '18'. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X 297) Li) (Please read the notes on the back before filling in this page)

Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 521088 Α7 Β7 V. Description of the invention (17) 2-hydroxy-4-methylthiobutyronitrile, the oscillating reaction was performed at 2 3 3C for 20 hours. After completion of the reaction, the reaction solution was centrifuged to remove bacteria, and the concentration of 2-hydroxy-4-methylthiobutyric acid in the centrifuged supernatant was subjected to high-speed liquid chromatography (column: TS KK gel 0 DS- 8 Ο Μ, vehicle: acetonitrile / water / trifluorobutyric acid = 20/80 / 0.1) quantitative. The concentration of 2-hydroxy-4-methylthiobutyric acid was 0.01 m. According to Japanese Patent Application No. 4-4088, the bacillus sclerotiorum-HR 4 strain, the culture method and hydrolysis reaction described above, can accumulate 2 -hydroxy-4-methylthiobutane of 5 5 μm. Therefore, it is clear that the bacillus arthritis N S S C 104 strain is different from the bacillus arthritis H R 4 strain. (Effects of the Invention) According to the present invention, by using microorganisms that are resistant to the concentration of α-hydroxyl [I] and / or α-acid base "U" and have durability that is active for a long period of time, they can be used at a high concentration. Accumulates α-hydroxy acid [II]. It is not possible to efficiently produce α-hydroxy acid [II] by using these blue bodies in heavy weight. Moreover, the addition of chloride due to the reaction system can efficiently produce α-hydroxy acid [II]. II]. Industrial Applicability: According to the above description, the present invention uses the durability that has concentration resistance to α-hydroxynitrile [I] and / or α-hydroxy acid [II] and has long-lasting activity. Microorganisms have obtained an industrially advantageous method for producing α-hydroxyacid [II] from cc-hydroxynitrile. Therefore, its industrial significance is of great significance. -19- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297) Li) (Please read the notes on the back before filling in this page)

Amendment 丨 Supplement 丨 971-3721 ^ 112, 〇3 ¥ Pleasei 1997/05 / 02th Case No. 86105867 Category (the above columns are filled by < 本 >) A4 C4 521088 ||| Patent Specification Chinese Use of Microorganisms Method for the production of α-hydroxy acid (as amended on December 3, 91)

A PRODUCTION METHOD 〇F α—HYDROXY ACID BY USING A MICROORGANISM Yanagida 4 Takachi City Co., Ltd., Hara-style, Dazhu, Cao, Xiaozhu County, Shangshang, Shenshen, and the same name (name) Nippon Soda Co., Ltd. Consumption cooperative prints national residence and residence (office) Name of representative: Benmoto, Tokyo, Chiyoda-ku, Otemachi, 2-chome, No.1, Shimomura. The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm).

Claims (1)

Date: 91.12,0 3, No. 丨 Announcement No. 6, Patent Application Range No. 86 1 05 867 "Method for the Preparation of α-Hydroxy Acids Using Microorganisms" (Amended on December 3, 91) Six Patent Application Scope: 1. A method for producing a compound of the formula [H], RCH (OH) C00H [Π] where R is a hydrogen atom, Ci-C6 alkylsulfanyl, hydroxyalkyl or phenyl, and the production method hydrolyzes the formula by the action of microorganisms The compound of [I] (formula [I] is RCH (OH) CN, where R has the same meaning as above), is transformed into a compound represented by formula [Π], which is characterized in that the compound represented by formula [I] and / Or the microorganism represented by the formula [Π] has concentrated tolerance and durability, and the compound represented by the formula [Π] is accumulated in a water-soluble solvent, and the microorganism is Variovorax paradoxus I AM 12374 or the bacillus NSSC 104 strain ( CCRC 910077). For example, the method for preparing a compound represented by formula [II] RCH (0H) C00H according to item 1 of the scope of patent application, wherein the compound represented by formula [I]: RCH (0H) CN is selected from lactonitrile, mandelonitrile, or 2-hydroxy -4 -methylthiobutyronitrile. A method for preparing a compound represented by formula [II] RCH (0H) C00H, which is characterized in that a compound represented by formula [I]: RCH (0H) CN (where R is the same as defined above) is hydrolyzed by a microorganism to produce formula [II ]: When the compound represented by RCH (0H) C00H (wherein R has the same meaning as above), the reaction system has the general formula [III]: Mm (CN) n (where M is hydrogen, ammonium or metal ion, m Η is an integer of 1 to 3). 521088 6. Scope of patent application 4. The method for preparing the compound represented by formula [II] RCH (0H) C00H according to item 3 of the scope of patent application, wherein R is a hydrogen atom, an alkylsulfanyl group, and (: a hydroxyalkyl group) Or a phenyl group. 5 · A method for preparing a compound represented by formula [II] RCH (0H) C00H according to item 3 of the scope of the patent application, wherein the formula [I]: RCH (0H) CN (where R is the same as defined above) The compound shown is selected from lactonitrile, mandelonitrile, or 2-hydroxy-4-methylthiobutyronitrile. 6. The method for preparing a compound represented by the formula [11] RCH (OH) C00H according to item 3 of the patent application scope, wherein The microorganism is a method belonging to Variovorax paradoxus I AMI 2374 〇7. The method for preparing a compound represented by formula [11] RCH (OH) C00H according to item 3 of the patent application scope, wherein the microorganism is Bacillus sp. NSSC 104 strain (CCRC 910077).