JP2007289062A - Method for producing α-hydroxy acid or α-hydroxy acid ammonium using biocatalyst - Google Patents

Abstract

Provided is a method for producing α-hydroxy acid or ammonium α-hydroxy acid with high productivity using a biocatalyst having nitrilase activity.
Impurity aldehyde contained in an aqueous solution of α-hydroxynitrile as a raw material when converting α-hydroxynitrile into α-hydroxyacid or α-hydroxyammonium ammonium using a biocatalyst derived from a microbial cell having nitrilase activity The manufacturing method of alpha-hydroxy acid or alpha-hydroxy acid ammonium characterized by controlling the density | concentration of 0 to 3000 ppm by weight.
[Selection figure] None

Description

  The present invention relates to a method for producing α-hydroxy acid or ammonium α-hydroxy acid using a biocatalyst having nitrilase activity.

  The method of synthesizing a target compound using a biocatalyst having enzyme activity can simplify the reaction process because the reaction conditions are mild, or can obtain a high-purity reaction product with few byproducts. Due to its advantages, it has recently been used in the production of various compounds. Among them, hydrolyzing enzymes such as nitrilase, which has the activity of converting nitrile compounds into carboxylic acids or ammonium carboxylates, and nitrile hydratase, which has the activity of converting nitrile compounds into carboxylic acid amides, have various reaction behaviors. Studies have been made on the production of carboxylic acids or ammonium carboxylates and carboxylic acid amides.

  Among them, many methods for producing α-hydroxy acid or α-hydroxy acid ammonium which convert α-hydroxy nitrile into α-hydroxy acid or α-hydroxy acid ammonium using a biocatalyst having nitrilase activity have been studied. ing. For example, a method for producing glycolic acid, lactic acid, 2-hydroxyisobutyric acid using Corynebacterium genus (Patent Document 1), a method for producing glycolic acid using Rhodococcus genus or Gordona genus (Patent Document 2), or Acidovorax genus A method for producing glycolic acid, 2-hydroxyisobutyric acid using Pt (patent document 3), a method for producing lactic acid using Brevibacterium genus (patent document 4), Pseudomonas genus, Arthrobacter genus, Aspergillus genus or Penicillium genus Alternatively, a method for producing lactic acid, 2-hydroxyisobutyric acid, 2-hydroxy-2-phenylpropionic acid, mandelic acid using Cochliobolus genus or Fusarium genus (Patent Document 5), Enterobacter genus, Arthrobacter genus or Caseobacter genus or Brevibacterium or Aureobacterium or Escherichia or Micrococcus or Streptomyces or Flavobacterium A method for producing D-lactic acid using a genus, Aeromonas genus, Nocardia genus, Mycoplana genus, Cellulomonas genus, Erwinia genus, Candida genus, Pseudomonas genus, Rhodococcus genus, Bacillus genus, Alcaligenes genus, Corynebacterium genus, Myclobacterium genus or Obsumbacterium genus (Patent Document 6), or a method for producing L-3-phenyllactic acid, L-4-phenyl-2-hydroxybutyric acid, D-mandelic acid and derivatives thereof using Gordona genus (Patent Document 7), Rhodococcus genus or A method for producing L-3-phenyllactic acid, L-4-phenyl-2-hydroxybutyric acid using Alcaligenes genus, Brevibacterium genus or Pseudomonas genus (Patent Document 8), or Rhodococcus genus, Alcaligenes genus, Brevibacterium genus or Pseudomonas genus To produce L-3-phenyllactic acid and L-4-phenyl-2-hydroxybutyric acid (Patent Document 9), or a method for producing L-3-phenyllactic acid, D-mandelic acid and derivatives thereof using Gordona genus (Patent Document 10), or Pantoea genus, Micrococcus genus, Bacteridium genus, Bacillus genus or Actinomadura Genus or Kitasatospora genus or Pilimelia genus or Achromobacter genus or Beijerinckia genus or Celluslomonas genus or Klebsiella genus or Actinopolispora genus or Actinopsynnema genus or Actinopulanes genus or Amycolata genus or Streptomyces genus or Novinioides R Rhodospirillum or Caceobcter or Pseudomonas or Alcaligenes or Corynebaterium or Brevibacterium or Nocardia or Rhodococcus or Arthrobacter or Torulopsis or Rhodopseudomonas or Acinetobacter or Mycobacterium or Candida Alternatively, a method of producing 2-hydroxy-4-methylthiobutyric acid using Agrobacterium genus, Aspergillus genus, Penicillium genus, Cochliobolus genus, Fusarium genus, Enterobacter genus, Xanthobacter genus, Erwinia genus, Citrobacter genus, Aeromonas genus or Gordona genus (patent 11), or a method of producing 2-hydroxy-4-methylthiobutyric acid using Alcaligenes genus (Patent Document 12), or producing lactic acid, 2-hydroxy-4-methylthiobutyric acid using Variovororax genus or Arthrobacter genus Method (Patent Document 13), or production method of R-mandelic acid using Escherichia genus, Pseudomonas genus or Alcaligenes genus (Patent Document 14), or producing 2-hydroxy-4-methylthiobutyric acid using Arthrobacter genus Method (Patent Documents 15 to 17), or using a genus Rhodococcus, 2-hydroxy-4- A method for producing tilthiobutyric acid (Patent Document 18), a method for producing mandelic acid using Corynebacterium genus (Patent Document 19), an Enterobacter genus, Arthrobacter genus, Caseobacter genus, Brevibacterium genus, Aureobacterium genus, Eschrichia genus or Micrococcus A method for producing L-lactic acid using the genus or Streptomyces genus, Flavobacterium genus, Aeromonas genus, Nocardia genus, Mycoplana genus, Cellulomonas genus, Erwinia genus, Candida genus, Pseudomonas genus, Rhodococcus genus or Bacillus genus (Patent Document 20), Alternatively, a method for producing R-mandelic acid and derivatives thereof using Aureobacterium genus, Pseudomonas genus, Caseobacter genus, Alcaligenes genus, Acinetobacter genus, Brevibacterium genus or Nocardia genus (Patent Documents 21 to 23), and using Rhodococcus genus Method for producing mandelic acid (Patent Documents 25, 2 7) Or a method for producing R-mandelic acid using Alcaligenes genus (Patent Document 26), transformation with recombinant DNA in which a gene DNA encoding a polypeptide having nitrilase activity derived from Alcaligenes genus is incorporated into a vector A method for producing R-mandelic acid using the transformed transformant (Patent Document 28), or a method for producing R-2-hydroxy-4-phenylbutyric acid using Alcaligenes genus or Corynebacterium genus (Patent Document 29) Or 3- (S) -tert. Method for producing carbonylamino-4-cyclohexyl-2- (R) -hydroxybutyric acid (Patent Document 30), or Rhodococcus Alternatively, a method for producing 2-hydroxyisobutyric acid using Pseudomonas genus, Arthrobacter genus or Brevibacterium genus (Patent Document 31), a method for producing 2-hydroxyisobutyric acid using Rhodococcus genus (Patent Document 32), or Achromobacter A method for producing 2-hydroxyisobutyric acid using a genus (Patent Document 33) is disclosed.

  In the methods for producing α-hydroxy acids disclosed in these prior documents, α-hydroxynitrile is hydrolyzed with a microbial catalyst (nitrilase) to obtain α-hydroxy acid ammonium first, which is well known to those skilled in the art. The free α-hydroxy acid is produced by a conventional method such as neutralization with an acid, ion exchange resin column, electrodialysis, thermal decomposition or the like.

  However, in the above-mentioned conventional technology, the hydrolase of various biocatalysts used, that is, nitrilase does not necessarily have an initial specific activity that is industrially satisfactory with respect to α-hydroxynitrile. There are problems such as low speed and very large reactor size, and further improvement in the initial specific activity of nitrilase has been demanded.

  For example, Patent Document 1 describes that the average production rate of ammonium lactate is 15.4 mmol / g-dry cells / Hr using the genus Corynebacterium. Further, in Patent Document 3, it is described that the average production rate of ammonium glycolate is 1.7 mmol / g-dry cells / Hr using Acidovorax genus. Patent Document 33 describes that the average production rate of ammonium 2-hydroxyisobutyrate is 1.8 mmol / g-dry cells / Hr using the genus Achromobacter. In Patent Document 17, using Arthrobacter, the average production rate of 2-hydroxy-4-methylthiobutyric acid is as low as 23.3 mmol / g-dry cells / Hr (35 ° C. × 18 Hr). Are listed. In Patent Document 2, it is described that the average production rate of ammonium glycolate is 18.5 mmol / g-dry cells / Hr (20 ° C. × 24 Hr) using Rhodococcus or Gordona. ing. None of these examples have an average production rate that is industrially satisfactory.

  The reason for the low initial production rate and / or average production rate is generally that α-hydroxynitrile is partially decomposed and dissociated into the corresponding aldehyde or ketone and hydrocyanic acid in an aqueous solution (Non-patent Document 1). Can inhibit the enzyme activity. (Non-patent document 2) It has also been pointed out that the enzyme may be deactivated in a short time by the action of the dissociated aldehyde. In addition, α-hydroxynitrile as a raw material is generally synthesized from a corresponding aldehyde aqueous solution and hydrocyanic acid through a cyanhydrinating reaction with an alkali catalyst, and either one is excessive from the viewpoint of increasing the degree of reaction completion. Therefore, it contains either aldehyde or hydrocyanic acid as a trace amount of impurities. Therefore, since it causes the initial activity of nitrilase to decrease for the same reason as the partial decomposition / dissociation of α-hydroxynitrile described above, a significant increase in the initial activity is expected by reducing the trace amount of hydrocyanic acid or aldehyde. The As described above, there has been an indication of inhibition or inactivation by hydrocyanic acid or aldehyde as described above. However, to what extent the reduction of hydrocyanic acid or aldehyde contained as an impurity can increase the initial specific activity, There was no description in the prior patents and literatures relating to the production method of α-hydroxy acid.

  Further, in addition to the aldehyde or cyanuric acid converted to glycolonitrile, aldehyde or cyanide contained in the raw material is considered to cause similar inhibition or deactivation. Alternatively, the effect of increasing the initial activity can be expected by reducing the concentration of the cyanide compound.

  In other words, a substantially high nitrilase ratio that can produce a large amount of α-hydroxy acid or ammonium α-hydroxy acid with a practical reaction time and a practical reactor size necessary for industrially sufficient productivity. In order to achieve an industrial production method of α-hydroxy acid or ammonium α-hydroxy acid using an active biocatalyst, there is no quantitative control technique for cyanide or aldehyde present as impurities in the raw material. Is real.

Japanese Patent Publication No. 3-38836 Japanese Patent Laid-Open No. 9-028390 JP 2005-504506 A U.S. Pat. No. 3,940,316 Japanese Examined Patent Publication No. 4-63675 US Pat. No. 5,234,826 JP-A-6-237789 JP-A-6-284899 European Patent Application No. 610049 European Patent Application 610048 JP-A-10-179183 JP-T-2001-502908 US Pat. No. 6,037,155 Special Table 2002-527106 JP 2002-34584 A International Publication No. 2002/052027 Pamphlet International Publication No. 2003/062437 Pamphlet JP 2004-81169 A JP 62-296883 A Japanese Patent Laid-Open No. 4-99497 Japanese Patent Laid-Open No. 4-99496 Japanese Patent Laid-Open No. 4-99495 JP-A-4-218385 JP-A-5-95795 JP-A-5-192189 JP-A-4-341185 US Pat. No. 5,296,373 JP-A-6-153968 Japanese Patent Laid-Open No. 5-192190 JP-A-6-319591 JP-A-4-40897 JP-A-5-219969 JP-A-6-237776 JP-A-5-192189 US Pat. No. 5,326,702 JP-A-7-213296 JP-A-8-131188 Chemical Reviews, 42,189-, (1948) Agricultural Biological Chemistry, 46, 1165-, (1982)

  An object of the present invention is to produce a practical reaction time and practical use in producing α-hydroxy acid or ammonium α-hydroxy acid from an α-hydroxy nitrile compound using a biocatalyst derived from a microbial cell having nitrilase activity. An object of the present invention is to provide an industrial production method of α-hydroxy acid or α-hydroxy acid ammonium capable of achieving production of a large amount of α-hydroxy acid or ammonium α-hydroxy acid with a reactor size.

  In the production of α-hydroxy acids or ammonium α-hydroxyacids from α-hydroxynitrile compounds using biocatalysts derived from microbial cells having nitrilase activity, the present inventors have practical reaction times and practical reactors. As a result of intensive investigations on an industrial production method of α-hydroxy acid or ammonium α-hydroxy acid capable of achieving production of a large amount of α-hydroxy acid or ammonium α-hydroxy acid by size, it is surprisingly used as a raw material. Among the impurities contained in α-hydroxynitrile, it was found that by controlling the concentration of aldehyde and cyanide ion, an unprecedented high nitrilase initial specific activity can be expressed, and the present invention has been completed.

That is, this invention has the structure as described below.
[1] When converting α-hydroxy nitrile to α-hydroxy acid or ammonium α-hydroxy acid using a biocatalyst derived from a microbial cell having nitrilase activity, impurities aldehyde contained in the raw material α-hydroxy nitrile aqueous solution A method for producing an α-hydroxy acid or an α-hydroxy acid ammonium, wherein the concentration is controlled to 0 to 3000 ppm by weight.
[2] Impurity cyanide contained in the raw material α-hydroxynitrile aqueous solution when α-hydroxynitrile is converted into α-hydroxyacid or α-hydroxyammonium ammonium using a biocatalyst derived from microbial cells having nitrilase activity A method for producing α-hydroxy acid or α-hydroxy acid ammonium, wherein the concentration is controlled to 0 to 5000 ppm by weight.
[3] The pH in the reaction system of the reaction for converting α-hydroxynitrile into α-hydroxy acid or α-hydroxy acid ammonium is controlled to 6 to 8 and the reaction temperature is controlled to 30 to 60 ° C. [1] A method for producing an α-hydroxy acid or α-hydroxy acid ammonium according to [2].
[4] The method for producing α-hydroxy acid or α-hydroxy acid ammonium according to [1] to [3], wherein the biocatalyst is a biocatalyst derived from a microbial cell belonging to the genus Acinetobacter.
[5] The microbial cell belonging to the genus Acinetobacter is Acinetobacter sp. AK226 or Acinetobacter sp. AK227 or a microbial cell derived from these cells [ 4] The production method of α-hydroxy acid or α-hydroxy acid ammonium according to [4].
[6] The α-hydroxy acid or α according to [5], wherein the microbial cell belonging to the genus Acinetobacter is Acinetobacter sp. AK226 or a microbial cell derived from these cells. -Method for producing ammonium hydroxy acid.
[7] A cyan compound represented by the general formula M _m (CN) _n (M represents H, NH ₄ or a metal element, and m and n represent integers) in an α-hydroxy nitrile aqueous solution containing an impurity aldehyde. The method for producing α-hydroxy acid or α-hydroxyammonium ammonium according to [1] to [6], wherein an α-hydroxynitrile aqueous solution with reduced impurity aldehyde concentration is used as a raw material.
[8] Using an α-hydroxynitrile aqueous solution in which the impurity cyanide concentration is reduced by allowing an equivalent aldehyde or ketone equivalent to or more moles of the cyanide to act on the α-hydroxynitrile aqueous solution containing the impurity cyanide compound. A method for producing an α-hydroxy acid or an α-hydroxy acid ammonium according to any one of [1] to [7].
[9] The method for producing glycolic acid or ammonium glycolate according to [1] to [8], wherein the α-hydroxynitrile used as a raw material is glycolonitrile.

  According to the method of the present invention, using a biocatalyst derived from a microbial cell having nitrilase activity, a large amount of α-hydroxy acid or α-hydroxy acid is converted from an α-hydroxynitrile compound with a practical reaction time and a practical reactor size. Ammonium hydroxy acid can be produced.

The present invention will be specifically described below.
The biocatalyst derived from a microbial cell having nitrilase activity referred to in the present invention is a catalyst having a nitrilase enzyme derived from a microbial cell having the ability to directly convert a nitrile group into a carboxylic acid or an ammonium carboxylate group. Any form may be used.

  Many microbial species are known. Examples of those having high nitrilase activity include Rhodococcus genus, Acinetobacter genus, Alcaligenes genus, Pseudomonas genus, Corynebacterium genus and the like. Of these, the genus Acinetobacter and Alcaligenes, which are gram-negative bacteria, are particularly preferred in the present invention, and more preferably the genus Acinetobacte. Specifically, Acinetobacter sp. AK226 (FERM BP-08590) and Acinetobacter sp. AK227 (FERM-BP-08591). These strains are disclosed in JP-A No. 2005-17639, JP-A No. 2004-305066, JP-A No. 2004-305058, JP-A No. 2004-305062, JP-A No. 2001-299378, JP-A No. 11-180971, JP-A No. 06-303991, and JP-A No. 63-209592, and Japanese Examined Patent Publication No. 63-2596.

    Further, for example, it may be a microorganism in which a natural or artificially improved nitrilase gene is incorporated by a genetic engineering technique, or a nitrilase enzyme extracted therefrom, but a microorganism having a low nitrilase expression level or α-hydroxynitrile To produce α-hydroxy acid (ammonium) using a small amount of microorganisms expressing nitrilase with low conversion activity from ammonium to α-hydroxy acid ammonium, more reaction time is required. It is desirable to use microorganisms that have been isolated and / or that have expressed nitrilase with high conversion activity, or nitrilase enzymes that have been removed therefrom.

    As a form of the biocatalyst, microbial cells may be used as they are in a dormant state, or those obtained by crushing treatment, or those obtained by removing the necessary nitrilase enzyme from the microbial cells may be used as they are. Those fixed by a comprehensive method, a crosslinking method, a carrier binding method or the like may be used. Examples of the immobilization carrier include, but are not limited to, glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, and photocrosslinking resin.

    When microorganisms are used as they are, they may be suspended only in water (distilled water and / or ion-exchanged water), but they are usually suspended in an inorganic salt buffer solution from the viewpoint of long-term retention of nitrilase activity. Similarly, when an immobilized product is used, it is usually used after being suspended in a buffer solution. The buffer solution concentration at this time is preferably as low as possible from the viewpoint of reducing impurities in the reaction solution, but it is usually less than 0.1 M from the viewpoint of biocatalytic stability and retention of nitrilase activity, and preferably is 0.1. It is 01-0.08M, More preferably, it is 0.02-0.06M.

    The α-hydroxynitrile referred to in the present invention is a compound represented by the general formula [I]: RCH (OH) CN (wherein R has a hydrogen atom, a C1-C6 alkyl group which may have a substituent, and a substituent. C2-C6 alkenyl group which may have, C1-C6 alkoxyl group which may have a substituent, aryl group which may have a substituent, aryloxy group which may have a substituent or a substituent An a-hydroxy nitrile represented by, for example, mandelonitrile, acetone cyanohydrin, glycolonitrile, lactonitrile, 2-hydroxy-4-methylthione butyronitrile. However, it is not limited to these.

  The impurity aldehyde referred to in the present invention refers to all aldehydes contained in the raw material α-hydroxynitrile. Aldehydes that are expected to be most abundant are aldehydes corresponding to the α-hydroxynitrile remaining when converted to the α-hydroxynitrile, specifically, aliphatics such as formaldehyde, acetaldehyde, propionaldehyde, and the like. Examples include aldehydes and aromatic aldehydes such as benzaldehyde, but are not limited thereto. In addition, examples of those which are not converted into the α-hydroxynitrile but are expected to exist include glutaraldehyde used as a crosslinking agent for enzyme immobilization.

  In the present invention, the concentration of the impurity aldehyde contained in α-hydroxynitrile is a very important factor. If the aldehyde concentration is too high, the initial specific activity decreases due to inhibition or inactivation of the nitrilase enzyme caused by the aldehyde, resulting in a decrease in the initial production rate and / or the average production rate. Therefore, the concentration of the impurity aldehyde in the raw material α-hydroxynitrile aqueous solution in the present invention is essentially 0 to 3000 ppm by weight, preferably 0 to 2000 ppm by weight, more preferably 0 to 1000 ppm by weight, and still more preferably 0 to 500 ppm by weight, most preferably not included at all.

In the present invention, the α-hydroxynitrile aqueous solution containing a large amount of impurity aldehydes has a general formula M _m (CN) _n (M is an alkali metal such as H, NH _4, Na, K or the like, or an alkalinity such as Ca, Mg, etc. The impurity aldehyde concentration can be reduced by the action of a cyanide compound represented by the following formula: metal or other metal such as Fe, where m and n represent integers. Usually, this operation can be carried out under the same conditions as the cyanohydrination reaction carried out using aldehyde or ketone and hydrocyanic acid as raw materials. Specifically, while controlling the pH and temperature of the reaction solution within an appropriate range, the impurity aldehyde in the α-hydroxynitrile aqueous solution and an equimolar amount or less of the cyanide compound are reacted with the impurity aldehyde and converted to α-hydroxynitrile. By doing so, the impurity aldehyde is reduced. In this case, if the pH and / or temperature is too high, the decomposition reaction of the raw material α-hydroxynitrile is promoted, so that diffusion of free hydrocyanic acid into the gas phase part and coloring due to polymerization of hydrocyanic acid become violent. Therefore, although not necessarily limited, the pH is preferably 2 to 5, more preferably 3 to 4. The reaction temperature is not necessarily limited, but it is preferably 10 to 50 ° C, more preferably 20 to 40 ° C.

  The impurity cyanide compound referred to in the present invention is all cyanide compounds contained in the raw material α-hydroxynitrile. The cyanide compound that is expected to be most abundant is hydrocyanic acid remaining when converted to the α-hydroxynitrile. In addition, when an alkali metal ion or the like is present in the reaction solution, it may be present in the form of cyan ion or sodium cyanide or potassium cyanide.

  In the present invention, the impurity cyanide concentration contained in α-hydroxynitrile is a very important factor. If the cyanide ion concentration is too high, the initial specific activity decreases due to the inhibition or inactivation of the nitrilase enzyme caused by the cyanide ion and / or its polymer, resulting in a decrease in the initial production rate and / or the average production rate. . Therefore, the impurity cyanide concentration in the raw material α-hydroxynitrile aqueous solution in the present invention is indispensable to be 0 to 5000 ppm by weight, preferably 0 to 1000 ppm by weight, more preferably 0 to 100 ppm by weight, and still more preferably 0. ˜50 ppm by weight, more preferably 0-10 ppm by weight, most preferably none.

  In the present invention, the α-hydroxy nitrile aqueous solution having an impurity cyan ion concentration of more than 5000 ppm by weight can be reacted with a corresponding aldehyde or ketone to reduce the impurity cyan ion concentration. The corresponding aldehyde or ketone as used herein refers to an aldehyde or ketone capable of synthesizing the α-hydroxynitrile by a cyanohydrination reaction with hydrocyanic acid. Usually, this operation can be carried out under the same conditions as the cyanohydrination reaction carried out using aldehyde or ketone and hydrocyanic acid as raw materials. Specifically, while controlling the pH and temperature of the reaction solution within an appropriate range, an impurity cyan ion in an α-hydroxy nitrile aqueous solution and an equimolar amount or more of aldehyde or ketone are reacted with the impurity cyan ion, and α-hydroxy By converting to nitrile, the impurity cyan ion is reduced. In this case, if the pH and / or temperature is too high, the decomposition reaction of the raw material α-hydroxynitrile is promoted, so that diffusion of free hydrocyanic acid into the gas phase part and coloring due to polymerization of hydrocyanic acid become violent. Therefore, although not necessarily limited, the pH is preferably 2 to 5, more preferably 3 to 4. The reaction temperature is not necessarily limited, but it is preferably 10 to 50 ° C, more preferably 20 to 40 ° C.

  In the present invention, the initial specific activity is a reaction temperature of 30 ° C., pH = 6.5-7.0, reaction substrate concentration: 1 wt%, and α-hydroxynitrile aqueous solution in biocatalyst phosphate buffer suspension. Is added, and the molar amount of α-hydroxyammonium produced in 5 minutes from the start of the reaction is divided by the weight of the dry biocatalyst used and further divided by the reaction time (5 minutes = 1/12 hours). Is defined as the amount of α-hydroxy acid ammonium produced per hour per dry biocatalyst weight. However, the amount of cells used should be adjusted so that the conversion rate of α-hydroxynitrile is 15 to 30%.

  The initial production rate in the present invention is such that the reaction is started by continuously or intermittently adding an α-hydroxynitrile aqueous solution to the biocatalyst suspension under any reaction conditions, and the α-hydroxy in 1 hour from the start of the reaction. It is defined as the amount of ammonium α-hydroxy acid produced per hour per dry biocatalyst weight obtained by dividing the ammonium acid production molar amount by the dry biocatalyst weight used. In the present invention, the initial production rate is 32 [mmol / g-dry biocatalyst / Hr] or more, preferably 50 [mmol / g-dry biocatalyst / Hr] or more, more preferably 100 [mmol / g-dry. Biocatalyst / Hr] or more, more preferably 200 [mmol / g-dry biocatalyst / Hr] or more, and most preferably 300 [mmol / g-dry biocatalyst / Hr] or more.

  The average production rate in the present invention is that the reaction is started by continuously or intermittently adding an α-hydroxynitrile aqueous solution to the biocatalyst suspension under any reaction conditions, and the product is an α-hydroxyacid ammonium salt. As a result of the decrease in activity caused as the accumulated concentration increases, this is the time-average production rate up to the point when the production rate is reduced to half of the initial production rate. Production of ammonium α-hydroxy acid up to that point It is obtained by dividing the molar amount by the reaction time. The average production rate in the present invention must be 16 [mmol / g-dry biocatalyst / Hr] or more, preferably 32 [mmol / g-dry biocatalyst / Hr] or more, more preferably 50 [mmol / g. -Dry biocatalyst / Hr] or more, more preferably 100 [mmol / g-dry biocatalyst / Hr] or more, and most preferably 150 [mmol / g-dry biocatalyst / Hr] or more.

  The pH of the reaction solution is determined from the viewpoint of 1) the optimum pH of the nitrilase specific activity of the biocatalyst used and 2) the suppression of decomposition of the substrate α-hydroxynitrile into HCN and aldehyde or ketone. Regarding 1), it is estimated that the alkaline region of pH = 7 to 11 is good from the hydrolysis activity behavior of nitriles other than α-hydroxynitrile, but from the viewpoint of 2), the acidic region of pH = 2 is good. I know that. In the present invention, the pH of the reaction solution is preferably 6 to 8, and preferably 6.5 to 7.

  Since α-hydroxynitrile as a raw material is a very unstable substance, it usually contains an acid component such as sulfuric acid, phosphoric acid or acetic acid as a stabilizer. Therefore, in order to adjust the pH in the reaction system, it is essential to add an alkali to the reaction system. In this case, the alkali used is not particularly limited as long as it does not affect the reaction, but it is desirable to use ammonia as one of the products. The form of ammonia may be a gas or ammonia water, but ammonia water is usually desirable for ease of handling.

  About reaction temperature, when reaction temperature is too low, reaction activity will become low, and more reaction time is required when manufacturing high concentration ammonium alpha-hydroxy acid. On the other hand, if the reaction temperature is too high, the biocatalyst is thermally deteriorated, and if the target ammonium α-hydroxy acid concentration is high, it is difficult to reach the concentration, resulting in the addition of a new biocatalyst, etc. Treatment is required and the catalyst cost is high. If the temperature is too high, the substrate α-hydroxynitrile will also be promoted to decomposition into hydrocyanic acid and aldehyde or ketone, causing further reduction in reaction activity such as reaction inhibition and deactivation. Therefore, the reaction temperature is usually 30 to 60 ° C, preferably 40 to 50 ° C.

  The reaction method for producing α-hydroxy acid or α-hydroxy acid ammonium may be any of a fixed bed, a moving bed, a fluidized bed, a stirring tank, etc., and may be a continuous reaction or a half-batch reaction. When microbial cells that are not present are used, a half-batch reaction using a stirring tank is preferable because of the ease of reaction. In that case, it is good to perform appropriate stirring from the viewpoint of reaction efficiency.

  In addition, when a half-batch reaction is performed, the biocatalyst may be disposable for one batch or may be repeatedly performed. However, when the reaction is repeated, the biocatalyst is rapidly changed from a high concentration of α-hydroxy acid ammonium to a low concentration, so that the specific activity may be reduced due to the influence of osmotic pressure, etc., so care must be taken.

  The steady concentration of the α-hydroxynitrile compound as the reaction substrate is preferably 2% by weight or less, more preferably 0.1 to 1.5% by weight, still more preferably 0.1 to 1.0% by weight, most preferably. Is preferably controlled to 0.2 to 0.5% by weight. If the concentration of the α-hydroxy nitrile compound is too high, product inhibition and / or deactivation, or substrate inhibition and / or deactivation, which become significant only at high product accumulation concentrations, rapidly increase and have progressed until then. Reaction may stop. Moreover, when the density | concentration of an alpha-hydroxy nitrile compound is too low, it will reduce reaction rate, Since alpha-hydroxy acid or alpha-hydroxy acid ammonium cannot be manufactured efficiently, it is disadvantageous. For the above reasons, it is very important to control the steady concentration of the α-hydroxynitrile compound during the reaction.

  The dry biocatalyst weight used for the α-hydroxy acid ammonium to be produced is preferably 1/100 or less, preferably 1/100 to 1/200, more preferably 1/200 to 1/300, and even more preferably 1/300 to 1/300. 1/500, most preferably 1/500 or less. If the amount of dry biocatalyst used relative to the produced α-hydroxy acid ammonium is too large, impurities from the biocatalyst suspension are often entrained in the reaction solution, which increases the purification cost and lowers the product quality. . Conversely, if the weight of the dry biocatalyst used relative to the α-hydroxy acid ammonium produced is too small, the productivity per reactor volume is reduced, and a large reactor size is required, which is economically disadvantageous.

  Hereinafter, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited by these Examples, A various change and modification are possible unless it exceeds the summary. In particular, in the examples, only experiments using glycolonitrile are shown, but in consideration of the gist of the present invention, it is important to control the concentration of aldehyde or cyanide compound present in the raw material. It can be easily estimated that the same phenomenon and result can be obtained for α-hydroxynitrile.

  Acinetobacter sp. AK226, which is a biocatalyst used in the present invention, has been internationally deposited by the present inventors at the Patent Organism Depositary (AIST) and has the international deposit number of FERM BP-08590.

  The method for measuring the weight of the dried biocatalyst in the biocatalyst suspension was performed as follows. First, an appropriate amount of a biocatalyst suspension having an appropriate concentration was taken, cooled to −80 ° C., completely dried using a freeze dryer, and the concentration of the biocatalyst suspension was calculated from the weight value. A biocatalyst suspension having a known concentration was diluted to a plurality of appropriate concentrations, and the transmittance was measured at room temperature using a UV meter (600 nm), and a calibration curve for the biocatalyst was prepared with the UV meter. . Thereafter, the dry biocatalyst concentration of the arbitrary biocatalyst suspension was calculated from the indicated value of the UV meter.

  The reaction solution was analyzed as follows. The substrate α-hydroxynitrile and the product α-hydroxy acid or ammonium α-hydroxy acid were measured as high-performance liquid chromatography as α-hydroxy acid. The column is an ion exclusion column (Shimadzu Shim-pack SCR-101H), the column temperature is 40 ° C., the mobile phase is phosphoric acid aqueous solution (pH = 2.3), the detector is UV (Shimadzu SPD-10AV vp, 210 nm) and RI (Shimadzu RID-6A).

[Example 1]
[Preparation of biocatalyst]
Sodium chloride 0.1 wt%, potassium dihydrogen phosphate 0.1 wt%, magnesium sulfate heptahydrate 0.05 wt%, ferrous sulfate heptahydrate 0.005 wt%, ammonium sulfate 0.1 wt% %, Potassium nitrate 0.1 wt% Manganese sulfate pentahydrate 250ml culture solution 250ml was placed in an Erlenmeyer flask, adjusted to pH 7 with sodium hydroxide and sterilized at 121 ° C for 20 minutes After that, 0.5% by weight of acetonitrile was added. This was inoculated with Acinetobacter sp. AK226 and cultured with shaking at 30 ° C. (preculture). Mist powder 0.3% by weight, sodium glutamate 0.5% by weight, ammonium sulfate 0.5% by weight, dipotassium hydrogen phosphate 0.2% by weight, potassium dihydrogen phosphate 0.15% by weight, sodium chloride 0. 1% by weight, magnesium sulfate heptahydrate 0.18% by weight, manganese chloride tetrahydrate 0.02% by weight, calcium chloride dihydrate 0.01% by weight, iron sulfate heptahydrate 0.003% by weight 3L of culture broth containing 0.002% by weight of zinc sulfate heptahydrate, 0.002% by weight of copper sulfate pentahydrate and 2% by weight of soybean oil was charged into a 5L jar fermenter and sterilized at 121 ° C for 20 minutes. After that, the pre-cultured solution was inoculated and aerated and stirred at 30 ° C. Soybean oil feed was started 10 hours after the start of the culture. The pH was controlled with phosphoric acid and aqueous ammonia so that the pH was 7, and finally about 5% by weight of Acinetobacter sp. AK226 suspension was obtained. Furthermore, it was washed twice with 0.06M phosphate buffer, and finally Acinetobacter sp. AK226 suspension (dry cell concentration 5 to 10% by weight) suspended in phosphate buffer was obtained.

[Analysis of raw glycolonitrile]
A composition analysis was performed using high performance liquid chromatography for 55 wt% glycolonitrile manufactured by Aldrich used as a raw material. The column is an ion exclusion column (Shimadzu Shim-pack SCR-101H), the column temperature is 40 ° C., the mobile phase is phosphoric acid aqueous solution (pH = 2.3), the detector is UV (Shimadzu SPD-10AV vp, 210 nm) and RI (Shimadzu RID-6A). Glycolonitrile: 53.8% by weight, glycolic acid: 1.57% by weight, formaldehyde: detection limit (5 ppm by weight) or less.

[Measurement of initial specific activity]
The hydrolysis reaction was carried out using the above-mentioned biocatalyst suspension using the above- analyzed 55% by weight glycolonitrile manufactured by Aldrich as a raw material. First, a suspension of Acinetobacter sp. AK226 with a known bacterial cell concentration that was refrigerated and stored in a suspension in 0.06 M phosphate buffer was charged in advance in a 50 mL eggplant flask to a concentration of 320 ppm by weight. The suspension was suspended in 20 ml of 06M phosphoric acid aqueous solution (the pH was adjusted in advance so that the pH was 6.8 when the aqueous solution of glycolonitrile was added). The flask was placed in a 30 ° C. constant temperature water bath and stirred with a stirrer, and held for a while until the internal temperature reached 30 ° C. Next, 0.36 ml of a starting 55% glycolonitrile aqueous solution was added using a micropipette to start the reaction. Sampling was performed after 5 minutes, the reaction was stopped by diluting with 2M aqueous hydrochloric acid, and the cells were removed by filtration, followed by high performance liquid chromatography analysis. The initial specific activity obtained was 340 [mmol / g-dry biocatalyst / Hr]. The results are shown in FIG.

[Examples 2 and 3]
The initial specific activity was evaluated by carrying out the hydrolysis reaction in the same manner as in Example 1 except that an appropriate amount of formaldehyde was added to 55% by weight glycolonitrile manufactured by Aldrich. When the formaldehyde concentration was 550 ppm by weight, it was 275 [mmol / g-dry biocatalyst / Hr], and when the formaldehyde concentration was 1129 ppm by weight, it was 242 [mmol / g-dry biocatalyst / Hr]. The results are shown in FIG.

[Comparative Examples 1 and 2]
The initial specific activity was evaluated by carrying out the hydrolysis reaction in the same manner as in Example 3 except that 55% by weight glycolonitrile manufactured by Aldrich was added with an amount of formaldehyde exceeding the range specified in the present invention. . When the formaldehyde concentration was 3966 wt ppm, it was 110 [mmol / g-dry biocatalyst / Hr], and when the formaldehyde concentration was 9900 wt ppm, it was 81 [mmol / g-dry biocatalyst / Hr]. The results are shown in FIG.

  According to the method of the present invention, the initial specific activity of the nitrilase can be sufficiently increased by controlling the impurity aldehyde or cyanide concentration in the raw material glycolonitrile, and as a result, the initial production rate and the average production rate are obtained. Therefore, industrially useful α-hydroxy acid or ammonium α-hydroxy acid can be produced from any nitrile compound with practical reactor size, reaction time and catalyst cost.

It is a figure which shows the relationship between the formaldehyde density | concentration in the raw material glycolonitrile aqueous solution in an Example and a comparative example, and the initial stage specific activity of a biocatalyst.

Claims (9)

  When α-hydroxy nitrile is converted to α-hydroxy acid or α-hydroxy acid ammonium using a biocatalyst derived from microbial cells having nitrilase activity, the concentration of impurity aldehyde contained in the raw material α-hydroxy nitrile aqueous solution is reduced to 0. A method for producing α-hydroxy acid or α-hydroxy acid ammonium, which is controlled to ˜3000 ppm by weight.   When converting α-hydroxynitrile to α-hydroxy acid or α-hydroxyammonium ammonium using a biocatalyst derived from a microbial cell having nitrilase activity, the impurity cyanide concentration contained in the raw material α-hydroxynitrile aqueous solution is reduced to 0. A method for producing α-hydroxy acid or α-hydroxy acid ammonium, which is controlled to ˜5000 ppm by weight.   The pH in the reaction system of the reaction for converting α-hydroxynitrile into α-hydroxy acid or α-hydroxy acid ammonium is controlled to 6 to 8, and the reaction temperature is controlled to 30 to 60 ° C. Alternatively, the method for producing α-hydroxy acid or α-hydroxy acid ammonium according to 2.   The method for producing α-hydroxy acid or ammonium α-hydroxy acid according to any one of claims 1 to 3, wherein the biocatalyst is a biocatalyst derived from a microbial cell belonging to the genus Acinetobacter.   5. The microbial cell belonging to the genus Acinetobacter is Acinetobacter sp. AK226 or Acinetobacter sp. AK227 or a microbial cell derived from these cells. A production method of α-hydroxy acid or ammonium α-hydroxy acid.     The α-hydroxy acid or α-hydroxy acid according to claim 5, wherein the microbial cell belonging to the genus Acinetobacter is Acinetobacter sp. AK226 or a microbial cell derived from these cells. A method for producing ammonium. A cyanide compound represented by the general formula M _m (CN) _n (M represents H, NH ₄ or a metal element, and m and n represent integers) is allowed to act on an α-hydroxynitrile aqueous solution containing an impurity aldehyde. The method for producing an α-hydroxy acid or an α-hydroxy acid ammonium according to any one of claims 1 to 6, wherein an α-hydroxynitrile aqueous solution with a reduced concentration of the impurity aldehyde is used as a raw material.   Using an α-hydroxynitrile aqueous solution in which the impurity cyanide concentration is reduced by reacting an equivalent aldehyde or ketone of equimolar or more with the cyanide compound on the α-hydroxynitrile aqueous solution containing the impurity cyanide compound. The manufacturing method of the alpha-hydroxy acid or alpha-hydroxy acid ammonium in any one of Claims 1-7 characterized by the above-mentioned.   The production method of glycolic acid or ammonium glycolate according to any one of claims 1 to 8, wherein α-hydroxynitrile used as a raw material is glycolonitrile.

Images