MXPA99003430A - Enzymatic production of vitamin - Google Patents

Abstract

A procedure for the enzymatic production of vitamin B6, which comprises the incubation of 1-deoxy-D-threo-pentulose and 4-hydroxy-L-threonine with an enzyme reaction system prepared from cells of a microorganism belonging to the genus Rhizobium, Sinorhizobium, Flavobacterium, Chryseobacterium, Lactobacillus, Arthrobacter, Bacillus, Klebsiella, Escherichia, Pseudomonas, Stenotrophomonas, Enterobacter, Serratia, Corynebacterium, Brevibacterium, Exiguobacterium, Saccharomyces, Yamadazma, Pichia or Candida, in the presence of NADP +, NAD +, ATP. The manganese and magnesium ions stimulate the previous reaction. This procedure provides high yields of vitamin B6, an essential vitamin for the nutrition of animals, plants and microorganisms, and useful as a medicine or food additive.

Description

g DESCRIPTION OF THE INVENTION This invention relates to a process for the enzymatic production of vitamin B, from l-deoxy-D-threo-pentulose (hereinafter, called DTP), and 4-hydroxy-L-threonine ( HT). The term "vitamin Bß" as used in the present invention, includes pyridoxol, pyridoxal and pyridoxamin. Vitamin Bt is one of the essential vitamins for the nutrition of animals, plants and microorganisms, and is also very important as a medicine or as a food additive for human beings. The object of the present invention is to provide a highly efficient process for the enzymatic production of vitamin Bβ from DTP and HT. There are many studies on the fermenting of vitamin B6. Several microorganisms belonging to the genus Saccharomyces [G.H. Scherr and M.E. Rafelson, J. Appl. Bacteriol. 25., 187-194 (1962)], Pichia [N. Nishino, K. Fujii, and T. Kamikubo, Agrie. Biol. Chem. 7, 553-559 (1973)], klebsiella [R. Suzue and Y. Haruna, J. Vitaminol. 1 £, 154-159 (1970)], AchromoJbacter [M. Ishida and K. Shimura, Agrie. Biol. Chem. 2 £, 327-334 (1970)], Bacillus [W. Pflug and F. Lingens, Hoppe-Seyler's Z. Physiol. Chem. 359. 559-570 (1978)] and Flavofcacterium [Y. Tani, T. Nakamatsu, T. Izumi and K. Ogata, Agrie. Biol. Chem. £, 189-197 (1972)] are known as producers of vitamin Bt. However, to date, no fermentation process has been known to be commercially attractive for the production of vitamin Bt. More recently, European patent application 0 756 938 A2 has described a process for obtaining by fermentation of vitamin Bβ by cultivating a microorganism belonging to the genus Rhizobium capable of producing said vitamin in a culture medium under conditions aerobic; the culture medium may contain, apart from sources of assimilable carbon and digestible nitrogen, inorganic salts and other nutrients, as well as substances that increase the titre of vitamin Bß, such as DTP and HT. Although this most recently known procedure produces vitamin Bß with high yield, there is room for improvement of its efficacy. The present invention makes possible the production of vitamin Bβ from DTP and HT with an efficiency superior to that achieved so far. It has been discovered that the cell-free extract prepared from cells of a microorganism of the genus RhizoJbium, Sinorhizobiu, Flavobac-teriu, Chryseobacterium, Lactobacillus, Arthrobacter, Bacillus, Klebsiella, Eecherichia, Pseudomonas, Stenotrophomo-nas, Enterobacter, Serratia, Corynebacteriu , Brevibacte-riu, Exiguobacterium, Saccharomyces, Yamadazma, Pichia 6 Candida, is able to produce vitamin Bβ from DTP and HT in the presence of nicotinamide adenine dinucleotide phosphate (NADP *), nicotinamide adenine dinucleotide (NAD *) and adenosine triphosphate (ATP). Thus, the present invention relates to a process for the enzymatic production of vitamin B6 from DTP and HT comprising the contacting of DTP and HT with an enzyme reaction system prepared from cells of a microorganism capable of producing vitamin B, from the DTP and the HT, making said contact in the presence of NADP *, NAD * and ATP. The present invention further relates to a process for the enzymatic production of vitamin B, comprising contacting DTP and HT with an enzyme reaction system containing the cell-free extract derived from a microorganism capable of producing vitamin Bß, belonging to the genus Rhizobium, Sinorhizobium, Flavobacteriupi, Chxyseojbacterium, Lactobacillus, Arthrobacter, Bacillus, Klebsiella, Escherichia, Pseudomonas, Stenotrophoonas, Enterobacter, Serratia, Corynebacterium, Brevibacteurium, Exiguobacterium, Saccharomyces, Yamadazma, Pichia 6 Candida, in the presence of NADP *, NAD * and ATP. The vitamin B content of a reaction mixture can be determined by a bioassay with Saccharomyces carlsbergensis ATCC 9080 according to the method of D.R. Osborne and P. Voogt [The Analysis of Nutrients in Foods], Academic Press, London, 224-227 (1978)]. To carry out the procedure of this Invention, cells of the microorganism, e.g. ex. belonging to the genus Rhizobium, Sinorhizobium, Flavobacterium, Chryseobacterium, Lactobacillus, Arthrobacter, Bacillus, Klebsiella, Escherichia, Pseudomonas, Stenotropomonas, Enterobacter, Serratia, Corynebacterium, Brevibacteurium, Exiguobacterium, Saccharomyces, Yamadazma, Pichia 6 Candida, cultivating said microorganism in a medium containing sources of assimilable carbon, sources of digestible nitrogen, inorganic salts and other nutrients necessary for the growth of microorganisms. As carbon sources, for example, glucose, fructose, lactose, galactose, sucrose, maltose, starch, dextrin and glycerin can be used. As nitrogen sources, for example, peptone, yeast extract, soybean meal, corn germ extract, meat extract, ammonium sulfate, ammonium nitrate, urea, and mixtures thereof can be employed. In addition, as inorganic salts, sulfates, hydrochlorides or phosphates of calcium, magnesium, zinc, manganese, cobalt and iron can be used. And if necessary, conventional nutrient factors or an antifoaming agent such as an animal oil, vegetable oil or mineral oil may also be included in the culture medium. The pH of the culture medium can be from about 5 to about 9, preferably from about 6 to about 8. The temperature range suitable for the culture is from about 10 ° C to about 45 ° C, preferably from about 25 ° C. ° C at approximately 40 ° C. The duration of the culture is usually from about 1 to about 5 days, preferably from about 1 to about 3 days. Aeration and agitation during cultivation usually gives favorable results. Microorganisms that can be used in the process of the present invention include all strains belonging to the genera JuizoJbium, Sinorhizobium, Fia-voJbacterium, Chryseobacterium, Lactobacillus, Arthrobacter, Bacillus, Klebsiella, Escherichia, Pseudomonae, Stenotrophoonas, Enterobacter, Serratia, Corynebacterium, Brevibacte-rium, Exiguobacterium, Saccharomyces, Yamadazma, Pichia and Candida. These microorganisms can be purchased in a public warehouse (crop collection) at the request of anyone, such as p. ex. the Institute of Fermentation ("Fermentation Institute"), Osaka, Japan (IFO); examples of said deposited strains are Rhizobium meliloti (also known as S'inorJuzoJbiupi jpeliloti) IFO 14782 (DSM nß 10226), Flavobacterium indologenes (also known as Chryseobacterium indologenes, IFO 14944, Lactobacillus Jbre-vis IFO 13110, Arthrobacter nicotianae IFO 14234, Bacillus subtilis IFO 3007, Klebsiella planticola IFO 3317, Escheri -chia coli IFO 13168, Pseudomonas putida IFO 3738, Stenotro-phomonas maltophilia (also known as Pseudomonas maltophilia or Xanthimonas maltophilia) IFO 12692, Enterobacter cloacae IFO 3320, Serratia marcescens IFO 12648, Corynebacterium ammoniagenes (also known as Brevibacterium ammoniagrenes) IFO 12612, Corynebacterium gruptainicum) (also known as Brevibacterium glutamicum) IFO 12168, Exigrubacteritum acetylicup? (also known as Brevibacterium acetylicum IFO 12146, Pichia guilliermondii (also known as Ya adazyma guilliermondii) IFO 10106, Saccha-romyces cerevisiae IFO 0304 and IFO 0306 and Candida tropicalis IFO 0199 and IFO 0587. Among these microorganisms, they are preferably used in the present invention: Rhizobium meliloti IFO 14782 (DSM No. 10226), Flavobacterium indologenee IFO 14944, Bacillus eubtilis IFO 3007, Escherichia coli IFO 13168, Serratia arceecens IFO 12648, Corynebacterium ammo-niagenes IFO 12612, Corynebacterium glutamicum IFO 12168, Pichia guilliermondii IFO 10106 , Saccharo? Yces cerevisiae IFO 0306. For the preparation of the cell-free extract of cells obtained by culture, general methods such as ultrasonic treatment and cell breakage in the presence of glass beads or by the French homogenizer under pressure can be applied If desired, treatment with a lytic enzyme such as lysozyme or zymolase from 15 ° C to 45 ° C, preferably at 20 ° C to 40 ° C for 1 to 3 hours, may also be applied, prior to disintegration in the above mentioned way. For example, after centrifugation of the culture broth, the resulting cells are washed with saline and suspended in a buffer such as Tris-HCl buffer (pH 7.5) containing sucrose, dithiothreitol (DTT) and fluoride. phenylmethylsulfonyl (PMSF) as general stabilizers of the enzymes. After cell disruption, the resulting solution is centrifuged to separate cell debris, and the supernatant can be used as a cell-free extract. The enzyme reaction system contains the cell-free extract as prepared above or those partially purified by general methods for the purification of enzymes such as precipitation with ammonium sulfate or gel permeation chromatography. Alternatively, the re-sidual cells or the growing cells of the microorganism can also be used. In addition to the cell-free extract, DTP and HT are added as substrates and also NADP *, NAD * and ATP as cofactors, to the reaction system. The amount of DTP, HT, NADP *, NAD * and ATP that must be added to the system may vary depending on the reaction system used. However, in general, the concentrations of DTP, HT, NADP *, NAD * and ATP in the enzyme reaction system are 0.1 mM or more, preferably from 1 mM to 10 mM for DTP and HT; preferably from 0.05 mM to 5 mM, and more preferably from 0.2 mM to 0.4 mM, for NADP * and NAD *; and preferably from 1 mM to 20 mM, and more preferably from 3 mM to 7 mM for ATP. The addition of manganese ions or magnesium ions to the enzyme reaction system stimulates the reaction, and the addition of both ions produces even better results. As salts supplying these ions, for example, the manganese and magnesium hydrochlorides, sulphates, nitrates or phosphates can be used. The amount of manganese ions and magnesium ions to be added may also vary depending on the reaction system employed. However, in general, the concentrations of manganese ions and magnesium ions are in the case of. manganese ions, from 0.1 mM to 100 mM, preferably from approximately 5 mM to 10 M, and in the case of magnesium ions, from 3 mM to 300 mM, preferably from 20 mM to 50 mM. To initiate the enzymatic reaction, a buffer solution that has no influence on the production of vitamin Bβ from DTP and HT can be used. The Tris-HCl buffer is preferably used for this purpose. The enzymatic reaction takes place effectively in a pH range of 6.0 to 8.5, and more preferably in the range of 7.0 to 8.0. The reaction temperature is suitable from 15 ° C to 45 ° C, more preferably from 20 ° C to 40 ° C. The incubation period can be varied depending on the reaction conditions, although it is generally from 30 minutes to 5 hours, more preferably from 2 hours to 4 hours. Vitamin Bt obtained from DTP and HT under the conditions described above, can be easily recovered as follows: For example, after the reaction, the proteins of the reaction mixture are precipitated by heat denaturation, acid, alkali or an organic solvent and separated by centrifugation. For this purpose, a procedure generally used for extracting a certain product from the superior supernatant, which is applicable to the various properties of vitamin Bß, can be applied. For example, the vitamin Bß in the supernatant is purified with an ion exchange resin. The desired product is subsequently recrystallized with a mixture of alcohol and water. The present invention will be illustrated in more detail by the following examples; however, it should be understood that the present invention is not limited to these particular examples.

Preparation of the cell-free extract The Rhizobium meliloti IFO 14782 (DSM No. 10226) was cultured in a seed medium containing 1% glucose, 0.5% polypeptone. { Nippon Seiyaku Co., Japan), 0.2% yeast extract (Difco), 0.05% MgSO4.7H20, 0.001% MnS04.5H20 and 0.001% FeS04.7H20 at 28 'C for 17 hours. The seed culture was transferred to a 500 ml flask containing 200 ml of a fermentation medium containing 4% glucose, 2% polypeptone, 0.2% yeast extract, 0.05% MgSO4.7H20, 0.05% of MnS04.5H20, 0.0001% of FeS04.7H20, and one drop of antifoam CA-115 (Nippon Yushi Co., Japan), and then the bottle was shaken on a shaker of flasks at 28 ° C . After cultivation for 72 hours, the cells were harvested from 400 ml of the culture broth by centrifugation at 10,400 xg for 10 minutes and washed twice with 0.85% NaCl solution and once with 10 mM. Tris-HCl buffer (pH 7.5) containing 15% sucrose, 0.1 mM PMSF and 1 mM DTT and stored at -30 ° C until needed for the preparation of the cell-free extract. The following operation was carried out completely in ice water or at 4 ° C. Cells stored at -30 ° C were thawed and suspended in 5 ml of 10 mM Tris-HCl buffer (pH 7.5) containing 15% sucrose, 0.1 M PMSF and 1 mM DTT. The cell suspension was passed through a French homogenizer under pressure (Ohtake Works Co., Ltd.) at 200 kg / cm2. The resulting homogenate was centrifuged at 34,800 x g for 30 minutes to separate cell debris. Next, ten milliliters of the supernatant ID was dialyzed overnight against 1 liter of 80% ammonium sulfate solution containing 15% sucrose, 0.1 mM PMSF and 1 mM DTT, and the precipitate was collected by centrifugation at 34,800 xg for 30 minutes. The precipitate was dissolved in 10 milliliters of 10 mM Tris-HCl buffer (pH 7.5) containing 15% sucrose and 0.1 mM PMSF, dialysed overnight against the same buffer, dialyzed solution it was stored at -30 ° C until it was needed for the enzymatic reaction The protein content of the cell-free extract was determined by Lowry's method [Lowry et al., J. Biol. Chem. 193. 265 (1951 )] and it was 11.4 mg / ml E1emolo 2 Enzymatic production of vitamin B, from DTP and HT The enzymatic reaction was carried out by means of incubation tubes containing 500 μl of the reaction mixture listed in the Table 1 to 28 ° C. A complete reaction system contained 2.5 mM DTP, 2.5 mM HT, 0.38 mM NADP *, 0.38 M NAD *, 5 mM ATP, 193.25 μl of the cell-free extract and 80 mM Tris-HCl buffer, pH 7.50 After an incubation of 2 hours, the reaction was stopped, heating in a boiling water bath for 3 minutes, it was centrifuged at 10,000 xg for 10 minutes and then the supernatant was treated with phosphatase by incubation of a tube containing 15 μl of the supernatant, 10 μl of 1 mg / ml of acid phosphatase (Boehringer Mannheim GmbH, Germany) and 10 μl of 100 mM acetate buffer (pH 5.0) at 37 ° C for 30 minutes. After incubation, 1,800 μl of water was added to the tube and determined by the microbiological method using the Saccharomyces carlsbergeneie ATCC 9080 as described below. The standard solutions of pyridoxol (0.2 μg per milliliter) were diluted to 1.21 x 10"2 in distilled water, 100 μl of the diluted standard solution or sample and 3 ml of the test medium for the vitamin were added. Bß (Ni-ssui Co., Japan) containing the Saccharomycee carlebergen-sis ATCC 9080, to the tubes in the order indicated and incubated at an angle of 30 ° to 28 ° C. After an incubation of 17 hours, interrupted cell growth by adding 5 ml of 0.1 N hydrochloric acid, and then the absorbance of the samples was measured at 660 nm The amount of vitamin B6 in a sample was determined by comparing the turbidity of the sample with the curve Growth standard of Saccharomycee carlebergeneie ATCC 9080. As a result, 97 ng of vitamin Bß / ml / mg protein / hour was obtained in the complete reaction system., no vitamin Bs were produced in the reaction systems in which a complete system factor was missing. In addition, 119, 123 or 587 ng of vitamin B6 / ml / mg of protein / hour were obtained in the system supplemented with 8.4 mM of MnCl2, 32 mM of MgCl2, or both, respectively, to the complete system (table 1) . The results indicated that the cell-free extract, NADP *, NAD * and ATP are essential for the production of vitamin B6 from DTP and HT, and that MnCl2 and MgCl2 stimulate production.

Table 1. Enzymatic production of vitamin Bß from DTP and HT Complete: 2.5 mM DTP, 2.5 mM HT, 0.38 mM NADP *, 0.38 mM NAD *, 5 mM ATP, 193.25 μL cell-free extract, and 80 mM of Tris-HCl buffer, pH 7.50 Example 3 In a manner similar to that described in example 1 and 2, the production of vitamin B6 was examined by cell-free extracts of various kinds of microorganisms. A loop filled with cells grown on an agar plate of each strain listed in Table 2 was grown in each seed medium at 28 ° C for 17 hours. Two milliliters of the seed culture were transferred into a 500 ml flask containing 100 ml of the bulk medium and one drop of antifoam, and then stirred in a flask shaker at 28 ° C. The compositions of the seeding medium and the bulk medium for the culture of each strain listed in Table 2 are summarized in Table 3. Table 2. Microorganisms and their culture medium Table 3. Media compositions After a 24-hour culture, the cells of each strain were harvested from 400 ml of culture broth by centrifugation and washed twice with 0.85% NaCl solution and once with 10 mM Tris-buffer. HCl (pH 7.5) containing 15% sucrose, 0.1 mM PMSF and 1 mM DTT. The resulting cells were suspended in 5 ml of the same buffer. Cells of Flavobacterium indologenes IFO 14944, Lactobacillus brevis IFO 13110, Arthrobacter nicotianae IFO 14234, Bacillus eubtilie IFO 3007, Klebeiella planticola IFO 3317, Eecherichia coli IFO 13168, Peeudomonas putida IFO 3738, Stenotrophomonae mal tophilia IFO 12692, Enterobacter cloacae IFO 3320 or Serratia arcescene IFO 12648, were disintegrated by passing them through a French homogenizer under pressure or by ultrasonic disintegration (Cosmo Bio Co., Ltd.) and others, treated with 2 mg of lysozyme (Sigma) or 200 units of zymolase (Sigma) / ml of cell suspension at 30 ° C for 1 hour before disintegration as shown in Table 4. The resulting homogenate was centrifuged to remove cellular debris, and the supernatant was dialyzed against 10 mM Tris-HCl buffer ( pH 7.5) containing 15% sucrose and 0.1 mM PMSF and used as a cell-free extract. The enzymatic reaction was carried out by incubating a tube with 500 μl of reaction mixture A containing 2.5 mM DTP, 2.5 mM HT, 0.38 mM NADP *, 0.38 M NAD * , 5 mM of ATP, 193.25 μL of cell-free extract, and 80 mM of Tris-HCl buffer, pH 7.50, or reaction mixture B supplemented with 8.4 mM of MnCl2 and 32 mM of MgCl2 28 ° C. After incubation for 2 hours, the reaction was stopped by heating in a boiling aryan bath for 3 minutes, the mixture was centrifuged at 10,000 x g for 10 minutes and the supernatant was treated with acid phosphatase at 37 ° C. After an incubation for 30 minutes, the vitamin Bs produced in the reaction mixture was determined by the bioassay method using the Saccharomy-ees carlebergeneie ATCC 9080. As a result, 7-23 and 33-139 ng of vitamin Bß were obtained / ml / mg protein / hour, in reaction mixture A and B respectively, as summarized in Table 4.

Table 4. Production of vitamin Bß by the F cell-free extract: Press French U: Ultrasonic disintegration *: Treatment with 2 mg of lysozyme (Sigma) / ml of cell suspension at 30 ° C for 1 hour before disintegration **: Treatment with 200 units of zymolase (Sigma) / ml of cell suspension ° C for 1 hour before disintegration. Reaction mixture A: 2.5 mM DTP, 2.5 mM HT, 0.38 mM NADP *, 0.38 mM NAD +, 5 mM ATP, cell-free extract, and 80 mM Tris buffer -HCl, pH 7.50, in a total volume of 500 μl. Reaction mixture B: 2.5 mM DTP, 2.5 mM HT, 0.38 mM NADP +, 0.38 mM NAD +, 5 mM ATP, 8.4 mM MnCl2, 32 mM MgCl2, cell-free extract, and 80 mM Tris-HCl buffer, pH 7.50, in a total volume of 500 μl.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (8)

CLAIMS Having described the invention as above, property as claimed in the following claims is claimed:

1. A procedure for the production of the vitamin Bß from 1-deoxy-D-threo-pentulose (DTP) and 4-r, hydroxy-L-threonine (HT), characterized in that it comprises contacting the DTP and the HT with a reaction system enzyme prepared from cells of a microorganism capable of producing vitamin Bβ from DTP and HT, said contact taking place in the presence of nicotinamide adenine dinucleotide phosphate (NADP *), nicotinamide adenine dinucleotide (NAD *), and adenosine triphosphate (ATP).

2. A method according to claim 1, characterized by the uuuai cres of DTP, HT, NADP *, NAD *, and

ATP in the enzyme reaction system is 0.1 mM or more, preferably from 1 mM to 10 mM for DTP and HT; preferably from 0.05 mM to 5 mM and more preferably from 0.2 mM to 0.4 mM for the NADP * and NAD *; and preferably from 1 mM to 20 mM, more preferably from 3 mM to 7 mM for ATP. 3. A process according to claim 1 or 2 characterized in that the enzyme reaction system also contains manganese ions, magnesium ions, or a mixture of both magnesium and manganese ions.

4. A process according to claim 3, characterized in that the concentrations of manganese ions and magnesium ions are, in the case of manganese ions, from 0.1 mM to 100 mM, preferably from 5 mM to 10 mM. mM, and in the case of magnesium ions, from 3 mM to 300 mM, preferably, from 20 mM to 50 mM.

5. A process according to any one of claims 1 to 4, characterized in that the enzymatic reaction for preparing the enzymatic reaction system is carried out in a pH range of 6.0 to 8.5, preferably 7.0 to 8. , 0, and in a temperature range of 15 ° C to 45 ° C, preferably, from 20 ° C to 40 ° C, for 30 minutes to 5 hours, preferably for 2 hours to 4 hours.

6. A process according to any one of claims 1 to 5, characterized in that the enzyme reaction system contains the cell-free extract derived from a microorganism belonging to the genus Rhizobium, Sinorhizobium, Flavobacterium, Chryeeobacteriu, Lactobacillue, Arthrobacter, Bacillue, Klebeiella, Eecheri -chia, Pseudomonas, Stenotrophomonas, Enterobacter, Serratia, Corynebacterium, Brevibacterium, Exiguobacterium, Saccharomycee, Yamadazma, Pichia or Candida.

7. A procedure according to the claim 6, characterized in that the enzyme reaction system contains a cell-free extract, derived from one or more of Rhizo-bium meliloti (also known as Sinorhizobium meliloti) IFO 14782 (DSM No. 10226), Flavobacterium indologenee (also known as Chryeeobacterium indologenee) ) IFO 14944, Lactobacillus brevie IFO 13110, Arthrobacter nicotianae IFO 14234, Bacillus eubtilie IFO 3007, Klebeiella planticola IFO 3317, Eecherichia coli IFO 13168, Peeudomonae putida IFO 3738, Stenotrophomonas maltophilia (also known as Peeudomonae maltophilia or Xanthomonae maltophilia) IFO 12692, Enterobacter cloacae IFO 3320, Serratia arcescene IFO 12648, Corynebacterium ammoniagenes (also known as Brevibacterium ammoniagenes) IFO 12612, Coxynebacterium glutamicum (also known as Brevibacterium glutamicum) IFO 12168, Exiguobacterium acetylicu (also known as Brevibacterium acetylicum IFO 12146, Pichia guilliermondii (also known as Yamadazyma guilliermondii) IFO 10106, Saccharomyces cerevieiae IFO 0304 and IFO 0306 and Candida tropicalie IFO 0199 and IFO 0587.

8. A process according to claim 7, characterized in that the enzyme system contains a cell-free extract derived from one or more of Rhizobium. meliloti IFO 14782 (DSM No. 10226), Flavobacterium indologenee IFO 14944, Bacillue eubtilie IFO 3007, Escherichia coli IFO 13168, Serratia marcescens IFO 12648, Corynebacterium ammo-niagenes IFO 12612, Corynebacterium glutamicum IFO 12168, Pichia guilliermondii IFO 10106, and Saccharomycee cerevieiae IFO 0306. SUMMARY] OF THE INVENTION A procedure for the enzymatic production of vitamin Bß, which includes the incubation of 1-deoxy-D-threo-pentulose and 4-hydroxy-L-threonine with an enzyme reaction system prepared from cells of a microorganism belonging to the genus Rhizobium, Sinorhizobium, Flavobacterium, Chryseobacterium, Lactobacillus, Arthrobacter, Bacillus, Klebsiella, Escherichia, Pseudomonas, Stenotrophomonas, Enterobacter, Serratia, Corynebacterium, Brevibacterium, Exiguobacterium, Saccharomyces, Yamadazma, Pichia or Candida, in the presence of NADP *, NAD *, ATP. The manganese and magnesium ions stimulate the previous reaction. This procedure provides high yields of vitamin Bß, an essential vitamin for the nutrition of animals, plants and microorganisms, and useful as a medicine or food additive.