Title of Invention

A PROCESS FOR PRODUCING RIBOFLAVIN GLUCOSIDE

Abstract

A process for producing riboflavin glucoside, which comprises cultivating a microorganism belonging to the genus Bacillus, which is capable of producing riboflavin glucoside, in an aqueous medium such as herein described containing starch under aerobic conditions, isolating and purifying riboflavin glucoside from the culture medium in a known manner. . 2. The process according to claim 1, wherein the microorganism belongs to the species Bacillus breivs, Bacillus cereus. Bacillus circulans. Bacillus coagulans, Bacillus licheniformis. Bacillus megaterium. Bacillus pumilus or Bacillus subtilis.

Full Text

This invention relates to a process for producing riboflavin glucoside from starch by fermentation. The term "riboflavin glycoside" as used in this specification embraces riboflavin glycosides featuring one or more glucose moieties per molecule of riboflavin. Riboflavin glycoside is known as one of the metabolites of riboflavin found in urine. It is more soluble in water than riboflavin. The solubility of riboflavin glucoside at 20°C and 37°C is 2.2 and 3.5 mg/ml, respectively. In comparison, riboflavin has a solubility of 0.1 and 0.2 mg/ml at these temperatures [see Methods in Enzymologist, Academic Press, 18B, 404-417 (1971)]. Riboflavin itself is widely used as an additive in drinks for coloring and/or nutrition, but the drinks become cloudy because of its low solubility in water. Riboflavin-containing solutions for intravenous drop injection also become turbid and tend to block the injection tubes. To solve such solubility problems the more soluble riboflavin glucoside could be used instead of riboflavin to prepare clear drinks and (injection) solutions. Riboflavin glucoside was first obtained by Whit by with the acetone-dried powder of rat liver [Biochem. J. 50, 433 (1952)]. Fluoridation of riboflavin occurs when riboflavin is incubated in a solution containing transglucosidase and glucosyl donors such as maltose, dextrin, starch, glycogen and salicin. Transglucosidase has been reported to be widely distributed in animal organs, microorganisms and plants such as rat liver, Aspergillus oryzae, Escherichia coli, Leuconostoc mesenteries, and cotylendons of pumpkin, Cucurbita pep, and of sugar beet. Beta vulgaris. However, the productivity of riboflavin glucoside by these enzymatic reaction methods or fermentation in media containing riboflavin and glucosyl donors has been rather low, and its purification procedure too complicated, for practical use [see J. Vitamin logy 6, 139-144 (1960) and Methods in Enzymology 18B, 404-417 (1971)]. By means of the process of the present invention, it is possible to produce riboflavin glucoside in a much higher yield, even without the addition of riboflavin, by the fermentation of a riboflavin glucoside-producing microorganism in a medium containing a starch. Said process comprises cultivating a microorganism belonging to the genus Bacillus which is capable Pa/So 18.8.98 of producing riboflavin glucosides in an aqueous medium containing a starch under aerobic conditions. The microorganisms which may be used in the present invention include all strains belonging to the genus Bacillus possessing amylase activity, e.g. Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus and Bacillus subtilis, and its recombinants which are capable of producing riboflavin. Some of these microorganism strains are deposited at the Institute for Fermentation, Osaka, Japan (IFO). These have the accession designations Bacillus brevis IFO 15304, Bacillus cereus IFO 15305, Bacillus circulans IFO 13626, Bacillus coagulans IFO 12583, Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308, Bacillus pumilus IFO 12092 and Bacillus subtilis IFO 13719, and are hsted in the IFO's "List of Cultures", Microorganisms, 10th Edition 1996. As such, samples of the microorganisms are publicly available from the IFO. Examples of the strains most preferably used in the present invention are Bacillus subtilis RB50::[pRF69]60::[pRF93]120Ade+, Bacillus subtilis RB50::[pRF69]60Ade+ and the like [European Patent Publications (EP) 405370 Al and 821063 A2] . The host strain, RB50, is a deregulated Bacillus subtilis strain resistant to roseoflavin. A plasmid pRF69 contains the SPOl-15 promoter and cat gene in the same direction as the rib operon. The details of the host microorganism and a plasmid pRF69 are given in EP 405370 Al. Said host microorganism RB50 and plasmid pRF69 have been deposited under the Budapest Treaty at the Agricultural Research Culture Collection (NRRL), Peoria, Illinois, and the American Type Culture Collection (ATCC), Rockville, Maryland, respectively, under the following deposit nos. on the given dates: Bacillus subtilis RB50: (NRRL) B-18502 (originally May 23, 1989; redeposited August 24, 1989) pRF69: ATCC 68338 (June 6, 1990) The former deposit was made by S.L. Misrock, c/o Pennie & Edmonds, 1155 Avenue of the Americas, New York, NY 10036, USA. As a result of various changes of responsibility for this deposit the current depositor is effectively Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, New Jersey 07110. The latter deposit was made by BioTechnica International, Inc., 85 Bolton Street, Cambridge, Massachusetts 02140, USA (the current depositor in this case is Omni Gene Bioproducts, Inc., 763-D Concord Avenue, Cambridge, Massachusetts 02138). RB50::[pRF69]60Ade+ is prepared by introducing pRF69 to the rib site of RB50 following by the gene amplification by selecting for colonies that grow in the presence of increasing level of chloramphenicol. A plasmid pRF93 is derived from pRF89 (see Fig. 14 of EP 405370 Al) by exchanging chloramphenicol-resistant gene for tetracycline-resistant gene (see Example 8, Second site Integration, of EP 405370 Al). RB50::[pRF69]60::[pRF93]120Ade+ is obtained by integrating the second plasmid, pRF93, at bpr site of the chromosome (EP 821063 A2). The recombinant strains possessing modified rib operon at the sites of chromosome are amplified by drug resistance. Bacillus subtilis RB50::[pRF69]60::[pRF93]120Ade+ is known to be capable of producing more than 14.0g/l of riboflavin under the optimized jar fermentation condition (EP 821063 A2). The preparation of a plasmid pRF93 is also described in the said European patent publication. In a preferred embodiment of the present invention, the production of riboflavin glucosides is effected by cultivating the last-mentioned microorganism strain in an aqueous culture medium containing a starch, especially a soluble starch and/or one or more other starches and supplemented with appropriate nutrients under aerobic condition. Said medium contains a soluble starch and/or one or more other starches at a (total) concentration from about 25 g/1 to about 400 g/1, preferably from about 200 g/1 to about 300 g/1. The amount of inoculums of microorganism is generally about 1% to about 30%, preferably about 5% to about 20%. The culture medium contains starch, of which in principle any sort can be used, such as soluble starch, potato starch, corn starch and wheat starch. It is usually required that the culture medium also contains nutrients. These may be digestible nitrogen sources, such as organic substances, for example, peptone, yeast extract, soybean meal, corn steep liquor, cottonseed refuse, dried yeast and meat extract; inorganic substances, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, potassium nitrate and potassium phosphate; vitamins; metals; amino acids; trace elements; and additional assimilable carbon sources, for example D-glucose, D-fructose, D-mannose, D-sorbitol, D-mannitol, sucrose, molasses, starch hydrolyzates, acetic acid and ethanol if necessary. The cultivation is conveniently conducted at a pH of about 4.0 to about 9.0, preferably about 4.5 to about 8.0. The cultivation period varies depending upon the particular microorganism and nutrient medium used, and is generally in the range of about 10 to about 150 hours. The temperature range for carrying out the cultivation is conveniently from about 20 to about 45°C, preferably from 25 to 40°C. The riboflavin glucosides thus accumulated consist of a mixture of those having one or more glucose moieties per molecule of riboflavin. If desired, the so-produced riboflavin glucosides can be easily concentrated to riboflavin monoglucoside, and this can be recovered, for example, by the following procedure: The culture broth containing riboflavin and riboflavin glucosides is first filtered or centrifuged to remove cells. Then the separated filtrate is treated with glucoamylase, whereby riboflavin glucosides are concentrated to riboflavin monoglucoside. About 1 unit of glucoamylase/mg riboflavin glucosides is usually sufficient for this purpose (one unit liberates 1.0 mg of glucose from starch in 3 minutes at pH 4.5 and 55°C). The amount of enzyme employed depends on the incubation temperature, period and other reaction conditions, e.g. pH. If the enzyme concentration and/or temperature are low, a long incubation period is required. Two to three days incubation at 37°C has been tried and showed good results. Considering these data, 0.001 to 100 units/mg, at 25 to 70°C for 1 minute to 100 hours, preferably 0.1 to 10 units/mg at 30 to 60°C for 3 minutes to 70 hours, are suitably employed. For further purification, if desired, the treated solution may be applied to an adsorbent resin. Regardless of whether riboflavin monoglucoside itself is to be obtained, the riboflavin glucoside accumulated in the fermentation can be isolated from the fermentation medium by standard techniques, preferably involving adsorbent resin and gel filtration resin for the separation of eac^ component. _^ ^O*" Accordingly the present invention provides a process for producing riboflavin glucoside, which comprises cultivating a microorganism belonging to the genus Bacillus, which is capable of producing riboflavin glucoside, in an aqueous medium such as herein described containing a starch under aerobic conditions, isolating and purifying riboflavin glucoside from the-culture medium in a known manner. The invention now having been described in general terms, the accompanying figure and examples are presented to illustrate the invention in more detail, without limiting it in any manner. Figure 1: HPLC analysis of a typical culture broth. Example 1 One loopful of Bacillus subtilis RB50::[pRF69]60::[pRF93]120Ade+ grown on an agar plate of Trytose Blood Agar Base (TBAB, DIFCO Laboratories, Detroit, USA) medium containing 60 |ig/ml of chloramphenicol and 120 μg/ml of tetracycline was inoculated into 8 ml of seed culture medium contained in a test tube. The contents of the test tube were incubated at 37°C for 2.75 hours using a tube shaker. The seed culture thus prepared (4 ml) was inoculated into a production medium made up to 40 ml after inoculation in a 500 mi Erlenmeyer flask with buffles. The composition of the seed culture and production medium was as follows. The production medium was incubated at 37°C and 240 rpm for 3 days. The broth was analyzed for the production level of riboflavin-related compounds by thin layer chromatography. One μ[ of the broth was spotted on a silica gel plate (Kieselgel 60F254, MERCK, Darmstadt, Germany) and developed by a solvent system consisting of acetone, n-butanol and water in a volume ratio of 5:4:1. At least three compounds other than riboflavin, designated component A, B and C and all yellow in colour, were detected. The Rf values for the components A, B, C and riboflavin were 0.27, 0.15, 0 (remaining at the spotted point after development) and 0.41, respectively. Accordingly, the component A is predominant. In direct comparison with flavin mononucleotide or flavin- adenin dinucleotide, it was demonstrated that none of the components corresponded to these nucleotides. HPLC analysis of the culture broth was effected under the conditions described in Fig. 1. The retention time of the component A was 8.6 minutes, while that of riboflavin was 9.6 minutes. The UV-Vis. absorption spectra of the components A, B and C coincided well with that of riboflavin. The total productivity of the components A, B and C measured by the UV absorption at 444 nm was 3.51 g/1 based on riboflavin. Example 2 A culture broth obtained in a similar manner to Example 1 was treated with glucoamylase (EC 3.2.1.3) of Aspergillus niger (Sigma Chemical Co., Missouri, USA) in sodium acetate buffer (pH 4.5) at 55°C for 2.5 hours. By thin layer chromatography analysis, the components A, B and C in the culture broth were observed to be centered at component A. Furthermore, a spot identical with authentic glucose was detected. Thus glucose was released from the components B and C by the treatment. The solution was then applied to a column packed with an adsorbent resin, Amberlite® XAD-7 (Rohm and Haas Co., Philadelphia, USA). Both the component A and riboflavin were adsorbed by the resin (glucose was not adsorbed) and eluted with a 1:1 aqueous acetone solution after washing with water. After concentration by evaporation to remove acetone, the eluate was freeze-dried. The resulting powder was dissolved in a small amount of sodium hydroxide solution and applied to a column packed with a gel filtration resin, Toyopearl HW-40F (TOYO SODA Mfg. Co. Ltd., Tokyo, Japan), to separate the component A and riboflavin. The eluted component A fraction was freeze-dried. The purity of the so-obtained powder was 97%. The molecular weight of the component A was determined by a mass spectrometer to be m/z 539. This value corresponds to the molecular weight of riboflavin monoglucoside. The component A was then hydrolysed by IN hydrochloric acid at 95°C for 2.5 hours to investigate the possibility that a glucose moiety is attached to riboflavin. The hydrolysate was then spotted on a thin layer chromatographic plate together with authentic samples of riboflavin and glucose, and developed. As a result, it was established that riboflavin and glucose had been released from the component A. Furthermore, analysis of the IH- and 13C-NMR spectra of the component A showed that the glucosidic bond consisted of an alpha-linkage at the 5'-position of riboflavin. From these results, the component A is concluded to be identical to 5'-D-riboflavin alpha-D-glucoside: 6,7-dimethyl-9-(5'-[a-D-glucopyranosyl]-D-ribityD-isoalloxazine. Example 3 Bacillus pumilus RLX3, a yellow coloured riboflavin-producing mutant strain derived from Bacillus pumilus RC15 (FERM-BP No. 2834, JP Kokai No. 203982/1995 by two N-methyl-N'-nitro-N-nitrosoguanidine treatments, was cultivated in the same manner as described in Example 1, except that no antibiotics were added for this strain. As a result, 0.25 g/1 of riboflavin glucoside (based on riboflavin measured by the UV absorption at 444 nm) was produced after 3 days of cultivation. We claim: 1. A process for producing riboflavin glucoside, which comprises cultivating a microorganism belonging to the genus Bacillus, which is capable of producing riboflavin glucoside, in an aqueous medium such as herein described containing starch under aerobic conditions, isolating and purifying riboflavin glucoside from the culture medium in a known manner. . 2. The process according to claim 1, wherein the microorganism belongs to the species Bacillus breivs, Bacillus cereus. Bacillus circulans. Bacillus coagulans, Bacillus licheniformis. Bacillus megaterium. Bacillus pumilus or Bacillus subtilis. 3. The process according to claim 2, wherein the microorganism is Bacillus brevis IFO 15304, Bacillus cereus IFO 15305, Bacillus circulans IFO 13626, Bacillus coagulans IFO 12583, Bacillus Hcheniformis IFO 12200, Bacillus megaterium IFO 15308, Bacillus pumilus IFO 12092 or Bacillus subtilis. IFO 13719. 4. The process according to claim 2, wherein the microorganism is Bacillus subtilis RB50:: (pRF69)60Ade+ or Bacillus subtilis RB50:: (pRF69)60(pRF93) 120Ade+. 5. The process according to any one of claims 1 to 4 , wherein an initial concentration of starch in the culture medium is about 25g/l to about 200g/l, preferably about 200g/l to about 300g/l. 6. The process according to any one of claims 1 to 5, wherein the cultivation is carried out at a pH of about 4.0 to about 9.0. preferably about 4.5 to about 8.0. 7. The process according to any one of claims 1 to 6, wherein the cultivation is carried out at a temperature from about 20 to about 45°C, preferable from 25 to 40 C. 8. A process tor producing riboflavin glucoside, substantially as hereinabove described.

Documents:

2713-mas-1998 abstract.pdf

2713-mas-1998 claims.pdf

2713-mas-1998 correspondence-others.pdf

2713-mas-1998 correspondence-po.pdf

2713-mas-1998 description (complete).pdf

2713-mas-1998 drawings.pdf

2713-mas-1998 form-1.pdf

2713-mas-1998 form-26.pdf

2713-mas-1998 form-3.pdf

2713-mas-1998 form-4.pdf

2713-mas-1998 petition.pdf