Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-09-28
2002-12-10
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S170000, C435S822000
Reexamination Certificate
active
06492141
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a fermentation process for the production of vitamin B12.
BACKGROUND OF THE INVENTION
Vitamin B12 is an important vitamin for humans and animals. It is used to treat pernicious anaemia and peripheral neuritis, and is used as a dietary supplement. Vitamin B12 is also an important animal feed supplement as growth enhancer.
The term vitamin B12 is used to describe compounds of the cobalt corrinoid family, in particular those of the cobalamin group. The most used compound of this group is cyanocobalamin and as such the term vitamin B12 is sometimes used to refer to cyanocobalamin. In this specification the term vitamin B12 should be attributed its broad meaning so as to include all the cobalt corrinoids of the cobalamin group, which include in particular cyanocobalamin, hydroxocobalamin, methylcobalamin and 5′desoxyadenosylcobalamin, characterised by a cyano, hydroxyl, methyl or 5′-desoxyadenosyl radical respectively. The methylcobalamin and 5′desoxyadenosylcobalamin compounds are known to be unstable to light in isolated form and are easily transformed to hydroxocobalamin in aqueous solution.
For this reason, almost all commercial vitamin B12 preparations consist of the stable cyanocobalamin, which as such is not the chemical form in which vitamin B12 can be found in nature. In this specification the term natural vitamin B12 is defined so as to comprise all chemical forms of vitamin B12 naturally occurring in nature, cyanocobalamin thus being excluded.
Vitamin B12 is produced industrially by microbial fermentation, using almost exclusively Pseudomonas denitrificans and Propionibacterium species (as reviewed by Spalla et al, 1989 “Microbial production of vitamin B12”, In: Biotechnology of vitamins, pigments and growth factors, E. J. Vandamme ed., Elsevier, London, N.Y., pp. 257-284). Contrary to Pseudomonas, Propionibacteria are food-grade. Processes using Propionibacterium species thus have the advantage that they allow to formulate natural vitamin B12 together with the biomass in which it is produced, as recently described in EP-A-0 824 152. Such processes avoid the conversion of natural vitamin B12 into the cyanocobalamin form by chemical processes including cyanidisation followed by extraction and purification steps using organic solvents. The chemical conversion step and any subsequent purification steps cause this production process to be expensive, unsafe to the operators and environmentally unfriendly.
Propionibacteria are Gram-positive bacteria capable of producing valuable compounds in a variety of industrial processes. Propionibacteria are, for instance, important in the production of cheese, propionic acid, flavours and vitamin B12.
Propionibacteria are, as the name suggested, characteristic in the production of propionic acid. Glucose is commonly used as carbon source, but other substrates, i.e. fructose, mannose, galactose, glycerol and milk, can be used for growth. Besides propionic acid, acetic acid is produced under anaerobic conditions, with a ratio of 2:1 for propionic acid : acetic acid. The production of propionic acid is a clear advantage over other species as this compound is toxic in low levels for many other organisms, like lactic acid bacteria, acetic acid bacteria and yeasts. As a result there is little chance of contamination with other microorganisms during fermentation. The upper tolerance level for propionic acid for Propionibacterium is approximately 20-40 g/l (with a fermentation around pH 7): this is the level where the propionic acid starts to inhibit growth. The undissociated propionic acid is the actual toxic component for Propionibacteria, as is shown by Nanba et al. (1983, J. Ferment. Technol., 61: 551-556) for
Propionibacterium shermanii
. The specific growth rate decreases rapidly for undissociated propionic acid concentrations above 5 mM. This effect is also demonstrated by Blanc et al. (1987, Bioproc. Eng., 2: 175-179) for
P. acidi-propionici
, where the growth rate is drastically reduced above a pseudo critical value of 4 mM propionic acid. This implies that in fermentations with a pH around 7.0 propionic acid concentrations above 40 g/l are only reached with very low growth rates. This maximum amount of propionic acid produced in such fermentations results in a maximum of 25-35 g/l biomass that can be reached. Propionic acid concentration is thus one limiting factor for biomass growth and thereby for high vitamin B12 yield.
Several Propionibacterium species are capable to produce vitamin B12 in large scale fermentation processes. The process is described as a two-stage fermentation with a 72-88 hours anaerobic fermentation followed by a 72-88 hours aerobic phase. The vitamin B12 concentration in the cells rapidly increases in the aerobic phase, with typical values of 25-40 mg vitamin B12/l (see e.g. DE 1 239 694, U.S. Pat. No. 3,411,991, or in: Biochemical engineering and biotechnology handbook, 1991, B. Atkinson ed., Macmillan Publishers Ltd, pp: 1213-1220). Anaerobic growth followed by an aerobic phase with limited growth is important for economic production of vitamin B12 using Propionibacterium species. This requirement, however, limits the amount of biomass to 25-35 g/l as described above. Several attempts have been made to overcome the barrier of propionic acid toxicity in order to increase biomass and thereby the yield of vitamin B12.
Alternated anaerobic-aerobic phases are e.g. suggested to reduce the amount of acids (Ye et al., 1996, J. Ferment. Bioeng. 85: 484-491). In the aerobic phase the propionic acid is converted to less toxic acetic acid, with simultaneous formation of vitamin B12. The relative yield of vitamin B12 has been increased, but the final titre is rather low. This is probably due to inhibition early in the synthesis of vitamin B12 and/or other oxygen related products limiting the synthesis of vitamin B12. The final vitamin B12 produced with this method is 9 mg/l compared to 4.5 mg/l with the fully separated anaerobic and aerobic phases. Both values are rather low for vitamin B12 production with Propionibacteria.
The suggestion to use immobilized cells is mainly focused on the production of propionic acid (Rickert et al., 1998, Enzyme Microb. Technol. 22: 409-414). The propionic acid production is greatly enhanced. Use of this option for vitamin B12 production (which is not mentioned by Rickert et al.) will imply harvesting of the vitamin B12-containing cells with the immobilization material. This is only feasible when the additional cost for the immobilization equipment as well as the immobilization material itself are competitive with the current technology. Yongsmith et al. presented the production of vitamin B12 with immobilised cells of Propionibacterium sp. strain arl AKU1251. The maximum vitamin B12 concentrations is in the range of 14-16 mg/kg, which is no improvement of the production with freely suspended cells, as described before (Yongsmith et al. 1983, J. Ferment. Technol. 61: 593-598).
Although Propionibacteria can grow under aerobic conditions, the production of corrinoids (i.e. the general name for vitamin B12 and its precursors) is absent above a dissolved oxygen concentration of 0.19 mM=6 mg O
2
/L. The lower the oxygen concentration the higher the corrinoid production is with a maximum corrinoid production under non-aerated conditions (Quesada-Chanto et al., 1998, Appl. Microbiol. Biotechnol. 49: 732-736). Oxygen concentration is a limiting factor for vitamin B12 synthesis.
A repeated fed-batch fermentation with an anaerobic phase followed by an aerobic phase and withdrawal of broth at the end of the aerobic phase is not possible. According to Quesada-Chanto et al. (1998), production of the corrinoids is optimal under anaerobic conditions, whereas small amounts of oxygen reduce the production of corrinoids. These findings are supported by the results obtained in example 1. A repeated fed-batch process with an aerobic and anaerobic phase in one fermenter therefore is, not economically feasible.
GB patent 846,149
DSM N.V.
Lilling Herbert J.
Morrison & Foerster / LLP
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