Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2000-10-12
2003-06-24
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S116000, C435S146000, C435S244000
Reexamination Certificate
active
06582939
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATION
This application is based on German Application DE 100 21 515.7, filed May 3, 2000, which disclosure is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to a process for the preparation of alkaline earth salts of D-pantothenic acid from fermentation broths.
BACKGROUND OF THE INVENTION
Pantothenic acid is a commercially important vitamin that is used in cosmetics, medicine, human nutrition and in animal nutrition.
Pantothenic acid can be prepared by chemical synthesis or via biotechnology by fermentation of suitable microorganisms in suitable nutrient solutions. DL-pantolactone is an important compound in the chemical synthesis, and is prepared in a multi-stage process from formaldehyde, isobutyl aldehyde and cyanide. In further process steps the racemic mixture is separated and D-pantolactone is condensed with &bgr;-alanine to yield D-pantothenic acid.
The advantage of a fermentative preparation using microorganisms is the direct formation of the correct stereoisometric form, namely the D-form of pantothenic acid free from L-pantothenic acid.
Various types of bacteria, for example
Escherichia coli, Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes
and also yeasts, for example Debaromyces castellii may, as demonstrated in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340, produce D-pantothenic acid under suitable fermentation conditions. Particularly suitable microorganisms are the derivatives, described in the citations, of
Escherichia coli
IF03547, for example the strains FV5069/pFV31 or FV5069/pFV202.
In the fermentative preparation of D-pantothenic acid as is described in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340, a microorganism capable of producing D-pantothenic acid is cultivated in a suitable nutrient medium and the D-pantothenic acid that is formed is then isolated according to a complicated expensive procedure, purified, and obtained as the calcium salt.
Suitable nutrient media contain a carbon source such as glucose or starch flour hydrolyzate, precursors such as &bgr;-alanine, D,L-pantoic acid or D,L-pantolactone, a nitrogen source such as ammonium sulfate, a phosphorus source such as potassium phosphate, and further salts, trace elements, amino acids and vitamins, and optionally complex media additives such as yeast extract or corn steep liquor. The microorganisms are then incubated in this medium at a suitable pH value under appropriate aeration and stirring, whereupon the microorganisms form D-pantothenic acid.
EP-A-0 590 857 describes, for example, a fed batch process for preparing pantothenic acid in a 5 liter reactor filled with 2.3 liter or 2.5 liter of culture medium. In this experimental example, solid calcium carbonate was added presumably to regulate the pH. The preliminary addition or subsequent addition of solid calcium carbonate is however extremely undesirable in a large-scale reactor having a volume of many cubic meters, because the material loading is increased by the calcium carbonate deposits on the stirrer blades, internal surfaces and seals, and the flow properties of the culture liquid and the sterile conditions are adversely affected.
According to the present prior art, which is outlined in W096/33283 and EP-A-0 590857, the calcium salt of D-pantothenic acid is obtained by a complicated and costly isolation and purification process starting from a fermentation broth containing pantothenic acid. After a first separation of the biomass by filtration or centrifugation, the filtrate is worked up further by purification by means of activated charcoal or by column chromatography. After adding calcium hydroxide to the pretreated filtrate or eluate, the batch is then purified by crystallization.
The purification method described in W096/33283 is carried out as follows. The filtrate is decolorized by means of activated charcoal in a first column. The pH is adjusted to 3.0 with concentrated hydrochloric acid and the liquid is then purified continuously through two columns packed with activated charcoal. The elution of the D-pantothenic acid is performed with methyl alcohol. A subsequent neutralization is carried out with Ca(OH)
2
powder while thoroughly mixing. The calcium D-pantothenate is obtained by subsequent crystallization at 5° C.
The purification method described in EP-A 0 590 857 is carried out as follows. The filtrate is first of all purified with the aid of cation exchange and anion exchange columns. Elution is performed with hydrochloric acid. The eluted fraction is then neutralized with Ca(OH)
2
, following which activated charcoal is added and the mixture is filtered. The resultant filtrate is extracted in a low molecular weight alcohol (methanol, ethanol, isopropanol) and the calcium D-pantothenate is obtained by crystallization.
The calcium D-pantothenate prepared in the aforementioned manner is used as a feed additive for animal nutrition.
SUMMARY OF THE INVENTION
An improved process for preparing alkaline earth salts, in particular the calcium and magnesium salts, of D-pantothenic acid, which are suitable for use as feed additives in animal nutrition, is provided.
The vitamin D-pantothenic acid is a commercially important product that is used in animal nutrition, medicine, human nutrition and in cosmetics. There is, therefore, a general interest in providing new processes for preparing pantothenic acid or its salts.
The present invention provides a process for preparing alkaline earth salts of D-pantothenic acid or mixtures containing the latter, from fermentation broths, which is characterized in that
(a) the fermentation is carried out in the presence of alkaline earth compound,
(b) after completion of the fermentation the biomass is optionally removed either in whole or in part,
(c) the thus worked up fermentation broth is concentrated, and
(d) the alkaline earth salt or salts of D-pantothenic acid is/are obtained from the latter in pure form or as a mixture containing the constituents of the fermentation broth.
The present invention also provides a process which is characterized in that one or more alkaline earth salt(s) of D-pantothenic acid is/are added in the desired amount to the constituents of the fermentation broth and mixture containing one or more of the alkaline earth salts of D-pantothenic acid.
The invention furthermore provides a process for preparing alkaline earth salts of D-pantothenic acid, characterized in that
a) the biomass is separated from a fermentation broth containing alkaline earth salt(s) of D-pantothenic acid,
b) the cell-free fermentation broth is concentrated,
c) a hydrophilic organic solvent, in particular ethanol, methanol or acetone, is added to the concentrate thus obtained, following which
d) the alkaline earth salt(s) of pantothenic acid is/are isolated, optionally washed with a hydrophilic organic solvent and then, if desired,
e) recrystallized in an aqueous solution of a hydrophilic organic solvent, optionally with the addition of activated charcoal, and obtained in a high state of purity.
All microorganisms that are capable of producing D-pantothenic acid and which produce D-pantothenic acid under appropriate fermentation conditions are suitable for the process according to the invention. The microorganisms can produce pantothenic acid from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol.
The microorganisms may be fungi or yeasts, for example
Debaromyces castells
or
Saccharomyces cerevisiae
or Gram-positive bacteria, for example of the genus Corynebacterium or may be Gram-negative bacteria, for example of the family of Enterobacteriaceae. Within the family of Enterobacteriaceae, the genus Escherichia as exemplified by the type
Escherichia coli
, in particular, should be mentioned. Within the type
Escherichia coli
there should be mentioned the so-called K-12 strains, for example the strains MG1655 or W3110 (Neidhard et al.:
Escherichia coli
and Salmonella. Cellular and Molecular Biology (ASM Press, Washington D.C.)
Becker Ulrich
Binder Michael
Hermann Thomas
Marx Achim
Pfefferle Walter
Degussa - AG
Marx Irene
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