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
1999-04-30
2001-08-28
Lilling, Herbert J. (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...
Reexamination Certificate
active
06280980
ABSTRACT:
The present invention relates to a process for the production of L-aspartic acid.
BACKGROUND OF THE INVENTION
Various processes for production of L-aspartic acid from fumaric acid and ammonia (to create diammonium fumarate) using microorganisms having aspartase activity have been published. For example see—Chibata, I; Tosa, T.; Sato, T.
Comprehensive Biotechnology Vol
. 3 Ed. Murray Moo-Young, Permagon, 1985. In these published processes, the L-aspartic acid is usually recovered by precipitation of L-aspartic acid crystals resulting from the addition of a mineral acid such as sulfuric acid to the reaction solution, and separation of the crystals. However in these published processes, a large amount of waste salt(s), such as ammonium sulfate, is generated as a by-product of the precipitation. The production of these salts is undesirable from an environmental perspective and causes the undesirable loss of ammonia.
U.S. Pat. No. 4,560,653 ('653) issued Dec. 24, 1985, discloses a process for the production of L-aspartic acid wherein aspartase or an aspartase-producing microorganism acts upon fumaric acid and ammonia. L-aspartic acid is precipitated by addition of maleic acid and filtered from the solution and the mother liquors are recycled. This '653 patent discloses the use of a chemical catalyst for maleic acid to fumaric acid isomerization.
As an alternative catalyst for maleate to fumarate isomerization, enzyme-containing organisms with maleate isomerase activity can be used. For examples see—Otsuka, K.
Agr. BioL. Chem
., 25(9), 1961, 726.—Scher, W. and Jakoby, W. B.
J. Biol. Chem
., 244(7), 1969, 1878. This catalyst reportedly gives high conversions of maleic acid salts to fumaric acid salts at basic pH.
U.S. Pat. No. 3,391,059 which issued Jul. 12, 1968, discloses an enzyme containing organism with maleate isomerase activity and aspartase activity to produce L-aspartic acid directly from maleic acid.
The art of preparing L-aspartic acid economically and efficiently with minimal or no environmental adverse impact continues to be of interest to industry.
OBJECTS OF THE INVENTION
It is an object of this invention to provide a highly efficient process for the production of L-aspartic acid using inexpensive starting substrates.
It is another object of this invention to provide a highly efficient process for the production of L-aspartic acid with minimal generation of ammonium salt as a by-product.
It is a still further object of this invention to provide a process for the production of L-aspartic acid having an improved yield while generating a minimum of an undesired side product.
These and other objectives are met in this invention which is hereinafter described in more detail.
BRIEF SUMMARY OF THE INVENTION
In the broadest aspect of this invention and in a preferred embodiment, L-ammonium aspartate is produced from maleic acid or maleic anhydride by enzymes or microorganisms. L-aspartic acid is preferably recovered by precipitation using maleic acid. An added benefit is that optionally the mother liquor can be recycled for reuse after recovering L-aspartic acid from the reaction solution. Optionally the pH of the mother liquor may be made basic.
This invention comprises a process for the production of L-aspartic acid comprising the steps of (1) contacting (A) an enzyme-containing material having maleate isomerase activity and aspartase activity, or (B) an enzyme-containing material having maleate isomerase activity and an enzyme-containing material having aspartase activity, with an aqueous substrate solution containing maleic acid and ammonia, and/or mono or di-ammonium maleate or mixtures thereof to form L-ammonium aspartate in a reaction solution, (2) adding said reaction solution at a controlled rate to an aqueous solution of maleic acid or aqueous slurry of maleic anhydride to crystallize L-aspartic acid in a mother liquor and (3) recovering said L-aspartic acid from said mother liquor, and (4) optionally recycling said mother liquor as a substrate solution for further reaction with said enzyme containing material of Step (1) above and (5) optionally adding ammonia or other alkali to said mother liquor before, during or after recycle.
In another preferred embodiment of this invention, a substrate solution at a temperature from about 10 C. to about 60 C. containing maleic anhydride, maleic acid and salts thereof and ammonia, or ammonium maleate wherein the molar ratio of ammonia to maleic acid is from about 1 to about 2 is contacted with microbial cells having both maleate isomerase activity and aspartase activity, or with microbial cells having maleate isomerase activity and microbial cells having aspartase activity, or alternatively disrupted cells or enzymes thereof or the equivalent thereof.
An enzyme-containing material such as microbial cells having maleate isomerase activity and an enzyme-containing material having aspartase activity may be used as a mixture if desired. Without being bound by theory it is believed that maleate isomerase-containing material converts ammonium maleate to ammonium fumarate, and then aspartase-containing material converts the ammonium fumarate to L-ammonium aspartate. These conversions preferably occur in the same vessel or container so as to minimize the concentration of fumarate salt formed during the reaction. When the activity of the prior-used enzyme-containing material decreases, preferably it may be totally or partially replaced with fresh species.
The ratio of maleate isomerase containing enzyme to aspartase enzyme may be adjusted. Preferably, the activity of the aspartase enzyme is sufficiently high to minimize any appreciable fumarate concentration being formed during the reaction. Highest yields of aspartate are believed to occur when the fumarate concentration is suppressed since it is believed to inhibit maleate isomerase activity.
L-ammonium aspartate is formed during the reaction and the resulting solution is added to a slurry of maleic anhydride or a solution of maleic acid at a controlled rate to crystallize L-aspartic acid. The L-aspartic acid crystals are separated from the solution. The recovered crystals are washed with water to reduce the amount of maleic acid and/or salt thereof. As used herein, the term “controlled rate” means the gradual addition i.e., addition in a controlled manner of the reaction solution to the maleic acid or maleic anhydride in order to minimize the formation of reaction byproducts and result in an increased yield of L-aspartic acid.
The mother liquor from which the crystals have been removed is preferably recycled and reused as a substrate solution for further production of more L-aspartic acid. If this optional step is carried out, it reduces raw material cost and avoids generating waste water containing significant ammonia which heretofore required disposal at a potential detriment to the environment.
In the process of this invention, maleic anhydride is used preferably as one of the starting materials to produce L-aspartic acid. The mother liquor may be recycled as the substrate solution without addition of chemical catalyst. The complete recycle of the soluble mother liquor results in very efficient production with minimal or zero waste and minimal ammonia usage. Thus, L-aspartic acid can be produced cheaply and efficiently.
DETAILED DESCRIPTION OF THE INVENTION
Without being bound by theory, illustrations of some chemical reactions believed to occur in parts of this process are shown in a Table below. Some steps are optional. Some steps occur to a greater or a lesser degree.
TABLE
Step
Action
Chemistry
1
Maleate Isomerase Enzymatic Maleate to Fumarate Isomerization
1
Aspartase Enzymatic Conversion of Fumarate to Aspartate
Dissolution of Maleic Anhydride
2
Acidification and precipitation of L-aspartic acid
3 & 4
Filtration of
precipitated L-
aspartic acid
Recycle mother
liquor
containing
ammonium
maleate
5
Neutralization of ammonium maleate to form diammonium maleate
Go Back to Step
1 optional
Any suitable microorganism having maleate isomerase activity and as
Lathrop & Gage LC
Lilling Herbert J.
Solutia Inc.
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