Production of aspartic and malic acids with microbacterium

Details Production of aspartic and malic acids with microbacterium Production of aspartic and malic acids with microbacterium

1996-12-19

2001-05-29

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...

C435S136000, C435S145000, C435S106000, C435S107000, C435S108000

Reexamination Certificate

active

06238895

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the production of organic acids and amino acids, and in particular of L-malic acid and L-aspartic acid, with the use of microorganisms, and in particular with the use of species of microorganisms of the genus Microbacterium.
BACKGROUND OF THE INVENTION
Organic Acids and amino acids are used for a variety of purposes in the pharmaceutical, health and food industries. Especially preferred are the L-isomer forms of organic acids and amino acids.
L-malic acid is an organic acid that is used primarily in pharmaceutical applications as an antidote for hyper-ammoniemia and as a component of amino acid infusion. L-malic acid also presents interest as a food acidulant in competition with citric acid for, for example, confectionary products. Further uses include that of a flavoring agent in a wide range of foods such as nonalcoholic beverages, candy and canned fruits and vegetables.
L-aspartic acid is an amino acid widely utilized in the food industry as a flavoring agent.
Organic acids and amino acids have conventionally been produced by chemical synthesis. However, chemically synthesizing amino acids and organic acids (for example, the chemical synthesis of malic acid from maleic anhydride and water) generally produces the amino acid or the organic acid as an optically inactive mixture (DL-isomer). Such mixtures must then be subjected to a separation process to remove the natural L-isomer from the DL-mixture.
To overcome the problems associated with such DL-isomer mixtures, resort has been made to the use of various microorganisms to synthesize (produce) organic acids and amino acids. Such use of microorganisms provides a biospecificity and selectivity to the enzymatic reaction to permit conversion of a substrate to the natural L-form of the organic acid or amino acid which is generally unavailable in chemical synthesis. This biospecificity and selectivity offers advantages of total product yield, product quality and a significant reduction in waste disposal, production of the natural L-form of the organic acid and/or amino acid produced thereby.
The use of various microorganisms to enzymatically convert fumaric acid to the natural L-form of malic acid has been disclosed. Such strains include those of the species
Lactobacillus brevis, Lactobacillus delbrueckii
and
E. coli
(U.S. Pat. No. 2,972,566) and the genus
Paracolobacterium aerogenoides
(U.S. Pat. No. 3,980,520). Furthermore, the use of a variety of fumarase-producing microorganisms, such as
Brevibacterium ammoniagenes, Corynebacterium egui, Xanthobacter flavus, Proteus vulgaris
and
Pichia farinosa
(U.S. Pat. No. 3,922,195), have all been disclosed for the conversion of fumaric acid to malic acid.
The use of various microorganisms to convert fumaric acid (ammonium salt) to L-aspartic acid has also been reported. Strains of
E. coli, Brevibacterium metalcoligenes,
Serratia and
Pseudomonas putida
have all been disclosed for this purpose. Further, the use of
Brevibacterium ammoniagenes
to convert ammonium fumarate to L-aspartic acid has also been described in U.S. Pat. No. 4,138,292.
Unfortunately, while the biospecificity and selectivity of the enzymatic reaction using the disclosed microorganisms offers certain advantages relating to the production of the natural L-form of malic acid, aspartic acid and/or other organic acids and/or amino acids, there nonetheless remains a need to improve the efficacy of these processes and to identify and use previously unconsidered species of microorganisms therefor.
Microorganisms of the genus Microbacterium are well known and described [see Bergey's Manual of Systematic Bacteriology, Volume 2, Section 15 at Pages 1320-1322 (1986)]. Even though strains of Microbacterium are fumarase producers, we are not aware of the use of microorganisms of the genus Microbacterium for the production of L-malic and/or L-aspartic acid.
Accordingly, it can be seen that there remains a need to identify and utilize other microorganisms which are capable of efficiently and effectively producing organic acids and/or amino acids, such as L-Malic acid and/or L-Aspartic acid, on an industrial scale.
SUMMARY OF THE INVENTION
It is a primary object of the present invention to identify and provide microorganisms which are capable of efficiently and effectively converting fumaric acid or a salt thereof for the production of organic acids and/or amino acids and, in particular of L-malic acid and/or L-aspartic acid.
It is a further primary object of the present invention to provide a method for efficiently and effectively producing organic acids and/or amino acids and, in particular, of L-malic acid and/or L-aspartic acid.
It is a still yet further primary object of the present invention to provide organic acids and amino acids, and in particular, L-malic acid and L-aspartic acid, which have been enzymatically synthesized (produced) by the use of microorganisms.
In accordance with the teachings of the present invention, identified and utilized as disclosed herein are microorganism strains of the genus Microbacterium which are useful for the production of organic acids and/or amino acids by the enzymatic conversion of fumaric acid or a salt thereof.
In further accordance with the teachings of the present invention, disclosed herein is a method for the production of organic acids and/or amino acids. This method is comprised of culturing a strain of the genus Microbacterium, whereby fumarase is produced (intracellularly) thereby. If desired, the cultivated strain may then be recovered from the cultivation media. Cultivation is followed by reacting a fumaric acid or a salt thereof with the cultivated strain of the genus Microbacterium, whereby the fumaric acid or salt thereof is enzymatically converted to either the L-organic acid or the L-amino acid. This method further comprises recovering the said L-organic acid or L-amino acid so produced.
Preferably, the method of the present invention further includes immobilizing the microorganism before the placement thereof in the conversion solution in the presence of a fumaric acid or a salt thereof. However, it is noted that the method of the present invention may also be performed with the microorganism in a “free” form within the conversion solution.
It is further preferred that the immobilized microorganisms be pretreated before the use thereof for enzymatically converting the fumaric acid or salt thereof. In this regard, the method of the present invention further comprises incubating the immobilized microorganism in a pretreatment solution that includes the L-organic acid or L-amino acid to be produced therewith and a buffer. It is further preferred that such pretreatment incubation be conducted with gentle agitation. Preferably, the method of the present invention comprises culturing one of the following strains of the genus Microbacterium:
Microbacterium imperiale, Micobacterium lacticum, Microbacterium ammoniaphilum, Micobacterium arborescens
and
Microbacterium laevaniformans.
Most preferably, the method of the present invention comprises culturing one of the following strains:
Microbacterium imperiale
ATCC 8365,
Microbacterium lacticum
ATCC 8180,
Microbacterium ammoniaphilum
ATCC 15354,
Micobacterium arborescens
ATCC 4358 and
Micobacterium laevaniformans
ATCC 15953.
It is further preferred that the fumaric acid to be used (enzymatically converted) in the method of the present invention is in a salt (fumarate) form thereof. Preferred is the use of either sodium fumarate or ammonium fumarate. In this regard, it is contemplated herein that the method of the present invention will comprise the enzymatic conversion of sodium fumarate by strains of the genus Microbacterium for the production of L-malic acid and the enzymatic conversion of ammonium fumarate by strains of the genus Microbacterium for the production of L-aspartic acid.
The organic acids and amino acids produced by the method of the present invention are in the natural L-isomer forms thereof. Most preferred is the use o

Affiliated with

Also associated with

Rating

Production of aspartic and malic acids with microbacterium does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Production of aspartic and malic acids with microbacterium, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Production of aspartic and malic acids with microbacterium will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2534640