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
1999-02-12
2001-04-10
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S177000, C435S180000, C435S232000, C435S252300
Reexamination Certificate
active
06214589
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for producing L-aspartic acid by using a microorganism with high aspartase activity.
BACKGROUND OF THE INVENTION
A method for enzymatically producing L-aspartic acid from ammonium fumarate by using a microorganism with aspartase activity,
Escherichia coli
, is known. Japanese Patent Publication No. 55-35110 (i.e., JP-B-55-35110) describes a method in which
E.coli
aspartase is immobilized on an ion-exchange resin Duolite A-7. Applied Biochemistry and Biotechnology vol. 13, pp.231-240 (1986) describes a method for continuously producing L-aspartic acid wherein a reactor is filled with
E.coli
cells immobilized in &kgr;-carrageenan by gel entrapment. These methods, however, are unsatisfactory in terms of industrial productivity since the immobilized aspartase has a low activity or since the gel entrapment immobilization causes diffusion barrier or rate-limiting diffusion.
Since conventional immobilized aspartases do not have a sufficiently high activity, the reaction has to be carried out at 30° C. or higher, in which case cooling is required because aspartase loses its activity by heat of reaction. Particularly, the activity is remarkably decreased at a temperature exceeding 40° C. (Applied Microbiology vol. 27, No. 5, pp.878-885 (1974); Applied Microbiology vol. 27, No. 5, pp. 886-889 (1974)). To prevent a rise in temperature in a reactor caused by heat of reaction, the reactor needs to be equipped with an internal cooling tube or a cooling device such as jacket, which renders the reactor complicate. Further, prior art immobilized aspartases cannot be used to carry out enzymatic reactions at a high LHSV due to their low activities. In methods that use aspartases immobilized by gel entrapment, it is also impossible to carry out an enzymatic reaction at a high LHSV (Liquid Hour Space Velocity) since diffusion barrier becomes greater.
The reaction at high LHSV is required to achieve the improved productivity of L-aspartic acid by the enzymatic reaction using immobilized aspartases. Such a reaction can be realized with an immobilized aspartase which has a sufficiently high activity to react at a low temperature and which leads to a low diffusion barrier and a low pressure loss. Unfortunately, such an immobilized aspartase has not yet been prepared in the art. In this situation, the present inventors have now prepared an immobilized aspartase with the above preferable properties and have now found that the reaction at high LHSV is achieved substantially without removing heat by introducing a substrate solution into a reactor while controlling a temperature of the substrate solution to be lower than a temperature of impairing the stability of aspartase by at least a rise in a temperature caused by heat of reaction, thereby ensuring the stable activity of the immobilized aspartase.
SUMMARY OF THE INVENTION
The present invention provides a method for producing L-aspartic acid, comprising the steps of: immobilizing microbial cells containing aspartase to produce an immobilized aspartase; feeding an ammonium fumarate solution into a reactor filled with the immobilized aspartase; and recovering the produced L-aspartic acid from the reaction mixture, wherein the immobilized aspartase has an activity of 250 U or more, and wherein the ammonium fumarate solution is fed into the reactor at the feed rate LHSV of 2 to 35. As used herein, “1U” means 1 &mgr;mole L-aspartic acid yielded per minute per milliliter of immobilized enzyme, and “LHSV” stands for “Liquid Hour Space Velocity” and refers to a volume of feeding substrate (in ml) per volume of filled catalyst (in ml) per hour.
The immobilized aspartase usable in the invention is, for example, an immobilized recombinant microbial cell with an aspartase gene introduced using recombinant DNA techniques.
The immobilized aspartase can be obtained by coating a spherical carrier with the above-mentioned microbial cell containing aspartase in combination with a polymer. More specifically, the immobilized aspartase can be obtained by coating a spherical styrene divinylbenzene copolymer type ion-exchange resin carrier with the above-mentioned aspartase-containing microbial cells admixed with a polymer represented by the general formula:
wherein Y is either a direct bond or a bifunctional group represented by the formula:
R
1
and R
2
are each independently a hydrogen atom or an organic residue, X
⊖
is an anion, and n is a number between 100 to 5000.
Exemplary organic residues include alkyl groups with not less than 10 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and ter-butyl groups, preferably methyl group. The organic residues may include at least one halogen or hydroxyl group as a substituent and such examples are 4-chloro-2-dimethylpentyl group, 3-ethyl-2,5-dichloroheptyl group and 2-hydroxy-3,5-dimethylnonyl group, preferably 3-chloro-2-hydroxypropyl group.
Examples of X
⊖
include halogen ions such as F
−
, Cl
−
, Br
−
and I
−
, preferably Cl
−
. Other monovalent anions, such as NO
3
−
, may also be used as X
⊖
.
The immobilized aspartase of this invention obtained by immobilizing the above-described recombinant microbial cell containing an aspartase gene by the above-described immobilization method has an extremely high activity and leads to a low pressure loss as well as to a low diffusion barrier. As a result, unlike the conventional immobilized aspartases, it becomes possible to carry out an enzymatic reaction at a high LHSV by using the immobilized aspartase of the invention.
Since an ammonium fumarate solution at a low temperature can be fed for the reaction, a lifetime of aspartase can be extended.
These and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description.
This specification includes all or part of the contents as disclosed in the specification of Japanese Patent Application No. 10-31809 which is a priority document of the present application.
DETAILED DESCRIPTION OF THE INVENTION
An aspartase-containing microorganism that can be used with the present invention is preferably
Escherichia coli, Pseudomonas fluorescens
, the genus Brevibacterium or the like, which are known to have a high aspartase activity. In particular, a recombinant cell having an aspartase gene introduced by recombinant DNA techniques is preferred.
Preferably, the aspartase gene used with the present invention can be obtained from
E.coli
and a microorganism which has an aspartase activity and whose gene is known to naturally cross with an
E.coli
gene, for example,
Pseudomonas fluorescens
, the genus Enterobacter, or the genus Citrobacter. However, an aspartase gene from any microorganism with aspartase activity can suitably be used.
For example, aspartase genes may be amplified by PCR using the chromosomal DNA from
E.coli
K-12 (IF03301) or
Pseudomonas fluorescens
(IFO3081) as a template and primers prepared based on the known aspartase gene sequence. Alternatively, aspartase genes may also be obtained by other usual methods such as the method where restriction fragments obtained from chromosomal DNA are electrophoresed to recover a fragment containing an aspartase gene.
A plasmid for incorporating the thus-obtained aspartase gene may be any plasmid that is capable of replicating in the cell of
E.coli
. Examples of such plasmids include, but are not limited to, pUC18 (Nippon Gene Co., Ltd.) and pKK223-3 (Pharmacia).
As a host microorganism for introducing the plasmid into which the aspartase gene has been incorporated,
E. coli
strain K-12 (Stratagene) or the like can suitably be used.
The aspartase-containing microbial cell is preferably immobilized by a method that gives a sufficient strength of the immobilized product and is less likely to cause pressure loss or diffusion barrier upon feeding a substrate solution.
Preferably, the microbial cell is immobilized by coating the surface of a sph
Komatsuzaki Satomi
Mukouyama Masaharu
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Nashed Nashaat T.
Nippon Shokubai Co. , Ltd.
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