Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-04-02
2002-03-05
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S212000, C435S227000, C435S228000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06352848
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority from German Application No. 198 14 813.5, filed on Apr. 2, 1998, the entire disclosure of which is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel recombinant (rec-) L-N-carbamoylase from
Arthrobacter aurescens
as well as to a method of producing L-arnino acids with this carbamoylase.
The L-amino acids which can be advantageously produced with the novel rec-carbamoylase are important chiral starting materials for the organic synthesis, for example, of drugs and have significance in human and animal nourishment.
2. Background Information
The advantageous possibility of the stereoselective splitting of the D,L-carbamoyl amino acids assessable from the corresponding hydantoins by hydantoinases by means of L-N- and/or D-N-carbamoylases resulted previously in the establishment of methods for the production of L- and D-amino acids on an industrial scale. L-specific carbamoylases have been detected in very different strains of microorganisms (Ogawa et al., J. Mol. Cal. B: Enzym. 1997, 2, 163-176; Syldatk et al. in Drauz, K. and Waldmann, H.: Enzyme Catalysis in Organic Synthesis, Weinheim: VCH-Verlag, 1995).
The presence of L-N-carbamoylase activity in cells of the strain
Arthrobacter aurescens
has been known for some time. This carbamoylase has been isolated in homogeneous form and the N-terminal sequence has been determined. (Müller, dissertation, Braunschweig TU Technical University, 1990). However, the gene coding for the enzyme was unknown and has therefore not been previously cloned or overexpressed. Thus, the recombinant enzyme and its amino acid sequence and the DNA coding the amino acid sequence were previously unknown.
Three L-carbamoylases produced in a recombinant manner have been described in the literature (Batisse et al., Appl. Environ. Microbiol. 1997, 63, 763-766; Mukohara et al., Biosci. Biotechn. Biochem. 1993, 57, 1935-1937; Watabe et al., J. Bacteriology 1992, 174, 962-969). However, the L-carbamoylases cited there are relatively unstable and are thus unsuitable for being used in an industrial process.
The L-specific carbamoylase from
Arthrobacter aurescens
(A.a.) was able to be used previously for the splitting of N-carbamoyl amino acids to free L-amino acids only as a predominantly homogeneously purified enzyme or in the form of free or immobilized, dormant [resting] whole cells. Several basic problems occur in the latter method. They include, among other things, transport limitations (especially for the N-carbamoyl amino acids), potential side reactions (degradation of the formed amino acids by other enzymes present) as well as contaminants in the product caused by lysis of the whole cells used, which make expensive purification processes necessary.
The L-carbamoylase from A.a. is very unstable in both forms used, which can be traced in the instance where dormant, immobilized cells are used to the natural protein turnover of the enzyme (Siemann, dissertation, Braunschweig TU, 1992). When used as a homogeneously purified enzyme, the lability is obviously affected by unavoidable changes in the protein concentration and the salt content. Moreover, the sensitivity of the wild enzyme to oxidation has been observed. In addition, only a very low yield of 2.7% is achieved in the purification of carbamoylase from the wild strain (Müller dissertation, Braunschweig TU, 1990). This low yield, the necessary complicated purification method, and the previously immanent lability of the enzyme prevent the use of this procedure in a competitive industrial method for obtaining L-amino acids.
The present invention therefore had the objective of obtaining an L-carbamoylase which is more stable than the L-carbamoylases of the prior art, and those previously producible from
Arthrobacter aurescens
, in a simpler form and with a better yield. However, the further requirements which a method places on the enzyme used on an industrial scale such as, for example, activity or selectivity regarding substrate and stereochemistry, etc. should not be negatively affected.
These and other problems not cited in detail but which will be evident to those of skill in the art are solved by rec-L-N-carbamoylase from
Arthrobacter aurescens
and mutants thereof, which are included in the invention. Their amino acid sequences and gene sequences derived therefrom are also included in the invention, as well as vectors modified with the gene sequences, corresponding plasmids, modified host organisms and cells of the host organisms.
In addition, the invention includes an advantageous method of producing L-amino acids using these enzymes of the invention.
As a result of the fact that L-N-carbamoylase is produced in a recombinant manner from
Arthrobacter aurescens
or its mutants, L-carbamoylases are obtained in very good yields of >90% and with a decidedly high purity which have a much greater stability over the previously known carbamoylases, yet have a good activity and very good stereoselectivity as concerns the regarded biotransformation. These characteristics are essential for the successful use of carbamoylases in an industrial method for the production of L-amino acids.
It resulted in stability studies that homogeneously purified carbamoylase has only a few minutes activity at 50° C. (Müller dissertation, Braunschweig TU, 1990), whereas on the other hand rec-carbamoylase is active for hours. At 37° C. the activity of rec-carbamoylase is almost unchanged for 100 h (FIG.
2
). This could in no way have been foreseen and is nevertheless all the more advantageous.
In addition thereto, the use of these recombinant carbamoylases opens up the possibility for the first time of obtaining industrial access to &bgr;-aryl-substituted L-amino acids by means of an enzymatic biotransformation via (D,L)-N-carbamoyl amino acids. The regarded carbamoylase is the only one of the previously known carbamoylases to also offer, in addition to the high L-enantioselectivity, the possibility of reacting [converting] &bgr;-aryl-substituted L-N-carbamoyl amino acids to an extent sufficient for an industrial method. (D,L)-formyl amino acids of this provenance are also suitable for being reacted with the enzymes in accordance with the invention. The free L-amino acid is also obtained with preference from the latter [formyl amino acids].
The amino acid sequences characterizing the recombinant L-N-carbamoylase from
Arthrobacter aurescens
and its mutants only have an agreement of maximally 38% with amino acid sequences of L-N-carbamoylases known from the state of the art. The novel and inventive gene sequences coding these amino acid sequences can be produced according to known biochemical methods. The vectors, plasmids and cells of host organisms containing these gene sequences are also novel and inventive. Preferred plasmids are pAW 16 and pAW 178-2 (FIG.
1
), a preferred vector is pJOE 2702. Host cells can be in principle all microorganisms known to the expert in the art and coming into consideration for this purpose; however,
E. coli
JM 109 or
E. coli
W3 110 is preferred.
The induction of the expression can be achieved in principle with all methods known to the expert in the art. However, rhamnose-, IPTG-and lactose systems are preferred.
The isolation and expression of the L-N-carbamoylase gene hyuC from
Arthrobacter aurescens
takes place as follows.
A gene bank representative for the entire genome of
Arthrobacter aurescens
and in an
E. coli
&lgr;TRESIII vector was prepared and screened with an oligonucleotide which was derived from the N-terminal amino acid sequence of the purified hydantoinase from
Arthrobacter aurescens
. Plasmid pAW16 was obtained thereby which contained a 7.6 kb DNA fragment of
Arthrobacter aurescens
. Its nucleotide sequence was completely determined. The hydantoinase gene was identified from the nucleotide sequence. Another reading frame was identified on the C-terminal end of the hydantoina
Altenbuchner Josef
Mattes Ralf
Pietzsch Markus
Syldatk Christoph
Wiese Anja
Degussa-Huls Aktiengesellschaft
Pillsbury & Winthrop LLP
Prouty Rebecca E.
Rao Manjunath N.
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