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
1995-04-03
2003-05-20
Prouty, Rebecca E. (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...
C435S320100, C435S252310, C435S252330, C435S231000, C536S023200
Reexamination Certificate
active
06566105
ABSTRACT:
The present invention relates to a process for the production of D-&agr;-amino acids by the stereospecific conversion of racemic mixtures of 5-substituted hydantoins with a microorganism transformed with a plasmid capable of espressing in high yields and without inducers an enzymatic system capable of directly converting said hydantoins into the corresponding D-&agr;-amino acids.
The term enzymatic system refers to a system consisting of D-hydantoinase and D-N-carbamylase enzymes.
D-&agr;-amino acids are extremely valuable compounds useful for the preparation of pharmacologically active substances (for example, D-phenylglycine and D-para-hydroxyphenylglycine are used in the synthesis of penicillins and cephalosporins), pesticides (D-valine for the synthesis of the insecticide fluvanilate) or sweeteners (D-alanine).
The preparation of D-&agr;-amino acids by the chemical and/or enzymatic hydrolysis of the corresponding 5-substituted hydantoins is known in the art.
For example patent FR 2.310.986 describes a process wherein 5-substituted hydantoins are chemically hydrolized into racemic mixtures of D,L amino acids which are subsequently subjected to a separation treatment of the isomer of interest.
Patent FR 2.317.357, on the contrary, describes a process wherein racemic mixtures of 5-substituted hydantoins are subjected to enzymatic hydrolysis and, subsequently, the products of this transformation (N-carbamyl-D-&agr;-amino acids) are chemically oxidized into the corresponding D-&agr;-amino acids.
The problems relating to these processes generally consist in the fact that they require complex procedures for the resolution and purification of the D-&agr;-amino acids. As a result these processes are not of economical interest from an industrial point of view.
Processes are described in the art wherein D-&agr;-amino acids are obtained directly from 5-substituted hydantoins by the treatment of these with enzymatic systems prepared from microorganisms such as Paeudomonas, Moraxella, Agrobacterium, Hansenula, Arthrobacter (EP-199.943, EP-309.310, U.S. Pat. No. 4,312,948, FR 2456728).
The preparation of these enzymatic systems, however, requires the use of efficient inducers capable of stimulating the production of these enzymes on the part of the microorganisms. It is, in fact, known that the expression level of the enzymes D-hydantoinase and D-N-carbamylase is constitutively very low (Syldatk et al. (1990), “Advances in Biochem. Engineering/Biotechnology (Fiechter, A. Ed.), 41, pages 29-75, Springer-Verlag, Berlin).
The inducers normally used are derivatives of hydantoins or nitrogenated cyclic compounds which are howevery easily metabolized by the microorganisms, or compounds such as uracil or thio-2-uracil or thymine which are not metabolized (Meyer et al., (1993), Fems Microbiol. Letters, 109: 67-74).
The use of inducers creates a series of drawbacks among which an increase in the production costs and a certain variability in the production yields of the enzymes. In addition, the expression level which can be obtained in most of the microorganisms following induction is insufficient for economical use in industrial processes (Syldatk et al. (1987), Biotechnol. lett., 9: 25-30; Yokozeki et al. (1987) Agric. Biol. Chem., 51, 715-722).
Recently the genes which encode the enzymes D-hydantoinase and D-N-carbamylase have been individually sequenced and cloned (U.S. Pat. No. 4,912,044 and EP-515-698).
More specifically, patent U.S. Pat. No. 4,912,044 describes the preparation of D-hydantoinase by the fermentation of a microorganism transformed with a hybrid vector containing the hydantoinase gene whose expression is induced by temperature variation. The enzyme thus obtained is used for the production of D-N-carbamyl derivatives from 5-substituted hydantoins.
Patent application EP-515.698 describes, on the other hand, the preparation of D-N-carbamylase by the fermentation of a microorganism transformed with a plasmid comprising the carbamylase gene whose expression is chemically induced with IPTG. The enzyme thus obtained is used for the production of D-&agr;-amino acids from N-carbamyl derivatives.
As industrial interest is directed towards the conversion of racemic hydantoins to D-&agr;-amino acids, the fact that the two enzymes are expressed in different strains involves the use of both and consequently the development of a process starting from two distinct fermentative processes.
This obviously increases the production costs and reduces the conversion kinetics. In fact, in order to complete the enzymatic reaction, the N-carbamyl derivative produced by the transformed microorganism containing the hydantoinase must pass through the bacterial membrane, spread into the reaction medium and then proceed in the opposite direction to reach the second enzyme (carbamylase) present in the other strain. All this is particularly penalizing from the point of view of kinetics considering the reduced permeability of the bacterial membranes to the carbamyl derivatives (Olivieri et al. (1981), Biotechnol. Bioeng., 23, 2173-2183) and the inevitable dilution of the carbamyl itself in the reaction mixture.
Finally, the use of a double volume of biomass has a negative influence on the yields and degree of purity of the final product.
In addition, the necessity of having to induce the expression of these enzymes creates a further problem thus making these processes of little interest for practical use.
The object of the present invention is to overcome the disadvantages of the known art described above.
In particular it has now been found, in accordance with the present invention, that the use of a particular plasmid which contains the genes of D-hydantoinase and N-carbamylase put under the control of an appropriate synthetic promoter, enables the high expression of these enzymes to be obtained without inducers.
It is therefore possible to prepare a single microorganism transformed with said plasmid containing the two enzymatic activities inside. This solution solves not only the problems relating to kinetics due to the limited permeability, as the two reactions occur inside the same cell where the concentration of the substrates is excellent, but also those relating to the requirement of inducers and treatment of the product and of the waste products.
In accordance with this, a first aspect of the present invention relates to a process for the production of D-&agr;-amino acids by the stereospecific conversion of racemic mixtures of 5-substituted hydantoins characterized in that, the conversion reaction is carried out in the presence of a microorganism transformed with a plasmid capable of expressing at high levels and without inducers an enzymatic system capable of converting said hydantoins into the corresponding D-&agr;-amino acids.
A further object of the present invention is the plasmid pSM651 comprising the genes which encode the enzymatic system.
Yet another object of the present invention is a microorganism transfored with the plasmid pSM651 capable of expressing with high efficiency and without inducers an enzymatic system capable of stereospecifically converting racemic mixtures of 5-substituted hydantoins into the corresponding D-&agr;-amino acids.
A further object of the present invention relates to the use of said microorganisms or enzymatic system isolated from said microorganisms for the production of D-&agr;-amino acids by the stereospecific conversion of racemic mixtures of 5-substituted hydantoins.
Further objects of the present invention will be evident from the description and examples below.
BRIEF DESCRIPTION OF THE FIGURES
FIG.
1
: Map of the plasmid pSM637 containing the carbamylase gene
FIG.
2
: Map of the plasmid pSM650 containing the hydantoinase gene
FIG.
3
: Map of the plasmid pSM651 containing the hydantoinase-carbamylase operon.
FIGS.
4
A-B: Nucleotide and amino acid sequence of carbamylase (SEQ ID NO:18-19).
FIGS.
5
A-C: Nucleotide and amino acid sequence of hydantoinase (SEQ ID NO:20-21).
FIGS.
6
A-B: SDS-PAGE (A) and Western-Blot (B) of the total proteins extracted fr
Frascotti Gianni
Galli Giuliano
Grandi Guido
Grifantini Renata
Eniricerche S.p.A.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Prouty Rebecca E.
Steadman David
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