JPH01160495A - Method for preparation of l-aspartyl-l-phenylalanine alkyl ester - Google Patents

Abstract

PURPOSE: To obtain the subject ester in high yield by allowing L-aspartic ester or amide to react with L-phenylalanine alkyl ester in the presence of a specific enzyme, a microorganism containing this enzyme or its immobilized enzyme.

CONSTITUTION: In the presence of (A) (i) an enzyme that can form L-aspartyl-L- phenylalanine alkyl esters by condensation of L-aspartic acid α-ester or L- aspartic acid α-amide with L-phenylalanine alkyl ester, (ii) a microorganism containing the enzyme (i), (iii) said enzyme containing the fractions of said microorganism and (iv) said enzyme immobilized on a solid support, L-aspartic ester or amide is allowed to react with L-phenylalanine alkyl ester.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing L-aspartyl-L-phenylalanine alkyl ester (hereinafter abbreviated as rAPMJ).

(Prior art and problems to be solved by the invention) APM is a peptide that has recently attracted attention as a sweetener. It is well known that methods for producing APM or L-aspartyl-phenylalanine (AP) include chemical synthesis methods and enzymatic synthesis methods.

The chemical synthesis method for APM production is N-terminally protected L
- APM protected at the N-terminus by condensing aspartic acid anhydride and L-phenylalanine methyl ester (PM)
It consists of obtaining. The protecting groups are later removed. Enzymatic synthesis methods enhance the potency of proteolytic enzymes with N-terminally protected L-
It consists of applying N-terminally protected APM or a PM adduct of N-terminally protected APM to aspartic acid and PM, and then removing the protecting group to form APM. However, both methods require complicated steps of introducing and removing protecting groups (GB2,09
2.161A).

Pseudomonas, Torulopsis, 0-dotorula (
Rhodotorula ) and Sporobolomyces (
A method for producing APM without using a protecting group (JP 83/128,796) is also known, which is a microbial synthesis method using one of the following: is not necessarily suitable for

A more direct, non-protective route has also been used in which APM can be formed directly from L-aspartate and PM by microorganisms or enzymes derived therefrom (
(Japanese Patent Application No. 58-75559). However, the problem with this method when producing APM using L-aspartic acid without using a protecting group is that L-aspartic acid and P
The reaction to form APM from M is an equilibrium reaction, and the equilibrium prevents sufficient conversion of the substrate to APM, resulting in low yields. Later literature (E P 154
．． Although the yield was improved somewhat by using the phenylalanine ester residue of the alcohol in 472), the reaction is still equilibrium dependent and thus results in relatively low yields.

SUMMARY OF THE INVENTION The present invention improves product yield without the problems of equilibrium in the unhindered pathway or in the pathway using underivatized aspartate.

That is, L-aspartic acid α ester (L
AE) or L-aspartic acid alpha amide (LAA)
an enzyme capable of forming L-aspartyl-di-phenylalanine alkyl ester (LPAE) by condensation with a microorganism containing the enzyme, a fraction of the microorganism containing the enzyme or the enzyme immobilized on a solid support; Reacting L-aspartic acid α ester (LAE) or L-aspartic acid α amide (LAA) with L-phenylalanine alkyl ester (LPAE) in an aqueous solvent, an organic solvent, or a mixture thereof in the presence of an enzyme. L- consisting of letting
The present invention relates to a method for producing aspartyl-L-phenylalanine alkyl ester.

The present invention relates to the production of APM using L-aspartic acid alpha ester or alpha amide and L-phenylalanine alkyl ester as starting materials. α-esters of L-aspartic acid are known in the art and are described, for example, in J. J., Kovacs et al. CJ, Kovacs et al.
al,, J,Org,Chem, 28.1084
(1981)). Preferred alpha esters include residues of alcohols, substituted or unsubstituted phenols, thiols or alkyl esters, although other forms of L-aspartic acid alpha
It is also possible to use esters or α-amides.

For example, N-terminally protected LAE or LAA, or those that form a β-ester or β-amide of LAE or LAA can be used.

Another preferred form would be L-aspartic acid cyakyl ester. Such compounds are known in the art. These additional groups can then be removed by methods known in the art.

Alpha amides are prepared by reacting aspartic acid alpha methyl ester with ammonia in methanol under conditions known in the art. Other methods for making amides are also known in the art and can be used. The amide group at the α position is, for example, NH3,
R-NH2 in which R contains an aromatic or aliphatic structure,
It can be derived from R2NH or R3N. A preferred amide is aspartic acid amide. A preferred alkyl ester is a methyl ester.

L-phenylalanine alkyl esters are well known in the art, particularly lower (1-6) alkyl esters. A preferred lower alkyl ester of L-phenylalanine is methyl ester.

The production method of the present invention comprises producing L-aspartic acid alpha esters or alpha amides in an aqueous organic medium in the presence of an enzyme capable of condensing the alpha esters or alpha amides of L-aspartic acid to form the condensation product APM. and L-
This is carried out by reacting with phenylalanine alkyl ester. The reaction is carried out at a suitable temperature, preferably 2
It is carried out at temperatures ranging from 5 to 45°C for about 1 to 10 days.

The reaction takes place at a pH of approximately 4-9. preferred pH
is approximately 8. The actual amount of starting reactant is of course proportional to the approximate stoichiometric amount that reacts to form the APM, and such amounts are preferred at any relative concentration for optimal results. . However, it is also possible to increase or decrease the amount of either reactant, and it is also possible to select multiple L-aspartic acid esters or amides or multiple L-phenylalanine alkyl esters. The enzyme selected preferably has specificity for the alpha ester or alpha amide bond of L-aspartic acid, preferably for the (especially ester) bond of L-phenylalanine alkyl ester and for other A preferred enzyme is one that does not hydrolyze the binding or APM product.
) and Staphylococcus aureus
cus aureus) strain V8 (G, R, Dra
peau, (197g) Canadian Jour
nal of Biochemistry 56534
-44) is an extracellular protease obtained from mutants and gene variants of . This enzyme has the following N-terminal amino acid sequence: Val lie Leu Pro Asn Asn
Asp Arg HisPro Val Thr Ty
r Ile Gin Val Glu AlaVal
Gly Lys Asp Thr Leu Leu T
hr AsnHis Ala Leu Lys Ala
Phe Pro Ser AlaThr Ala G
lu Glu lie Thr Lys Tyr Se
r 1Pro Asn Glu Glu Asn Ly
s Hls Ile Gly 1Ala Glu Th
r Glu Val Asn Glu Asn Ile
'Gin Ile Thr Asp Thr Thr
Asn Gly) Iis Thr AlaPro
Thr Gly Thr Phe Ile Ala S
er Gly Val Val ValLys His Val
Val Asp Ala Thr His Gly
Asp Pr.

11e Asn Gln Asp Asn Tyr P
ro Asn Gly Gly PheGly Glu
Gly Asp Leu Ala lie Val
Lys phe 5erGlu Val Val Ly
s Pro Ala Thr Met Ser Asn.
AsnThr Val Thr Gly Tyr P
ro Gly Asp Lys Pro Va1Asp
Leu Ser Thr Thr Thr Gly Gly
Asn Ser 11ie Gly lie l1is
Trp Gly Gly Val Pr.

Asn Val Arg Asn Phe Leu L
ys Gln AsnPro Asn Asn Pro
Asp Asn Pro Asp AsnGlu P
ro Asn Asn Pro Asp Asn Pr
oAsn.

Asn Ser Asp Asn Pro Asp A
la AlaGly Ser Pro Val Phe
Asn Glu Lys Asn Gly ValA
sn Pro Asp Asn Pro Asp As
It has n Gly Asp Asn Asn.

In addition, specific activity, reaction rate, reaction specificity Kcat %Km
, site-specific changes in codons that can alter their stability to organic solvents and temperature, etc.
It is also known in the art that changes other than spontaneous or random mutations or modifications can be made to enzymes. Such modifications for site-specific changes in particular codons are known in the art and are intended to be part of this invention in selecting an enzyme. Important site-specific changes occur at or near the active site, eg, near the catalytic residue of the enzyme, at the face of the enzyme, at other amino acids, or at external portions of amino acids. Additionally, disulfide bonds can be introduced to stabilize the enzyme. It is also possible to use enzyme-containing microorganisms or fractions of enzyme-containing microorganisms. Furthermore, immobilized enzymes can also be used.

Enzymatic reaction of LAE or LAA with PAE in the presence of an appropriately selected enzyme yields the condensation product APM.

In other embodiments, the same reactions and conditions are used, but an L-aspartic acid alpha ester or alpha amide with an N-terminal protecting group or a beta ester or beta amide is used. Protecting groups or beta esters or amides can be removed from the APM product.

When using enzymes derived from microorganisms or fractions of microorganisms, such microorganisms can be obtained using customary media. Furthermore, it is also possible to add the reactants at the beginning or during the cell growth process.

The media used for the microorganisms are conventional, containing conventional carbon and nitrogen sources and inorganic ions. Additionally, the addition of trace amounts of organic nutritional substances such as vitamins and amino acids often provides desirable results.

Carbon sources suitable for use herein include carbohydrates such as glucose and sucrose, organic acids such as acetic acid, and alcohols. Suitable nitrogen sources for use herein include ammonia gas, aqueous ammonia and ammonium salts. The inorganic ion is appropriately selected from, for example, magnesium ion, phosphate ion, potassium ion, and iron ion, if necessary.

Cultivation is carried out under aerobic conditions at pH 4-9, preferably at about pH 8, at a suitable temperature within the range of 25-45°C.
It will be held for ~10 days.

Microorganisms that can be used in the present invention include the whole culture solution obtained after completion of culture, microorganisms separated from the culture solution, or washed microorganisms. In addition, usable microorganisms can be obtained by freeze-drying, drying with acetone, contacting with toluene, surfactants, etc., exposing to ultrasound, mechanically crushing, or using these cell-treated materials. , an enzyme protein fraction having enzymatic activity. Furthermore, immobilized cells of these microorganisms, insolubilized substances of treated cells, etc. can also be used.

As an aqueous medium, one containing water, a buffer, and an organic solvent such as ethanol can be used. In addition, nutrients and antioxidants necessary for the growth of microorganisms,
Surfactants, coenzymes, hydroxylamine, metal ions, and organic solvents such as DMSO can also be added to the aqueous medium as needed.

When the whole culture solution, cultured cells or treated cell material of said microorganism is brought into direct contact with the reactant so as to affect said reactant, the aqueous medium contains the reactant and the culture solution,
It is prepared by dissolving or suspending cultured cells or treated cell material, preferably adjusted to a temperature of 10 to 70°C and a pH of 4 to 9, and allowed to stand for a while or stirred.

APM produced in this manner can be separated and purified by known separation methods. The obtained APM is measured with an amino acid analyzer.

The present invention will now be described by way of examples, but these examples are included for illustrative purposes only and are not intended to limit the invention. Therefore, those skilled in the art can select other enzymes or adopt appropriate reaction conditions without unnecessary experimentation.

(Example) Example I Aqueous solutions of L-phenylalanine methyl ester and L-aspartic acid α-methyl ester were prepared and adjusted to pH 8.0. These solutions were then mixed with dimethyl sulfoxide (DMSO) to give a final concentration of L-phenylalanine methyl ester, L-aspartic acid alpha methyl ester, and DMSO of 0.1 each.
M, 0.5 M and 50%. Next, the v8 enzyme solution described above was added to a final concentration of 0.5 #Ig/rrdl, and the reaction mixture was left standing at room temperature for 24 hours. The product based on PM converted to APM was approximately 33% as analyzed by HPLC.

Claims (7)

[Claims] (1) An enzyme capable of forming L-aspartyl-L-phenylalanine alkyl ester by condensation of L-aspartic acid α ester or L-aspartic acid α amide and L-phenylalanine alkyl ester, a microorganism containing the enzyme, L-aspartate alpha ester or L-aspartate alpha amide is reacted with L-aspartate alpha ester or L-aspartate alpha amide in a solvent medium in the presence of the enzyme containing a fraction of the microorganism or the enzyme immobilized on a solid support.
A method for producing L-aspartyl-L-phenylalanine alkyl ester, which comprises reacting with phenylalanine alkyl ester. (2) Amino acid sequence of the enzyme: [There is a gene sequence. 10. The method according to claim 1, wherein the N-terminus is: (3) The manufacturing method according to claim L, characterized in that L-aspartic acid methyl ester is used. (4) The manufacturing method according to claim 1, characterized in that L-phenylalanine methyl ester is used. (5) The L-aspartic acid α ester or L-
2. The method according to claim 1, wherein the aspartic acid α-amide is N-terminally protected, and the protecting group is removed from the product. (6) The L-aspartic acid α ester or α amide is also a β ester or β amide, and the β ester or β amide is removed to produce L-aspartyl-L-phenylalanine alkyl ester. A manufacturing method according to claim 1. (7) The enzyme is more preferably the L-aspartic acid α ester or α than the L-phenylalanine alkyl ester.
The manufacturing method according to claim 1, characterized in that it has a high specificity for amides.