Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2001-06-22
2003-09-09
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S108000, C435S109000, C435S129000, C435S227000, C435S228000
Reexamination Certificate
active
06617127
ABSTRACT:
The invention relates to a method for synthesis of &agr;-L-aspartyl-L-phenylalanine methyl ester (&agr;-APM; aspartame) involving enzymatic deformylation of an N-formyl-&agr;-L-aspartyl-L-phenylalanine compound. An N-formyl-&agr;-L-aspartyl-L-phenylalanine compound as meant herein is understood to be either N-formyl-&agr;-L-aspartyl-L-phenylalanine or its methyl ester (F-&agr;-AP or F-&agr;-APM). The invention also relates to a method for preparation and recovery of &agr;-APM from either (i) a mixture of N-formyl-&agr;- and N-formyl-&bgr;-L-aspartyl-L-phenylalanine or (ii) a mixture of N-formyl-&agr;- and N-formyl-&bgr;-L-aspartyl-L-phenylalanine methyl ester (F-&agr;&bgr;-AP or F-&agr;&bgr;-APM) by enzymatic deformylation. And finally, the invention relates to a simple method for one-pot enzymatic synthesis of &agr;-APM from N-formyl-L-aspartic acid (F-Asp) and L- or D,L-phenylalanine methyl ester (L- or D,L-PM) also involving an enzymatic deformylation reaction. The latter combination of simultaneous enzymatic coupling and deformylation reactions has wider and more general applicability.
Aspartame (&agr;-APM, L,L-form) is known to be a high intensity artificial sweetener, having a sweetness which is about 200× as potent as the sweetness of sucrose, whereas its tast properties are close to those of sucrose. The &bgr;-form of APM does not have sweet taste properties. &agr;-APM is used for the sweetening of various edible materials. Synthesis methods for &agr;-APM include chemical syntheses routes (e.g. by coupling of the anhydride of F-Asp with L-Phe or L-PM) which are invariably leading to mixtures of &agr;- and &bgr;-forms of F-APM in ratios of about 70/30 to 80/20 wt./wt.). Apart from the required deformylation, these chemical methods therefore also require separation of the &agr;-APM from &bgr;-APM and large recycle streams for recovery of &agr;-APM in high purity and adequate yield. The synthesis methods for &agr;-APM also include enzymatic coupling methods (e.g. by coupling of F-Asp or N-benzyloxy-carbonyl-L-Asp, also known as Z-Asp, with D,L-PM or L-PM). The enzymatic methods have the clear advantage that they selectively yield the &agr;-coupled L,L-product in protected form. Although still needing a deprotection step, the enzymatic coupling routes therefore do not require difficult separation of &agr;-APM from &bgr;-APM nor large recycle streams for recovery of &agr;-APM in high purity and adequate yield. In the state of the art processes for the synthesis of &agr;-APM many of those processes involve formyl-protection routes, and thus will need a deformylation step in one of the final stages of the process. Chemical deformylation, which is often performed in aqueous medium containing methanol and a strong acid, has the disadvantage that also demethylation of the phenylalanine methyl ester part of the molecule will occur, and mixtures of many compounds will be obtained in any subsequent methylation step. Enzymatic deformylation is believed to take place under mild conditions, and thus without simultaneous demethylation. However, so far no suitable enzymatic deformylation process seems to be available.
Enzymatic deformylation of an N-formyl-&agr;-L-aspartyl-L-phenylalanine compound, namely of F-&agr;-APM, is disclosed in Example 11 of U.S. Pat. No. 4,668,625. This patent teaches that penicillin-acylases are not suitable for removal of formyl-groups from oligopeptides. For instance, in Example 11 the yield is described to be 20% after 36 hours of reaction at an extremely high concentration of active enzyme as compared to the F-&agr;-APM (namely 50 U of enzyme and 2 g of F-&agr;-APM). However, even these results could not be reproduced by the present applicants. This is, amongst other things, because no clear disclosure is given of the Pseudomonas strain used and of the method of isolating the enzyme having penicillin-acylase activity therefrom.
Thus, there is need for an improved method of preparation for synthesis of &agr;-L-aspartyl-L-phenylalanine methyl ester (&agr;-APM; aspartame) involving enzymatic deformylation of an N-formyl-&agr;-L-aspartyl-L-phenylalanine compound, without the disadvantages as mentioned above.
It now surprisingly has been found that enzymes having formylmethionyl peptide deformylase activity (hereinafter for convenience also represented by PDF or by PDF enzyme) can be used in the synthesis of aspartame. The method for synthesis of &agr;-L-aspartyl-L-phenylalanine methyl ester by enzymatic deformylation of an N-formyl-&agr;-L-aspartyl-L-phenylalanine compound according to the present invention comprises treating N-formyl-&agr;-L-aspartyl-L-phenylalanine or its methyl ester with an enzyme having formylmethionyl peptide deformylase activity and having as a co-factor bivalent metal ions chosen from the group of group 5 to 11 metals from the periodic system of elements.
The periodic system of elements (new IUPAC version) and the group numbers as meant herein are presented in the Handbook of Chemistry and Physics, 70th edition, CRC Press, 1989-1990, inner page of cover. Bivalent metal ions from the group of group 5 to 11 metals are, for instance, V
2+
, Cr
2+
, Mn
2+
, Fe
2+
, Co
2+
, Ni
2+
, Cu
2+
, Pd
2+
, and Pt
2+
. Mn
2+
, Fe
2+
, Co
2+
and Ni
2+
are preferred.
Preferably the amount of the bivalent metal ions should be about equivalent to the number of moles of enzyme. Suitably the molar ratio between these bivalent metal ions and the number of PDF molecules is in the range of 0.6 to 1.4, preferably of 0.8 to 1.2, and most preferred the amount of bivalent metal ions is equimolar to the enzyme.
In case F-&agr;-AP is used as a starting material, of course, a final methylation step of the phenylalanine carboxylic acid group needs to be carried out in order to obtain the desired aspartame final product. Methods for such methylation are known to the skilled man, e.g. from U.S. Pat. No. 4,946,988 and EP-A-0468063.
Recovery of &agr;-APM from the reaction mixture can be done by any method known to the skilled man. Various methods of crystallisation of aspartame have been described in the literature, e.g. in EP-A-0091787, EP-A-0399605 and EP-A-0582351. Preferably the recovery of &agr;-APM is done at a pH near the iso-electric point of &agr;-APM, i.e. at a pH in the range of 3 to 7.
The present invention is in particular surprising as there has not been any indication in the state of the art so far that PDF enzymes are also suitable for deformylating terminal N-formyl-L-aspartic acid residues in oligopeptides or dipeptides.
Enzymes having formylmethionyl peptide deformylase activity (PDF's) are widely available in nature. Mostly they are being described as formylmethionine deformylases. It should be noticed, however, that in the literature also other names are being used instead of the name formylmethionine deformylase; in particular the following names may be mentioned here: (poly)peptide deformylase, N-formylmethionylaminoacyl-tRNA deformylase, N-formyl-L-methionine amidohydrolase, N-formylmethionyl-aminoacyl-tRNA amidohydrolase.
The native PDF's in nature, e.g. in eubacteria, catalyse the deformylation of the formyl group from the terminal N-methionine residue in nascent polypeptides; more specifically, the PDF's catalyse the hydrolysis of the N-formyl group from the N-terminal L-methionine residue of nascent polypeptides synthesized by the ribosomal protein synthesis machinery. However, no other practical applications of these enzymes are known so far. On the contrary, Rajagopalan et al. (Biochem. 36, 13910-8 (1997)) have established that deformylases have strong sequence preference for methionine at the N-terminus of peptide substrates and to a lesser extent for norleucine at that site. It is thus surprising that these enzymes can be used favourably in the synthesis of &agr;-APM.
The PDF's are obtainable, for instance, from eubacteria, for example
Escherichia coli, Bacillus subtilis, Clostridium beijerinckii, Clostridium acetobutylicum, Thermotoga maritima,
Quaedflieg Peter J. L. M.
Sonke Theodorus
Wagner Adolf F. V.
Holland Sweetener Company V.O.F.
Lilling Herbert J.
Pillsbury & Winthrop LLP
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