Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1997-02-21
1999-11-16
Marx, Irene
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
434212, C12N 902, C12N 948
Patent
active
059856327
DESCRIPTION:
BRIEF SUMMARY
The invention is relative to a process for obtaining microorganisms containing peptide amidase, microorganisms obtained therewith, peptide amidases contained in them and the use thereof.
The invention is relative in particular to a screening process for microorganisms exhibiting peptide amidase activity in accordance with the generic part of claim 1; microorganisms obtained according to this process and deposited in conformity with claims 2-4; peptide amidases which can be isolated from the microorganisms according to claims 5-7 and the use thereof.
The following publications are cited regarding the state of the art:
A peptide amidase is an enzyme which catalyzes the selective hydrolysis of a C-terminal amide function in a peptide amidase, that is, accelerates the following conversion: ##STR1##
Here, R' signifies a protective group for n=0 and for n>0 any amino acid, a protective group or H; n stands for zero or any whole number, R.sub.x are the side chains of the amino acids for n>0 whereas R.sub.1 signifies the side chain of the C-terminal amino acid.
The selective splitting off of the C-terminal amino group of peptide amides is generally difficult to achieve by a chemical conversion since the peptide bond is also subject to a hydrolytic attack. This results in mixtures which are difficult to separate and in low yields.
(1) teaches amidases for an enzymatic splitting off of the acid amide group which can only be used, however, on account of their .alpha.-amino acid amidase activity for the production of L-amino acids from .alpha.-unprotected D,L-amino acid amides. Peptide amides are not accepted.
(4) teaches the continuous production of N-Ac-L-Met from N-Ac-D,L-methionine amide in an enzymatic process using Erwinia carotovera.
Erwinia carotovera does contain an amidase activity; however, it is limited exclusively to amides of methionine. Thus, the enzyme from Erwinia carotovera only "splits off amino acid amide" and is not a peptide amidase. Furthermore, the enzyme from Erwinia carotovera can obviously only convert N-acetylated amino acid amides, in which conversion it is a disadvantage that the Ac protective group can only be split off with difficulty or not at all again.
On the other hand, peptidases are known which catalyze the hydrolytic splitting of the peptide bonds and of which it is only known that they have a certain secondary activity for splitting off the C-terminal amide protective group. An example for this is the carboxy peptidase Y, especially in chemically modified form (see (2)).
Thus, all these processes have serious disadvantages.
The state of the art according to (3) is also a peptide amidase which can be isolated from the flavedo of citrus fruits, especially of oranges. The peptide amidase described does not attack the peptide bond and catalyzes the splitting off of the free amino group from peptide amides. The peptide amidase known from (3) is characterized by the following parameters:
Splitting off of the C-terminal amino group from peptide amides and N-terminally protected amino acid amides;
No splitting of peptide bonds;
Optimum pH at 7.5.+-.1.5;
Good stability in the pH range between pH 6.0 and pH 9.0;
The optimum temperature is 30.degree. C. at a pH of 7.5;
Slight inhibition by inhibitors from serine proteases, especially phenylmethane sulfonyl fluoride;
The molecular weight is 23,000.+-.3,000;
Aggregate formation is occasionally observed;
The isoelectric point is approximately pH 9.5;
The enzyme does not accept any D-amino acid groups in C-terminal position and the rate of hydrolysis thereby is distinctly less than in the case of L-amino acid groups.
However, the isolated enzyme can be obtained from flavedo only in slight amounts and as a function of the season. More extensive studies also did not succeed, in spite of an approximately 500-fold enrichment, in preparing the protein in homogeneous form, so that molecular and genetic studies for improving the enzyme production were not able to be included due to lack of data.
However, this also renders the suggestion given i
REFERENCES:
patent: 5215897 (1993-06-01), Sakashita
patent: 5238838 (1993-08-01), Kula
Nawaz et al., Can. J. Microbiol., 1993, 39(2), 207-12.
Stelkes-Ritter, et al., "Characterization of a Newly Screened Microbial Peptide Amidase", Proceedings of the 5th Akabori Conference, May 1994, pp. 9-12.
Bommarius Andreas
Drauz Karlheinz
Kula Maria-Regina
Schwarm Michael
Stelkes-Ritter Ursula
Degussa - Aktiengesellschaft
Marx Irene
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