Process for obtaining microorganisms containing peptide...

Details Process for obtaining microorganisms containing peptide... Process for obtaining microorganisms containing peptide...

1999-08-25

2001-12-25

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using a micro-organism to make a protein or polypeptide

C435S189000, C435S212000

Reexamination Certificate

active

06333176

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a process for obtaining microorganisms containing peptide amidase, microorganisms obtained therewith, peptide amidases contained in them and the use thereof.
The invention relates 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.
2. Background information
The following publications are cited regarding the state of the art:
(1) DE-OS 36 29 242,
(2) K. Breddam, Carlsberg Res. Commun. 49 (1984) 535-554,
(3) DE patent 40 14 564 and
(4) Y. Nishida et al., Enzyme Microb. Technol., 6 (1984), 85-90.
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:
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
x
are the side chains of the amino acids for n>0 whereas R
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.
Reference (1) teaches amidases for an enzymatic splitting off of the acid amide group which, on account of their a-amino acid amidase activity, can only be used, however, for the production of L-amino acids from a-unprotected D,L-amino acid amides. Peptide amides are not accepted.
Reference (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.
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 of this is the carboxy peptidase Y, especially in chemically modified form (see reference (2)).
Thus, all these processes have serious disadvantages.
The state of the art according to reference(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° 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 in reference (3) moot—that a microbial production of the enzyme can be achieved in a known manner by gene technology manipulation. The problems in the presentation of the homogeneous form do not allow manipulations of gene technology.
SUMMARY OF THE INVENTION
Therefore, in view of the problems associated with the state of the art, the invention has the object of making available a process for isolating microorganisms containing peptide amidase which makes possible a rapid selection of suitable strains. A further object is also a stable peptide amidase which is more readily available than the known peptide amidase from flavedo at an equally high selectivity of the hydrolytic splitting off of the free amino group on the C-terminal end of peptide amides.
These objects and others not cited in detail are achieved by a process with the features recited in claim
1
.
Microorganisms can be produced at practically any time of the year in any desired amount. Therefore, collection strains and isolated strains which can mobilize amide nitrogen as a source of nitrogen are analyzed for peptide amidase activity in a limited screening. Z-Gly-Tyr-NH
2
was used as test substrate and the expected hydrolysis product Z-Gly-Tyr-OH determined by HPLC.
It is possible, by first incubating specimens containing microorganisms in a “double screening” in a nutrient medium containing amide nitrogen as nitrogen source and inoculating colonies subsequently produced onto a nutrient medium containing N-acetyl-D,L-methionine amide, then incubating them and selecting the microorganisms which grow in both nutrient media, to find strains with an unusually good rate of success which are both relatively stable and also selective and active.
For the screening for microorganisms which can utilize amide nitrogen, samples of soil were suspended 4-6 hours in isotonic solution of common salt. The samples of soil were of any origin, including garden soil, forest soil, loamy or sandy soil. The solids were separated off at 2000 rpm by centrifugation. The supernatant was spread out onto agar plates and used to inoculate liquid media in Erlenmeyer flasks. The plates were incubated 3-7 days at 30° C. and the Erlenmeyer flasks agitated at the same temperature at 120 rpm. Then, individual cultures were isolated from the plates and brought into pure culture by being multiply spread out. After this time aliquots were taken from the incubated Erlenmeyer flasks and fresh medium inoculated therewith. This process was repeated up to five times before specimens of the culture liquid were spread out after a suitable dilution onto plates. The nutrient medium for the solid and also for the liquid medium had the following composition:
K
2
HPO
4
2.50
g/l
KH
2
PO
4
1.95
g/l
NaCl
1.00
g/l
CaCl
2
*2H
2
O
0.05
g/l
MgSo
4
*7H
2
O
0.3
g/l
Yeast extract
0.50
g/l
DL-carnitine amide
5.00
g/l
Trace saline solution
0.80
ml/l
Vitamin solution
2.5
ml/l
(Agar for solid media
18.0
g/l)
pH 7.2
CaCl
2
*2H
2
O, MgSO
4
*7H
2
O as well as DL-carnitine amide and the vitamin solution (see below) were sterilized by filtration and added to the autoclaved, cooled-down medium. The trace saline solution was composed as follows:
H
3
BO
3
75.0
mg
MnCl
2
*4H
2
O
50.0
mg
ZnCl
2
187.0
mg
CuSO
4
*5H
2
O
50.0
mg
FeCl
3
*6H
2
O
625.0
mg
(NH
4
)
8
Mo
7
O
24
*4H
2
O
25.0
mg
CoSO
4
*7H
2
O
37.5
mg
H
2
O demin.
ad. 0.2
l.
Individualized microorganisms grown on this medium were used for screening for organisms with peptide amidase activity.
For screening for microorganisms with peptide amidase activity, a part of the organisms obtained above were spread out onto agar nutrient media and incubated 2 days at 30° C. In order to obtain higher cell masses, the cultures were enriched in 100 ml Erlenmeyer flasks with 20 ml me

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