Enzymes and micro organisms with amidase activity which...

Details Enzymes and micro organisms with amidase activity which... Enzymes and micro organisms with amidase activity which...

1998-04-03

2001-01-30

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S263000, C435S252300, C435S252330, C435S320100, C536S023200

Reexamination Certificate

active

06180388

ABSTRACT:

TECHNICAL FIELD
The present invention relates in general terms to the enzymatic hydrolysis of amides, especially secondary amides.
More precisely, the invention relates first of all to a process for the enzymatic hydrolysis of substrates of the polyamide 6.6 type to give the two comonomers, A and B, of said substrates. The invention further relates to enzymes and/or microorganisms which are capable of being used in the enzymatic hydrolysis of amide groups, preferably on substrates containing at least one amide group, for example polyamides (PA). The invention further relates to the genetic tools expressing these enzymes.
PRIOR ART
In this field, SMITH R. et al. are the authors of an article published in “Journal of Biomedical Materials Research (1987), vol. 21, p. 991-1003” and disclose the bringing of samples of polyamide
66
labeled with carbon
14
into contact with enzymes of the papain, trypsin and &agr;-chymotrypsin type. These known polypeptides degrade polyamide
66
slightly, but the hydrolysis is not sufficiently significant to be able to be exploited on the industrial scale.
It is also known, through the article by KINOSHITA et al. (Eur. J. Biochem. 116, 547-551, 1981), that Flavobacterium sp KI 72 is able to produce a first enzyme (E
1
) which catalyzes the hydrolysis of cyclic dimers of 6-aminohexanoic acid to linear dimers of this same acid, and a second enzyme (E
2
) which is capable of converting this linear dimer to two molecules of 6-aminohexanoic acid or aminocaproic acid. The enzymatic pathway in question is summarized below.
The activity of the linear amidase E
2
is optimal for the dimers and decreases as the degree of polymerization increases, no longer being significant beyond oligomers with a degree of polymerization (DP
n
) of 7.
An enzyme E
3
active towards cyclic and linear oligomers of 6-aminohexanoic acid of DP
n
≧3 (PA 6) is also known. E
3
is described by NEGORO et al. in “Journal of Bacteriology, Dec. 1992, vol. 174, no. 24, p. 7948-7953”. E
3
also originates from Flavobacterium sp KI 72.
In their article published in “Journal of Bacteriology, June 1989, p. 3187-3191, vol. 171 no. 6”, TSUCHIYA et al. teach that a high degree of homology exists between the enzymes E
1
from Flavobacterium sp KI 72 and one of the enzymes derived from Pseudomonas sp NK 87. These enzymes E
1
and homologs, and the enzymes E
2
, more particularly the latter, are said to be active on oligomers or polyamides (PA 6) of the formula
—CO&Brketopenst;NH—(CH
2
)
5
—CO&Brketclosest;
n
NH—
where: 2≦n≦20.
The disadvantage of these enzymes derived from Flavobacterium or Pseudomonas is that they have relatively low specific activities towards oligomers, said activities amounting to at most only 1.05 micromol of aminocaproic acid produced per minute and per milligram of protein from a substrate consisting of a trimer. Furthermore, these enzymes are specific for homo-oligomers.
It is thus apparent that the prior art does not comprise means for the enzymatic hydrolysis of amide groups which have a high performance, are viable and can be applied to amides, including especially secondary amides, of a variety of types, particularly of the co-oligomer and/or homo-oligomer type.
DISCLOSURE OF THE INVENTION
Therefore, after lengthy and laborious research, the Applicant succeeded in isolating and characterizing a novel enzyme of the amidase type formed by one or more polypeptides which, in particular, can be derived from novel microorganisms isolated from the biotype and/or novel recombinant microorganisms obtained from these natural microorganisms.
Consequently, the present invention relates firstly to an enzymatic hydrolysis process and secondly to an enzyme.
The process according to the invention for the hydrolysis of (poly)amides characterized in that:
enzymatic hydrolysis is carried out on substrates comprising (poly)amides of the following formula (I):
in which:
A and B are monomer units,
R
1
and R
3
are identical or different—preferably different—divalent radicals representing a substituted or unsubstituted, linear or branched (cyclo)alkylene, an arylene or an arylalkylene, the aromatic radicals optionally being polycondensates and the number of carbons in the alkylenes being greater than or equal to 4, preferably between 4 and 12,
R
2
corresponds to identical or different—preferably identical—radicals selected from hydrogen and/or alkyl radicals advantageously having from 1 to 6 carbons,
X is:
either X
1
=OH, OM or OR
4
, where M is selected from metals, preferably alkali metals and alkaline earth metals, and R
4
is a linear or branched alkyl containing from 1 to 6 carbon atoms,
or X
2
=
where R
2
and R
3
are as defined above and R
5
and R
6
, which are identical or different, have the same definition as that given above for R
2
,
Y is:
either Y
1
=hydrogen,
or Y
2
=
where R
1
is as defined above and Z is hydrogen, M
1
defined in the same way as M, or R
4
,
with the following conditions:
-a- if X=X
1
,then Y=Y
1
or Y
2
,
-b- if X=X
2
, then Y=Y
2
,
and, finally, p is between 1 and 4, preferably between 1 and 3; and
it is capable of converting the above-mentioned substrates (I) to monomers A, on the one hand, and monomers B, on the other.
The present invention further relates to a family of enzymes of the amidase type which are capable (inter alia) of being used in the process defined above.
The enzyme belonging to the family according to the invention is an amidase characterized in that:
-{circle around (5)} →it is active, especially towards substrates of the (poly)amide type having the following formula:
in which:
A and B are monomer units,
R
1
and R
3
are identical or different—preferably different—divalent radicals representing a substituted or unsubstituted, linear or branched (cyclo)alkylene, an arylene or an arylalkylene, the aromatic radicals optionally being polycondensates and the number of carbons in the alkylenes being greater than or equal to 4, preferably between 4 and 12,
R
2
corresponds to identical or different—preferably identical—radicals selected from hydrogen and/or alkyl radicals advantageously having from 1 to 6 carbons,
X is:
either X
1
=OH, OM or OR
4
, where M is selected from metals, preferably alkali metals and alkaline earth metals, and R
4
is a linear or branched alkyl containing from 1 to 6 carbon atoms,
or X
2
=
where R
2
and R
3
are as defined above and R
5
and R
6
, which are identical or different, have the same definition as that given above for R
2
,
Y is:
either Y
1
=hydrogen,
or Y
2
=
where R
1
is as defined above and Z is hydrogen, M
1
defined in the same way as M, or R
4
, with the following conditions:
-a- if X=X
1
, then Y=Y
1
or Y
2
,
-b- if X=X
2
, then Y=Y
2
,
and, finally, p is between 1 and 4, preferably between 1 and 3;
-{circle around (2)} →and it is capable of converting the above-mentioned substrates (I) to monomers A, on the one hand, and monomers B, on the other.
Among this family of enzymes according to the invention, it is advantageous to isolate an enzyme with amidase activity which comprises the peptide sequence as shown in the attached sequence SEQ ID NO : 1, or a homologous peptide sequence having a homology of at least 80%, preferably at least 90% and particularly preferably at least 95% with SEQ ID NO: 1.
According to one advantageous characteristic of the invention, this enzyme has amidase activity particularly towards substrates of the (poly)amide type having the following formula (II):
in which:
R
2
and R
3
are as defined above,
U and V respectively have the same definitions as those given above for X
1
and Y
1
in formula (1) given in claim
1
,
and q=1 to 8.
The invention initially arose from the isolation of a wild-type strain which produced the enzyme, namely:
Comamonas acidovorans
N 12.
The identification of this wild-type strain is the result of a long-winded screening operation. This identification was effected on the basis of the morphological,

Affiliated with

Also associated with

Rating

Enzymes and micro organisms with amidase activity which... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Enzymes and micro organisms with amidase activity which..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Enzymes and micro organisms with amidase activity which... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2518291