Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Reexamination Certificate
1998-04-03
2001-04-10
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
C435S227000, C435S228000
Reexamination Certificate
active
06214592
ABSTRACT:
TECHNICAL FIELD
The present invention relates in general terms to the enzymatic hydrolysis of amides, especially secondary amides.
The invention relates more precisely 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 material in their production and to the microorganisms which contain this genetic material and exhibit this amidase activity.
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.
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
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 amninocaproic acid produced per minute and per milligram of protein from a substrate consisting of a trimer. Furthermore, these enzymes are specific for homo-oligomers and are found to have a poor activity towards co-oligomers.
Moreover, the strain Flavobacterium sp KI 72 was at the center of the discovery of another gene, called nyl-c, coding for a polyamidase E
3
active towards substrates of the PA 6 type.
The following is one of the literature references referring to E
3
produced under the control of nyl-c: “NEGORO et al. (1992)—Journal of Bacteriology, vol. 174, p. 7948-7953”.
It appears that E
3
has a relatively low specificity for oligomers of nylon PA 66 compared with oligomers of nylon PA 6. The resulting ratio
Ra
=
enzymatic activity towards PA 66
enzymatic activity towards PA 6
is of the order of 2, the activities being expressed as the amount of substrate hydrolyzed per unit time and per unit amount of hydrolyzing substance used. Examples of the substrates in question are tetramers and trimers of PA 66 and PA 6.
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 type.
DISCLOSURE OF THE INVENTION
One of the essential objects of the present invention is thus to propose novel enzymes with amidase activity, genetic material for producing them, and microorganisms containing this genetic material, said enzymes and microorganisms being simultaneously characterized by satisfactory yields in the hydrolysis of amides from substrates of the co-oligomer and homo-oligomer type and by a pronounced specificity towards co-oligomers (e.g. PA 66). Such an activity is capable of providing access to an industrially valuable method of regenerating monomers, especially of PA 66 (recycling).
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.
This enzyme is used either as such or, preferably, in the form of recombinant microorganisms which generate them.
The present invention consequently relates to an enzyme with amidase activity, particularly towards substrates of the polyamide type having at least one of the following formulae:
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
,
b—if X=X
2
, then Y=Y
2
or Y
1
,
and, finally, p is between 1.5 and 10, preferably between 1.5 and 5;
or
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 (I),
and q=1 to 20.
One very advantageous feature of the enzyme according to the invention is the fact that its specific enzymatic activity (U
s
) is much greater towards the co-oligomers of formula (I) than towards the homo-oligomers of formula (II).
Thus the enzyme according to the invention—also called PAM I—is characterized by a ratio
Ra
=
Enzymatic
Activity
towards
(
poly
)
amide
substrates
of
formula
(
I
)
Enzymatic
Activity
towards
(
poly
)
amide
substrates
of
formula
(
II
)
which is:
greater than 2;
preferably greater than or equal to 10;
and particularly preferably greater than or equal to 50.
In the ratio Ra, the enzymatic activity is expressed in mol of hydrolyzed substrate x h
−1
×g
−1
of enzyme or dry cells producing the enzyme used.
The conditions of measurement of these activities are given below:
reaction medium=100 mM phosphate buffer,
volume=400 &mgr;l,
temperature=30° C.,
pH=7,
concentration of dry cells=2.5 g/l.
The enzyme according to the invention is also characterized by its primary structure, which is given by the attached amino acid sequence SEQ ID NO: 2.
This also embraces any polypeptide having a degree of homology of at least 50% with this sequence SEQ ID NO: 2.
The primary or even quaternary structure of the enzyme according to the invention constitutes one of its numerous novel characteristics.
In addition to this structural characterization, however, it is possible to envisage the enzyme according to the invention through its activity towards some of its
Crouzet Joel
Favre-Bulle Olivier
Jourdat Catherine
Le Coq Anne-Marie
Petre Dominique
Burns Doane Swecker & Mathis L.L.P.
Patterson Jr. Charles L.
Rhone-Poulenc Fibres et Polymeres S.A.
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