Biocatalysts with amine acylase activity

Details Biocatalysts with amine acylase activity Biocatalysts with amine acylase activity

1998-11-12

2001-05-22

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S183000, C435S106000, C435S228000, C435S128000, C435S280000, C435S826000, C435S893000, C435S908000

Reexamination Certificate

active

06235516

ABSTRACT:

There is a high and continuous need for enantiomerically pure pharmaceutical and agrochemical drugs avoiding the side effects triggered by the therapeutically ineffective enantiomer. The present invention helps to fulfill this need by providing novel enzymes capable of stereoselectively hydrolysing a racemic acylamide.
Up to now enzymatic reactions on amines or amine-derivatives have been reported in the literature only for penicillins, cephalosporins, glucosamines, amino acids, and their derivatives. Among the known examples in this field figure the resolution of racemic 2-aminobutanol via the hydrolysis of appropriate N-acyl-derivatives by acylases of microbial origin (Japanese Kokai JP 58-198,296 and JP 59-39,294) and the enantiomeric enrichment of different amines possessing the amino group on a secondary carbon atom through a process involving the action of omega amino acid transaminases (Stirling et al., U.S. Pat. No. 5,300,437). In JP 06-253,875 the stereospecific transfer of the amino group of L-alanine to acetophenone in the presence of e.g. Acinetobacter sp. MBA-15 (FERM P-13432) for the production of S-1-phenyl ethylamine is disclosed. U.S. Pat. No. 5,360,724 discloses the production of optically active 1-aryl-2-amino propane by enantioselective transfer of the amino group in the racemate by means of amino acid transaminase from
Bacillus megaterium
. In DE 4332738 the production of optically active primary and secondary amines is disclosed; the process uses enantioselective acylation of a racemic amine with an activated ester as acyl donor in the presence of a hydrolase.
However, the present invention provides novel biocatalysts for enantioselective hydrolysis
OBJECT OF THE INVENTION
It is an object of the invention to provide novel biocatalysts for stereoselective hydrolysis of enantiomers in a racemic acylamide. Such biocatalysts are microorganisms capable of producing enzymes (acylases) capable of stereoselectively hydrolysing enantiomers in a racemic acylamide, or the enzymes themselves.
Moreover, it is an object to provide a process for the stereoselective hydrolysis of acylamides.
SUMMARY OF THE INVENTION
The invention concerns a biocatalyst, i.e. a dead or living microorganism or a polypeptide, preferably in isolated form, which exhibits acylase enzymatic activity without lipase- or esterase-activity. The biocatalyst is capable of stereoselectively hydrolysing a racemic acylamide which has an aliphatic acyl residue and which is not a derivative of a natural amino acid.
The invention accordingly also concerns microbial strains capable of producing an enzyme of the invention. Included are both genetically modified microorganisms and naturally occurring ones. The naturally occurring strains according to the invention are microorganisms obtainable by a selection process comprising inoculating a selection medium with a natural sample.
Another aspect of the invention is a process for the hydrolysis of a racemic N-acylamide which has an aliphatic acyl residue and which is not a derivative of a natural amino acid, characterized in that an enzyme according to the invention is used.
The process of the invention can be performed with free or immobilized enzyme, which can be used in enriched or purified form or in the form of a crude cell extract. In another embodiment of the invention a microorganism expressing an acylase of the invention is used for performing the reaction, i.e. the enzyme is in cell-bound form.
The process of the invention can be used for separating racemates, if one stereoisomer of the racemic acyl amide is specifically hydrolyzed. However, if the hydrolysis is not stereoselective (e.g. since the acylamide is not a racemate or since the enzyme hydrolyses both enantiomers of a certain racemic acylamide), the process of the invention can also be used for other purposes, e.g. in the chemistry of protecting groups.
DETAILED DESCRIPTION OF THE INVENTION
The invention concerns a biocatalyst exhibiting amine acylase enzymatic activity without lipase- or esterase-activity, which biocatalyst is capable of stereoselectively hydrolysing a racemic acylamide of formula (1)
wherein R
2
is aryl or C
1
-C
4
aryl; or R
1
and R
2
together are forming a 5 to 7 membered ring substituted by or fused to aryl;
R
3
is an aliphatic acyl residue; and
R
1
and R
3
together are chosen in order to form a compound of formula (1) that is not a derivative of a natural amino acid; wherein each of the residues can be substituted or unsubstituted.
A preferred embodiment is a biocatalyst, the substrate of which can be hydrolyzed to a primary amine. The biocatalyst according of the invention is preferably selected from the group of (a) a polypeptide with said enzymatic activity and (b) a living microorganism or dead microorganism containing a polypeptide with said enzymatic activity or a cell extract of such a microorganism. A dead microorganism in context with the present invention is e.g. a microorganism in a disintegrated form in which the cell wall and/or cell membrane is mechanically or chemically disrupted or removed.
The term without lipase- or esterase-activity in context with the present invention means that no enzymatic activity can be detected in the Api Zym Test (Bio Mérieux SA, Marcy-L'Etoile, France). In this test the C4 esterase activity is tested with 2-naphtyl butyrate, C8 esterase activity with 2-naphtyl caproate, and C14 lipase activity with 2-naphtyl myristate.
In particular, the invention concerns a enzyme with amine acylase activity and without lipase- or esterase-activity and mutants, variants, and fragments of said acylase, which exhibit acylase activity and without lipase- or esterase-activity. A preferred embodiment is a polypeptide, the substrate of which can be hydrolyzed to a primary amine.
The polypeptide of the invention is herein also named “acylase of the invention” or “enzyme of the invention”. If not stated otherwise, all these terms include not only the naturally occurring, authentic sequence of a polypeptide of the invention, which are the preferred embodiments of the invention, but also all mutants, variants and fragments thereof which exhibit acylase enzymatic activity, preferably the same stereoselective activity as the natural enzyme. The polypeptide of the invention can be used in enriched or, preferably, purified form.
In a preferred embodiment of the invention a polypeptide of the invention is particularly capable of catalyzing the following stereoselective reaction (A)
wherein the racemic acylamide of the formula (1) is stereoselectively hydrolyzed with an amine acylase of the invention, which is a polypeptide with amine acylase enzymatic activity but without lipase- or esterase-activity, and which is capable of stereoselectively hydrolysing the racemic acylamide of formula (1), and wherein said hydrolysis results in the R- (or S-) amine of the formula (2) and the acid of formula (3) and the S- (or R-) amide of formula (4), whereby
whereby in the formula (1) to (4)
R
1
is hydrogen, C
1
-C
8
alkyl, C
2
-C
8
alkenyl, C
1
-C
8
alkoxy, C
2
-C
8
alkylcarboxy or carboxy; preferably C
1
-C
4
alkyl, C
2
-C
4
alkenyl, C
1
-C
4
alkoxy or C
2
-C
4
alkylcarboxy; more preferably C
1
-C
3
alkyl, C
2
-C
3
alkenyl, C
1
-C
3
alkoxy or C
2
-C
3
alkylcarboxy; and most preferably selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, iso-propyl, propyl-1-en, propyl-2-en, prop-2-ylen, and methoxy;
R
2
is aryl or C
1
-C
4
aryl; unsubstituted or substituted by C
1
-C
4
alkyl, C
1
-alkoxy, C
1
-C
4
hydroxyalkyl, C
1
-C
4
aminoalkyl, C
1
-C
4
haloalkyl, hydroxy, amino, halogeno, nitro, sulfo, or cayno;
or wherein R
1
and R
2
together are forming a 5 to 7 membered ring substituted by or fused to aryl, wherein the rings may contain one or two heteroatoms selected from nitrogen, sulfur and oxygen; preferably indane, tetraline or chromane, unsubstituted or substituted with methoxy, ethoxy, halogeno, methyl, ethyl, nitro, cyano, amino, hydroxy, trifluoromethyl; and
R
3
is an aliphatic acyl residue; preferably C
1
-C
4
alkyl; and more p

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