Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2001-01-02
2003-04-22
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S041000, C435S134000, C435S155000, C435S177000, C435S180000, C435S182000, C435S280000
Reexamination Certificate
active
06551806
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel enzyme-containing polymers comprising at least one of the following structural elements:
Structural Element 1
Structural Element 2
where the abbreviations and symbols have the following meanings:
E enzyme
X
1
, X
2
independently of one another oxygen, sulfur or NH from a functional group of the enzyme
Q hydrogen or the group:
A
1
, A
2
, A
3
, A
4
independently of one another oxygen or sulfur
p 0 or 1, with the proviso that A
1
, A
2
, A
3
, A
4
are oxygen when p is 0
w 1-4
q 1-4
v 1-100
R
1
an alkane, alkene, cycloalkane, cycloalkene, arene, arylalkane, diaryl ether, diaryl thioether or diarylamine group which is bonded 2 to 5 times, it being possible for the aromatic or nonaromatic cyclic groups in turn to be substituted by one to four C
1
-C
4
-alkyl, C
1
-C
4
-haloalkyl and/or halogen radicals,
R
2
an alkane, alkene, cycloalkane, cycloalkene, arene, arylalkane, dialkyl ether, dialkyl thioether, diaryl ether, diaryl thioether, diarylamine or piperazinedialkanediyl group which is bonded 2 to 5 times, it being possible for the aromatic or nonaromatic cyclic groups in turn to be substituted by one to four C
1
-C
4
-alkyl, C
1
-C
4
-haloalkyl and/or halogen radicals,
R
4
und R
5
independently of one another hydrogen, a C
1
-C
4
-alkyl, aryl or alkylaryl group.
The present invention also relates to the preparation and use of the enzyme-containing polymers.
The enzyme-containing polymers are employed as enzyme catalysts in chemical reactions. Compared with the free enzymes, the immobilized enzymes are distinguished by an increased stability and useful life when reactions are carried out continuously and batchwise, and by easy recovery of the catalytically active species in the case of batchwise reactions.
2. Description of the Related Art
It is known to incorporate enzymes into polymers by covalent bonding with retention of the activity. It is further known to use polyurethanes, polyureas and polyamides as polymeric carrier material. U.S. Pat. No. 5,482,996 describes protein-containing polyurethanes, polyureas, polyamides and polyesters. In this case, the proteins are transferred from the aqueous solvent system into the organic solvent system, with retention of the activity, by attaching an amphiphilic spacer (polyalkylene oxide) which has the terminal functional group suitable for the type of polymerization. The monomers are then reacted with the functional group of the spacer. This method makes only low coverage possible and is moreover very elaborate and costly because there is no direct incorporation of the enzymes into the polymer, but the attachment of the amphiphilic spacer unit comprises a preceding additional step.
U.S. Pat. No. 3,672,955 discloses the preparation of enzymes immobilized in polyurethane, where there is initial preparation of amphiphilic isocyanate prepolymers bridged with polyether and polyesterpolyol units, and these are emulsified and reacted in a water-immiscible solvent with the aqueous enzyme solution. The contact with water converts the isocyanates which have not reacted with the functional groups of the enzymes into unstable carbamic acid groups which then decompose into the corresponding amines with elimination of CO
2
. The resulting amino groups react with isocyanate groups which are still present, to give crosslinking, and the CO
2
produced leads to foaming of the polymer mass. U.S. Pat. No. 4,342,834 follows the analogous prepolymer strategy of transferring the polymerization into aqueous systems, the difference from U.S. Pat. No. 3,672,955 being that the reaction is completely carried out in aqueous solution.
The use of the amphiphilic prepolymers Hypol® which is analogously bridged with polyalkylene oxide units (produced by reacting a polyether- or polyesterpolyol with polyisocyanates in the presence of linking reagents) and PU-3® (prepolymer saturated with two terminal TDI units and containing polyethylene oxide and polypropylene oxide units as spacer) for immobilizing a number of enzymes from aqueous solution has been described in scientific articles.
It was possible to use these amphiphilic prepolymers to immobilize phosphotriesterases from aqueous solution for breaking down neurotoxins (K. E. Lejeune et al., Biotechnology and Bioengineering 1997, 54, 105-114). Lipases immobilized from aqueous solution with prepolymers have been used for the acylation (S. F. Dias et al., Biocatalysis 1991, 5, 21-34.) and for the enantioselective acylation (S. Koshiro et al., Journal of Biotechnology 1985, 2, 47-57) of alcohols in organic solution.
In all these processes, the polymerization (immobilization of the enzyme) is carried out in the presence of water with prepolymers. This has several disadvantages:
The amphiphilic prepolymers must be prepared separately.
Only short useful lives are achieved with prior art enzyme-containing polymers.
Only low loading densities with active enzyme species are achieved. The typical loading of a polymer with enzyme in the abovementioned studies is a maximum of 1% of the total mass. Low space-time yields (STY) result from this.
SUMMARY OF THE INVENTION
It is an object of the present invention to remedy the deficiencies described and to provide novel enzyme-containing polymers which have been prepared by a simplified process and have optimized properties, such as longer useful life and higher loading density with catalytically active enzyme species.
We have found that these objects are achieved by the enzyme-containing polymers according to the invention described at the outset.
The structural elements are produced by reacting the functional groups (amino, hydroxyl, mercapto) located on the enzyme surface with monomers having at least bifunctionally reactive groups, and subsequently adding at least bifunctional amines.
DETAILED DESCRIPTION OF THE INVENTION
The enzymes are to be regarded as at least monofunctional, usually polyfunctional, amines, alcohols and/or thiols. They can be obtained, for example, from organisms such as fungi, bacteria (Gram
+
or Gram
−
), yeasts or mammals, and have an enzymatic activity in organic solvents.
Examples which may be mentioned are hydrolases such as esterases, lipases, amidases, proteases and haloperoxidases, aminotransferases, aspartate aminotransferases, pyruvate decarboxylases, lyases/laccases, benzene dioxygenase, aspartases, dehydrogenases, fumarases, dehalogenases, amino-acid dehydrogenases, oxygenases, aminopeptidases, aminoamidases, alkylaminopeptidases and racemases.
Preference is given to lipases from
Aspergillus niger, Aspergillus oryzae, Candida antarctica, Candida cylindracea, Candida lipolytica, Candida utilis, Candida rugosa, Mucor javanicum, Mucor miehei, Rhizomucor miehei, Rhizopus arrhizus, Rhizopus delemar, Rhizopus niveus, Penicillium acylase, Penicillium roqueforti, Thermus aquaticus, Thermus flavus, Thermus thermophilus, Chromobacterium viscosum, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas cepacia,
and pig pancreatic lipase (PPL) and wheat germ lipase, esterases from
Bacillus subtilis, Bacillus stearothermophilus, Bacillus thermoglucosidasius, Candida lipolytica, Mucor miehei,
equine liver, porcine liver,
Saccharomyces cerevisiae, Thermoanaerobium brockii, Elektrophorus electricus,
proteases such as subtilisin, thermolysin, subtilisin Carlsberg, nagarse or from
Bacillus subtilis, Tritirachium alba, Aspergillus oryzae,
Aspergillus sp., benzene dioxygenase from Pseudomonads, Alcaligenes sp., Micrococcus sp.,
Pseudomonas oleovorans,
dehalogenases from
Pseudomonas putida,
oxygenases from
Pseudomonas oleovorans, Corynebacterium equi, Nocardia carallina,
Mycobacter, Xanthobacter, aminoamidases and alkylaminopeptidases from Mycobacterium, dehydrogenases from
Pseudomonas putida
and nitrilases and nitrile hydratases from
Aspergillus niger
JCM 1925, Fusarium sp. MY-2,
Rhodococcus rhodocrus
J 1, K22, PA 34 and NCIB 11216,
Pseudomonas chlororaphis
B 23, Corynebacterium sp. N-774.
The lipase from
Burkholderia plantarii
is
BASF - Aktiengesellschaft
Keil & Weinkauf
Naff David M.
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