Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-07-06
2003-07-22
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S128000, C435S132000, C435S134000, C435S136000, C435S155000, C435S180000, C435S198000, C435S280000, C435S874000, C435S875000
Reexamination Certificate
active
06596520
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a process for preparing immobilized lipase, to the immobilized lipase itself and to a process for enzyme-catalyzed conversion in the presence of the immobilized lipase.
BACKGROUND OF THE INVENTION
Lipases can be used in solution as enzymatic catalysts for converting substrates. Immobilized lipases are distinguished from free lipases by having an increased stability and useful life on carrying out the reaction continuously and batchwise, and by easy recovery of the catalytically active species in batchwise reactions.
It is known to immobilize lipases by adsorption onto a solid support. It is also known to prepare immobilized lipases by contacting polyolefin particles with an aqueous solution of a purified lipase.
EP 232 933 describes the immobilization of a purified lipase from an aqueous solution by adsorption onto hydrophobic thermoplastic polymers such as, for example, aliphatic polyolefins. The immobilized lipase is used for fat hydrolysis.
WO 90/15868 discloses the immobilization of purified
Candida antarctica
lipase from an aqueous solution by adsorption onto aliphatic polyolefins which have been pretreated with organic solvents. The immobilized lipase is used for ester synthesis.
In WO 94/28118 a nonionic surface-active substance is added before immobilization of the purified lipase on hydrophobic support materials.
All prior art processes have the disadvantage that the lipase is purified to remove other proteins, enzymes and other cell constituents from the crude lipase solution before the immobilization on the solid support. This purification step by precipitation and chromatographic processes is time-consuming and costly.
In addition, the immobilized lipases prepared according to the prior art have a greatly reduced activity compared with the free lipases and must be reactivated by adding, for example, surface-active substances.
Addition of surface-active substances is also necessary to activate free lipases in organic solution which have been purified from a crude lipase solution according to the prior art (WO 95/17504).
In addition, the useful life of the immobilized lipases prepared according to the prior art is still not optimal.
SUMMARY OF THE INVENTION
It is an object of the present invention to remedy the described deficiencies and provide a novel simplified process for preparing immobilized lipase and novel immobilized lipases which have optimized properties and have been prepared by a simplified process.
We have found that these objects are achieved by a novel process for preparing immobilized lipase, in which a crude lipase solution is contacted with polyolefin particle.
DETAILED DESCRIPTION OF THE INVENTION
A crude lipase solution means, for example, a lipase solution which contains more than 2% by weight, preferably more than 5% by weight, particularly preferably more than 15% by weight, of impurities such as, for example, other proteins, other cellular constituents of the lipase-producing organism or residues of nutrient media. The lipase can be present in aqueous solution or else in aqueous buffer systems or in organic solvents such as, for example, in optionally halogenated aliphatic or aromatic hydrocarbons such as, for example, toluene. An aqueous crude lipase solution is preferred.
Preferred aqueous crude lipase solutions are, for example, culture broths obtained by cultivation of a lipase-producing organism in an aqueous nutrient medium, or obtainable by dispersing and/or homogenizing a lipase-producing organism or a lipase-producing cellular tissue, such as, for example, of an animal organ or of a plant, in an aqueous solvent containing, where appropriate, a buffer or other lipase-stabilizing ingredients.
The crude lipase solution is preferably purified, before contacting with the polyolefin particles, to remove cells by methods known per se, such as centrifugation or filtration.
The contacting takes place, for example, by introducing the polyolefin particles into the crude lipase solution.
When the crude lipase solution is contacted with the polyolefin particles, the lipase is adsorbed onto the polyolefin particles. It was surprising in this connection that polyolefin particles have a very high selectivity for lipases so that there is adsorption from the crude lipase solution onto the polyolefin only of the lipase and, where appropriate, its fragments and not—or only to a very small extent (usually <2% by weight)—the other proteins.
The adsorption step is thus also a step to purify the lipase from the other proteins and enzymes in the crude lipase solution, so that another purification step before the immobilization on the solid support can be omitted for the crude lipase solution. Besides simplified preparation, the immobilized lipases prepared in this way have the following advantages over prior art immobilized lipases:
The immobilized lipase has a longer useful life.
The addition of activated substances such as, for example, oleic acid is no longer necessary and brings about no increase in activity.
It is possible in principle also to use further purified crude lipase solutions for the process according to the invention. The crude lipase solution can be purified, for example, up to the point where addition of oleic acid to the immobilized lipase again brings about a jump in activity.
Suitable purification steps are all conventional processes for protein purification such as, for example, ion exchange chromatography, molecular sieve chromatography, hydrophobic chromatography and precipitation methods.
However, it is preferred to use an unpurified, cell-free crude lipase solution in the process according to the invention.
Accordingly, a preferred process for preparing immobilized lipase is one in which the crude lipase solution is a cell-free culture broth which is obtainable by
a) cultivating a lipase-producing organism,
b) where appropriate subsequently dispersing and/or homogenizing the organism in a solution and
c) subsequently removing the cells.
A lipase-producing organism means an organism which is able by nature or through genetic modification, for example by insertion of a lipase gene into the genome of the organism, to produce a lipase. Organism means microorganisms, plants and animals, as well as cellular tissue of animal or plant origin.
Preferred bacterial and fungal lipases are derived from organisms of the genus Aspergillus, Arthrobacter, Alcaligenes, Bacillus, Brevibacterium, Pseudomonas, Chromobacterium, Candida, Fusarium, Geotrichum, Humicola, Mucor, Pichia, Penicillium, Rhizomucor, Rhizopus or Thermus.
Particularly preferred bacterial and fungal lipases are lipases from the genera and species Arthrobacter sp., Alcaligenes sp.,
Aspergillus niger, Aspergillus oryzae, Bacillus cereus, Bacillus subtilis, Bacillus coagulans, Brevibacterium ammoniagenes, Burkholderia plantarii, Candida antarctica, Candida cylindracea, Candida lipolytica, Candida utilis, Candida rugosa, Chromobacterium viscosum, Fusarium solani, Geotrichum candidum, Humicola lanuginosa
, Mucor sp.,
Mucor japonicus, Mucor javanicum, Mucor miehei, Pichia miso, Rhizopus nigricans, Rhizopus oryzae, Rhizopus arrhizus
, Rhizopus sp.,
Rhizomucor miehei, Rhizopus arrhizus, Rhizopus delemar, Rhizopus niveus, Penicillium acylase, Penicillium roqueforti, Thermus aquaticus, Thermus flavus, Thermus thermophilus, Chromobacterium viscosum
, Pseudomonas sp.,
Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas cepacia, Pseudomonas burkholderia
or
Pseudomonas aeruginosa.
Preferred animal and plant lipases are pig pancreatic lipase (PPL) and wheatgerm lipase.
Particular preference is given to the lipase from
Pseudomonas burkholderia
(former name:
Burkholderia plantarii
) or
Pseudomonas aeruginosa
, and the use of
Pseudomonas burkholderia
or
Pseudomonas aeruginosa
as lipase-producing organism.
The cultivation of microorganisms or plant or animal cell cultures can take place in a manner known per se, for example by fermentation in a nutrient medium which, besides nutrients, trace elements and, where appropriate,
Friedrich Thomas
Stürmer Rainer
BASF - Aktiengesellschaft
Keil & Weinkauf
Naff David M.
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