Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-05-09
2003-10-07
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S049000, C435S051000, C435S176000, C435S177000, C435S180000, C435S816000
Reexamination Certificate
active
06630338
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a method for preparing a bioreactor. More particularly, the present invention relates to a method for purifying an enzyme by the use of a surfactant and a method for regenerating an immobilized enzyme carrier by the use of a protease.
DESCRIPTION OF THE BACKGROUND
A bioreactor is a system technology to reproduce a biochemical reaction in an artificial container, and what is meant by this term has been gradually expanding. As used herein, however, a bioreactor means a reactor wherein an enzyme itself is used as a catalyst. For this end, the enzyme should be immobilized in some way for an economical use of a catalyst. Thus, an immobilized enzyme plays the principal part of a bioreactor. One of the important factors that determine the superiority of an immobilized enzyme is the purity of the enzyme used.
An enzyme protein is purified from a biological sample or cell culture supernatant by appropriately combining the conventionally-known various protein separation techniques according to the properties of the objective enzyme and contaminant protein. At present, predominant separation techniques include, for example, a method utilizing difference in solubilities, such as salting out, solvent precipitation method and the like; a method utilizing difference in molecular weights, such as dialysis, ultrafiltration, gel filtration, polyacrylamide electrophoresis and the like; a method utilizing electric charges, such as ion exchange chromatography and the like; a method utilizing specific affinity, such as affinity chromatography and the like; a method utilizing difference in hydrophobicities, such as reversed phase high performance liquid chromatography and the like; and a method utilizing difference in isoelectric points, such as isoelectric point electrophoresis and the like.
In not a few cases in practice, however, contaminant protein cannot be removed completely from the objective enzyme. This is attributable to the absence of noticeable difference between the objective enzyme and contaminant protein in various physico-chemical properties that the conventional protein separation methods utilize. For example, cephalosporin C acylase derived from Pseudomonas [enzyme that converts cephalosporin C and glutaryl 7-aminocephalosporanic acid (GL7-ACA) to 7-aminocephalosporanic acid (7-ACA); hereinafter to be abbreviated as CC acylase] can be purified up to an approximately 95% purity by repeatedly separating a crude cell extract solution as a starting material by steps such as dialysis, ammonium sulfate fractionation, anion exchange chromatography and the like (Japanese Patent Unexamined Publication No. 5-84078). The contaminant deacetylase, however, cannot be removed by a conventional method because it shows nearly the same behavior on anion exchange resin column as CC acylase. A deacetylase deactylates 7-ACA to produce deacetyl 7-ACA, causing less yield of 7-ACA. Thus, there remains a strong demand for a novel enzyme purification method capable of separating and removing such undesirable proteins.
Another factor determining the superiority of an immobilized enzyme is the life of an immobilized enzyme carrier. In general terms, enzymes tend to be unstable to heat, strong acid, strong alkali, organic solvent and the like, and easily lose activity even under the conditions preferable for enzyme reactions. An immobilized enzyme shows decreasing enzyme activities with the repeated use thereof, thereby lowering the production efficiency of the objective substance. A degraded immobilized enzyme is generally disposed, but when an ion exchange resin is used as a carrier, a recycled use of the carrier is desirable from environmental and economical considerations.
Conventional methods for regenerating an immobilized enzyme carrier include use of a strong acid or strong alkali to liberate and remove the enzyme from the carrier. However, the enzyme cannot be removed completely from the carrier by this method, which causes drastic decrease in the activity of the immobilized enzyme upon repeated regenerations of carrier and reimmobilizations of enzyme. Particularly when the carrier has fine pores, the enzyme clogs in the fine pores, resulting in appreciable degradation due to regeneration and reimmobilization.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method for selectively separating and removing a contaminant enzyme which is undesirable for the objective enzyme and which cannot be removed by conventional separation techniques, thereby to enable production of a highly pure objective enzyme. Another object of the present invention is to provide a method for regenerating an immobilized enzyme carrier, which is capable of efficiently removing an enzyme from a carrier.
The present inventors have conducted intensive studies in an attempt to achieve the above-mentioned objects and had a conception to utilize selective aggregation and precipitation of protein caused by surfactants. Thus, they investigated from various aspects using the system of CC acylase solutions contaminated by deacetylase. The results revealed that the addition of a surfactant, particularly a cationic surfactant, leads to selective aggregation and precipitation of deacetylase, and using this action, the present inventors have succeeded in preparing a standard CC acylase product having a high purity and a high production efficiency of 7-ACA.
The present inventors have also succeeded in removing CC acylase efficiently by allowing a protease to act on an immobilized enzyme obtained by adsorbing CC acylase onto an ion exchange resin carrier and crosslinking CC acylase with glutaraldehyde. Moreover, they have found that, in case of an immobilized enzyme using the above-mentioned enzyme and crosslinking agent, the use of an acidic protease is particularly effective for the removal of enzymes, which resulted in the completion of the present invention.
Accordingly, the present invention provides a method for purifying an enzyme, comprising causing selective aggregation and precipitation of contaminant enzyme by the use of a surfactant. The present invention also provides the above-mentioned method for purifying an enzyme, particularly acylase, wherein the surfactant is cationic. The present invention also provides a method for purifying the above-mentioned acylase, wherein the acylase is cephalosporin C acylase and the contaminant enzyme is deacetylase. The present invention further provides a method for purifying the above-mentioned acylase, wherein the surfactant is a methyl type or benzyl type cationic surfactant, particularly, alkyl(palm)dimethylbenzyl ammonium chloride. In addition, the present invention provides a method for purifying the above-mentioned acylase, wherein the cationic surfactant is used in a concentration of 0.1-0.6%.
The present invention moreover provides a method for regenerating an immobilized enzyme carrier, comprising allowing a protease to act on an immobilized enzyme to remove the enzyme from the carrier, said immobilized enzyme being prepared by binding the enzyme with the carrier, such as a synthetic adsorbent and an ion exchange resin, and optionally crosslinking the enzymes by the use of a crosslinking agent after binding. Particularly, the present invention provides a method for regenerating the above-mentioned carrier, wherein said carrier has fine pores. Moreover, the present invention provides a method for regenerating the above-mentioned carrier, wherein the enzyme is CC acylase. Furthermore, the present invention provides a method for regenerating the above-mentioned carrier, wherein said protease is an acidic protease.
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patent: 4055469 (1977-10-01), Snoke et al.
patent: 4250260 (1981-02-01), Rohrbach et al.
patent: 4634673 (1987-01-01), Johnson et al.
patent: 4931498 (1990-06-01), Pidgeon
patent: 5190864 (1993-03-01), Giese et al.
patent: 0 475 652 (1992-03-01), None
“Separation and Purification of
Fujisawa Pharmaceutical Co. Ltd.
Naff David M.
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