Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an organic...
Patent
1999-02-12
2000-12-26
Naff, David M.
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
435 49, 435 51, 435176, 435177, 435195, 435816, C12N 1108, C12N 1114, C12N 914, C12P 3506, C12P 3502
Patent
active
061657581
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE 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 method 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 immobiliz
REFERENCES:
patent: 4055469 (1977-10-01), Snoke et al.
patent: 4250260 (1981-02-01), Rohrbach et al.
patent: 4931498 (1990-06-01), Pidgeon
patent: 5190864 (1993-03-01), Giese et al.
Fujisawa Pharmaceutical Co. Ltd.
Naff David M.
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