# US 5,352,601 A · Method for recovering purified 52,000 dalton fraction of human pancreatic cholesterol esterase using deae-cellulose, hydroxyapatite and heparin-sepharose

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- Number: US 5,352,601 A
- Document ID: US5352601A
- Status: Granted
- Kind: A
- Application number: 07/936,103
- Filed: 1992-08-26
- Granted: 1994-10-04
- Earliest claimed: 1988-03-15
- Assignees: Louis G. Lange, III
- Inventors: Louis G. Lange, III; Curtis A. Spilburg; Paula M. Kinnunen
- Web page: https://patentgrep.com/patent/US5352601A

## CPC

- A61K 31/715: Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K 38/51: Lyases (4)
- C12N 9/18: Carboxylic ester hydrolases {(3.1.1)}
- Y10S 435/814: Enzyme separation or purification
- Y10S 435/815: by sorption
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

A method for recovering a purified 52,000 dalton fraction of human pancreatic cholesterol esterase is disclosed. The 52,000 dalton fraction of human pancreatic cholesterol esterase is purified by passing a solution having a pH of about 8.2 which contains two or more fractions of human pancreatic cholesterol esterase including the 52,000 dalton fraction over DEAE-cellulose to produce an effluent containing said 52,000 dalton fraction. Since the 52,000 dalton fraction of the esterase does not bind to the DEAE-cellulose whereas the other esterase fractions do bind to the DEAE-cellulose, the 52,000 dalton fraction is then collected in the effluent. The method can be further carried out by passing the purified 52,000 dalton fraction over both hydroxyapatite and heparin-Sepharose to obtain a homogeneous solution of the 52,000 dalton fraction.

## Claims

### Claim 1

A method for recovering a purified 52,000 dalton molecular weight fraction of a human pancreatic cholesterol esterase comprising passing a solution having a pH of about 8.2 and containing two or more molecular weight fractions of human pancreatic cholesterol esterase including a 52,000 dalton molecular weight fraction over DEAE-cellulose to produce and effluent containing said 52,000 dalton fraction of the esterase, said 52,000 dalton fraction of the esterase not binding to the DEAE-cellulose and the other esterase fractions binding to the DEAE-cellulose, and collecting the 52,000 molecular weight dalton fraction of human pancreatic cholesterol esterase in the effluent.

### Claim 2

A method for producing a purified 52,000 dalton molecular weight fraction of human pancreatic cholesterol esterase comprising the steps of:
(a) passing a dialyzed solution of human pancreatic cholesterol esterase over DEAE-cellulose to produce and effluent containing said 52,000 dalton fraction of the esterase, said 52,000 dalton fraction of the esterase not binding to the DEAE-cellulose and the other esterase fractions binding to the DEAE-cellulose;
(b) collecting liquid that passes through the DEAE-cellulose; and
(c) directing the liquid that passes through the DEAE-cellulose over a hydroxyapatite column and then over a heparin-Sepharose column to produce a homogeneous solution of a 52,000 dalton molecular weight human pancreatic cholesterol esterase.