Method of purifying enzymes using boronic acids covalently bonded to an insoluble support

• US 3,912,595 A
• Filed: 02/25/1974
• Issued: 10/14/1975
• Est. Priority Date: 02/25/1974
• Status: Expired due to Term

• Current Assignee: Manfred H.W. Philipp, Myron L. Bender, Pablo V. Valenzuela
• Original Assignee: Manfred H.W. Philipp, Myron L. Bender, Pablo V. Valenzuela
• Inventors: Philipp, Manfred H. W., Bender, Myron L., Valenzuela, Pablo V.
• Primary Examiner(s): Shapiro, Lionel M.
• Application Number:
  US05/445,665
• Time in Patent Office: 596 Days
• Field of Search: 195/66R,63,68,DIG.11
• US Class Current: 435/197
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12N 9/50 Proteinases , e.g. Endopeptidases (3.4.21-3.4.25)
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12N 9/6427 Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
• CPC Class Codes: Y10S 435/815 by sorption

Abstract

Soluble serine-hydrolytic enzymes are purified by passing a solution at pH 7.5 through a column packed with a granular support to which is covalently bonded a boronic acid to reversibly complex the enzyme; washing the column with a first buffer solution; freeing the enzyme by washing the column with a second buffer solution; and isolating the freed enzyme.

Claims

1. A METHOD FOR THE PURIFICATION OF A SOLUBLE SERINEHYDROLYTIC ENZYME CONSISTING OF PASSING AN AQUEOUS SOLUTION OF THE SOLUBLE SERINE-HYDROLYTIC ENZYME AT PH FROM 7.0 TO 8.5 THROUGH A COLUMN PACKED WITH A GRANULAR WATER-INSOLUBLE SUPPORT TO WHICH IS COVALENTLY BONDED A BORONIC ACID GROUP ATTACHED BY AN ALKYL HYDROCARBON RESIDUE HAVING AT LEAST ONE ADDITIONAL FUNCTIONAL GROUP CAPABLE OF FORMING A COVALENT BOND WITH THE GRANULAR WATER-INSOLUBLE SUPPORT TO REVERSIBLY COMPLEX SAID ENZYME.

2. The method of claim 1, wherein said enzyme is chymotrypsin.

3. The method of claim 1, wherein said enzyme is subtilisin.

4. The method of claim 1, wherein said enzyme is cholinesterase.

5. The method of claim 1, wherein said boronic acid group is m-aminophenylboronic acid.

6. The method of claim 1, wherein said granular water-insoluble support is a higher carbohydrate.

7. The method of claim 1, wherein said second buffer solution is at a pH different from the pH of the first buffer solution.

8. The method of claim 1, wherein said boronic acid group is covalently bonded to a higher carbohydrate water-insoluble support material by treating the higher carbohydrate material in sequence with cyanogen bromide, an oligopeptide, and a water-soluble carbodiimide and an aminophenylboronic acid.