One-pot enzymatic sulfation process using 3'-phosphoadenosine-5'-phosphosulfate and recycled phosphorylated adenosine intermediates

• US 5,919,673 A
• Filed: 03/22/1995
• Issued: 07/06/1999
• Est. Priority Date: 03/22/1995
• Status: Expired due to Term

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Lin, Chun-Hung, Wong, Chi-Huey, Shen, Gwo-Jenn
• Primary Examiner(s): Rollins, John W.
• Assistant Examiner(s): PRATS, FRANCISCO CHANDLER
• Application Number:
  US08/408,774
• Time in Patent Office: 1,567 Days
• Field of Search: 435/130, 435/101, 435/52, 435/92
• US Class Current: 435/130
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/13 transferring sulfur containing groups (2.8)
• CPC Class Codes: C12P 11/00 Preparation of sulfur-containing organic compounds
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12P 19/30 Nucleotides

Abstract

A process for the enzymatic synthase of sulfate esters is disclosed in which intermediate phosphorylated adenosine compounds are recycled to minimize enzyme inhibition. Exemplary enzymes include ATP sulfurylase, APS kinase and a sulfotransferase.

Claims

1. A process for using 3'"'"'-phosphoadenine-5'"'"'-phosphosulfate (PAPS) in an enzyme-catalyzed sulfation of an acceptor with recycling of phosphorylated adenosine intermediates that comprises the steps of:

(a) admixing the following ingredients in an aqueous medium containing magnesium and potassium ions within a single vessel to form an aqueous reaction medium

(i) 3'"'"'-nucleotidase or 3'"'"'(2'"'"'),5'"'"'-bisphosphate nucleotidase;

(ii) ATP sulfurylase;

(iii) APS kinase;

(iv) pyrophosphorylase;

(v) a sulfotransferase;

(vi) at least one adenine-containing compound selected from the group consisting of ATP, ADP, AMP, APS, PAPS and PAP;

(vii) sulfate ion;

(viii) an ATP-regenerating system comprising a phosphate donor and a phosphorylating enzyme; and

(ix) a sulfate acceptor for said sulfotransferase

the concentration of said sulfate ion being greater than the concentration of all of said adenine-containing compound, and the activity of said 3'"'"'-nucleotidase being less than that of the enzymes of (ii)-(v); and

maintaining said aqueous reaction medium at a pH value of about 5 to about 10 at a temperature of about zero degrees C to about 40°

C. for a time period sufficient for said acceptor to be sulfated.

2. The process according to claim 1 including the further step of recovering the sulfated acceptor.

3. The process according to claim 1 wherein said ATP-regenerating system comprises myokinase, pyruvate kinase and phospho(enol)pyruvate.

4. The process according to claim 1 wherein said sulfotransferase is chondroitin transferase.

5. The process according to claim 1 wherein said sulfotransferase is hydroxysteroid sulfotransferase.

6. The process according to claim 1 wherein said sulfotransferase is NodH sulfotransferase.