Processes for the digestion of colanic acid

Abstract

The present disclosure generally relates to processes employing polypeptides having colanic acid-degrading activity. The processes generally involve contacting a biological material with a polypeptide capable of digesting colanic acid. Additional process steps, such as chromatographic separation steps, are also described.

What is claimed is: 
     
         1 . A process for digesting colanic acid in a biological material, the process comprising contacting the biological material with a polypeptide capable of digesting colanic acid. 
     
     
         2 . The process of  claim 1  wherein the biological material is a bacterial slime. 
     
     
         3 . The process of  claim 1  wherein the biological material is selected from a crude bacterial lysate, a partially purified bacterial lysate, and an aqueous solution containing extracted bacterial nucleic acid. 
     
     
         4 . The process of  claim 1  wherein the biological material is a crude bacterial lysate. 
     
     
         5 . The process of  claim 1  wherein the biological material is a partially purified bacterial lysate. 
     
     
         6 . The process of  claim 1  wherein the biological material is an aqueous solution containing extracted bacterial nucleic acid. 
     
     
         7 . The process of  claim 1  wherein the polypeptide is a recombinant polypeptide. 
     
     
         8 . The process of  claim 2  wherein the colanic acid is present in the cellular membrane of the bacteria. 
     
     
         9 . The process of  claim 1  wherein the biological material is a biofilm. 
     
     
         10 . The process of  claim 1  wherein the polypeptide comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof. 
     
     
         11 . The process of  claim 1  wherein the polypeptide comprises an amino acid sequence having at least 98% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof. 
     
     
         12 . The process of  claim 3  wherein the polypeptide is SEQ ID NO: 1. 
     
     
         13 . The process of  claim 3  wherein the polypeptide is SEQ ID NO: 2. 
     
     
         14 . A process for the removal of endotoxin from an aqueous composition containing bacterial macromolecules, the process comprising digesting colanic acid in the aqueous composition and thereafter combining the aqueous composition with a chromatographic material to separate endotoxin from the bacterial macromolecule. 
     
     
         15 . The process of  claim 14  wherein the aqueous composition is derived from a bacterial lysate. 
     
     
         16 . The process of  claim 14  wherein the bacterial lysate is pre-treated to partially remove endotoxin from the lysate prior to digesting colanic acid. 
     
     
         17 . The process of  claim 14  wherein the pre-treatment comprises combining the bacterial lysate with a chromatographic material. 
     
     
         18 . The process of  claim 14  wherein the bacterial macromolecule comprises plasmid DNA. 
     
     
         19 . The process of  claim 16 , wherein the plasmid DNA is a gram negative bacterial plasmid DNA. 
     
     
         20 . The process of  claim 14  wherein colanic acid is digested by treating the aqueous composition with a polypeptide having colanic acid-degrading activity. 
     
     
         21 . The process of  claim 14  wherein the chromatographic material is selected from the group consisting of an anion exchange chromatography resin, a cation exchange chromatography resin, a hydrophobic interaction chromatography resin, and an affinity chromatography resin. 
     
     
         22 . The process of  claim 17  wherein the chromatographic material is an anion exchange resin. 
     
     
         23 . The process of  claim 22  wherein the anion exchange resin comprises a quaternary ammonium resin. 
     
     
         24 . The process of  claim 21  wherein the affinity chromatography resin comprises a boronic acid- or boronate-based resin. 
     
     
         25 . The process of  claim 14  further comprising filtering the aqueous composition to further separate endotoxin from the biological macromolecule after eluting the biological macromolecule from the chromatography material. 
     
     
         26 . The process of  claim 14  wherein the aqueous composition is first combined with an affinity chromatography resin, and thereafter combined with a hydrophobic interaction chromatography resin. 
     
     
         27 . The process of  claim 26  wherein the affinity chromatography resin comprises a boronic acid- or boronate-based resin. 
     
     
         28 . The process of  claim 20  wherein the polypeptide is a recombinant polypeptide. 
     
     
         29 . The process of  claim 20  wherein the polypeptide comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof. 
     
     
         30 . The process of  claim 20  wherein the recombinant polypeptide comprises an amino acid sequence having at least 98% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof. 
     
     
         31 . The process of  claim 20  wherein the polypeptide is SEQ ID NO: 1. 
     
     
         32 . The process of  claim 20  wherein the polypeptide is SEQ ID NO: 2.