PROCESSES FOR THE DIGESTION OF COLANIC ACID
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.
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.