IP Library Granted Patent US 10,703,775
Granted Patent B2
US 10,703,775 · App. 16/080,453 · Granted Jul 7, 2020

Method of purifying antifungal compounds and exopolysaccharides from a microbial cell culture

Inventors: Chad Kimmelshue (Ames, IA); Yan Li (Citrus Heights, CA); Colleen S. Taylor (Folsom, CA); Hong Zhu (West Sacramento, CA)
Assignee: Bayer CropScience LP
C07K1/36C07K1/30C07K7/00C07K7/64C12P19/04C12P21/02
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Quick Facts
Patent No.
US 10,703,775
App. No.
16/080,453
Granted
Jul 7, 2020
Kind
B2
Abstract

The present invention provides a method of enriching a lipopeptide in a microbial cell culture, the method comprising mixing an amphiphilic sulfonate and/or an amphiphilic sulfate with the cell culture to induce the formation of aggregates containing the lipopeptide, centrifuging the cell culture to generate a supernatant fraction and a pellet fraction, separating the pellet fraction from the supernatant fraction, and mixing the pellet fraction with a polyoxyethylene glycol alkyl ether to release the lipopeptide from the aggregates. Also provided is a method of purifying an exopolysaccharide from a microbial cell culture, the method comprising mixing an amphiphilic sulfonate and/or an amphiphilic sulfate with the cell culture to induce the formation of aggregates, centrifuging the cell culture to generate a supernatant fraction and a pellet fraction, separating the supernatant fraction from the pellet fraction, adding alcohol to the supernatant fraction to precipitate the exopolysaccharide; and removing the precipitated exopolysaccharide from the supernatant fraction.

Claims (31)

1. A method of enriching a lipopeptide in a microbial cell culture, the method comprising:

a) mixing an amphiphilic sulfonate and/or an amphiphilic sulfate with the cell culture to induce the formation of aggregates containing the lipopeptide;

b) centrifuging the cell culture to generate a supernatant fraction and a pellet fraction;

c) separating the pellet fraction from the supernatant fraction; and

d) mixing the pellet fraction with a polyoxyethylene glycol alkyl ether to release the lipopeptide from the aggregates.

2. The method of claim 1 , wherein the lipopeptide is a fusaricidin-type compound selected from the group consisting of fusaricidin A, fusaricidin B, fusaricidin C, fusaricidin D, LI-F03, LI-F04, LI-F05, LI-F06, LI-F07, LI-F08, Paeniserine A1, Paeniserine A2, Paeniserine A3, Paeniserine A4, Paeniserine B1, Paeniserine B2, Paeniserine B3, Paeniserine B4, Paeniserine C1, Paeniserine C2, Paeniserine C3, Paeniserine C4, Paeniprolixin A1, Paeniprolixin A2, Paeniprolixin B1, Paeniprolixin B2, Paeniprolixin C1, Paeniprolixin C2, Paeniprolixin D1, Paeniprolixin D2, Paeniprolixin E1, Paeniprolixin E2, Paeniprolixin F1, Paeniprolixin F2, and combinations thereof.

3. The method of claim 1 , wherein an amphiphilic sulfonate is mixed with the cell culture and the amphiphilic sulfonate is a compound of formula (I):

wherein

R 1 and R 2 are independently a linear or branched C 1-20 alkyl or a linear or branched C 2-20 alkene; and

M is H + , Li + , Na + , K + or (C 1-8 alkyl) 4 N + .

4. The method of claim 3 , wherein the sulfonate is dioctyl sulfosuccinate; 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate; or a H + , Li + , Na + , K + or (C 1-8 alkyl) 4 N + salt thereof.

5. The method of claim 1 , wherein an amphiphilic sulfate is mixed with the cell culture and the amphiphilic sulfate is an alkyl sulfate of formula (II):

wherein

R 1 and R 2 are independently H, a linear or branched C 1-20 alkyl, or a linear or branched C 2-20 alkene; and

M is H + , Li + , Na + , K + or (C 1-8 alkyl) 4 N + ;

with the proviso that R 1 and R 2 are not both H.

6. The method of claim 5 , wherein the alkyl sulfate is 7-ethyl-2-methyl-4-undecanyl sulfate, 2-ethylhexyl sulfate, or a Li + , Na + , K + or (C 1-8 alkyl) 4 N + salt thereof.

7. The method of claim 1 , wherein an amphiphilic sulfonate is mixed with the cell culture and the amphiphilic sulfonate is a linear alkylbenzenesulfonate or a branched alkylbenzenesulfonate.

8. The method of claim 7 , wherein the amphiphilic sulfonate is a linear alkylbenzenesulfonate of formula (III):

wherein

R 1 and R 2 are independently H, a linear or branched C 1-20 alkyl; and

M is H + , Li + , Na + , K + or (C 1-8 alkyl) 4 N + ;

with the proviso that R 1 and R 2 are not both H.

9. The method of claim 8 , wherein the linear alkylbenzenesulfonate is dodecylbenzesulfonate or a Li + , Na + , K + or (C 1-8 alkyl) 4 N + salt thereof.

10. The method of claim 1 , wherein the polyoxyethylene glycol alkyl ether is a compound with a molecular formula of C n H 2n+1 (OCH 2 CH 2 ) m OH,

wherein

m is an integer between 1 and 120; and

n is an integer between 1 and 20.

11. The method of claim 10 , wherein n is 12 or 18 and/or m is 25 or 100.

12. The method of claim 1 , wherein the microbial cell culture comprises a strain of Paenibacillus sp., Bacillus sp., and/or Pseudomonas sp.

13. The method of claim 12 , wherein the microbial cell culture comprises Paenibacillus sp. strain NRRL B-50972, Bacillus subtilis strain NRRL B-21661, and/or a fungicidal mutant strain thereof having all the identifying characteristics of the respective strain.

Assignments (2)
CHANGE OF ADDRESS Recorded Sep 12, 2019
From: BAYER CROPSCIENCE LP
To: BAYER CROPSCIENCE LP
Reel/Frame 050370/0245 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 28, 2018
From: KIMMELSHUE, CHAD; LI, YAN; TAYLOR, COLLEEN S.; ZHU, HONG
To: BAYER CROPSCIENCE LP
Reel/Frame 046725/0809 →
Continuity (2)
Provisional Application 62303171 · Mar 3, 2016
Related Publication 20190062370A1 · Feb 28, 2019
Cited By (1)
US 12,408,673