The IP.com Prior Art Database
English (United States)
16 pages / 75.0 KB
USE OF CLOUD POINT EXTRACTION
The present invention deals with extracting a polypeptide of interest from a whole or clarified fermentation broth in a very efficient way.
Surfactant based extraction for recovering enzymes is a field of active research both in academia and industry.
Phase separation using non-ionic surfactants but high amounts of salts (2 to 30 % w/v) is claimed in, e.g., US 6,105,786. Although salts are inexpensive, these processes may not be commercially attractive for large scale recovery of commodity products, primarily because of the huge environmental load such high amounts of salt would impose. Furthermore, corrosion could potentially be an issue needing an expensive material of construction.
An alternative is to induce phase separation using temperature. Traditional cloud point extraction (heating above the cloud point of non-ionic detergent with or without the aid of the centrifuge) do not work well for extracting proteins directly from fermentation broth containing high starting protein concentrations (typically obtained in industrial fermentations for commodity products). This is because of phase separation/disengagement problems due to high viscosity and low density difference of phases.
A new extraction process based on non-ionic surfactants has been developed and is described herein that is useful for recovering proteins in high yields directly from fermentation broth in a single step.
SUMMARY OF THE INVENTION
The inventors have found that it is possible to recover in high yields and simultaneously concentrate polypeptides of interest in one step directly from a fermentation broth by using the following method:
i) adjusting the pH close to pI of the polypeptide of interest;
ii) adding a non-ionic surfactant with a hydrophile-lipophile balance (HLB) of 12 or lower;
iii) cooling the mixture for solubilization and incubating at above cloud point for extraction;
iv) phase separating at below cloud point to obtain liquid-liquid-solid fractions; and
v) recovering the surfactant-rich top phase containing the polypeptide of interest.
DETAILED DISCLOSURE OF THE INVENTION
The present invention deals with extracting a polypeptide of interest from a fermentation broth; in particular from a whole or clarified fermentation broth.
When using a whole fermentation broth as the starting material for the method of the invention, the fermentation broth may or may not be diluted. Often a dilution of 0 to 100% w/w (broth basis) with water is preferred; if the dilution is higher it may result in spending energy in concentrating the product later in the downstream processing.
When using a clarified fermentation broth, the fermentation broth is typically diluted with water (0 to 100% w/w broth basis) and flocculated; the cells are separated by any conventional means such as drum filtration or centrifugation, and the clarified fermentation broth is then used as the starting material for the method of the inve...