Whole cell and cell-debris polysaccharide

Details Whole cell and cell-debris polysaccharide Whole cell and cell-debris polysaccharide

2001-04-19

2003-08-05

Prats, Francisco (Department: 1651)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S101000, C435S084000, C435S072000

Reexamination Certificate

active

06602997

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a method of freeing a polysaccharide from a bacterial culture and the bacterial culture free polysaccharide obtained by the method. Specifically, the present invention relates to a method of freeing polysaccharide S-7 from a bacterial culture and the bacterial culture free polysaccharide S-7 obtained by the method.
Polysaccharide S-7 (hereinafter referred to as “S-7”) was initially described as a polymer secreted into a culture broth by a bacterium designated as
Azotobacter indicus
var.
myxogenes
which was deposited at the American Type Culture Collection as ATCC21423. See U.S. Pat. No. 3,960,832 issued to Kang et al. on Jun. 1, 1976 which discloses a single composition of matter and U.S. Pat. No. 3,915,800 issued to Kang et al. on Oct. 28, 1975 which discloses the growth of the naturally occurring bacterial strain
Azotobacter indicus
in a submerged aerated culture in a nutrient medium and the recovery of the polysaccharide. The bacterial strain ATCC21423 was recently cultured and it was determined that the bacteria in the culture belonged to the genus Sphingomonas. See Pollock, T. J., “Gellan-related Polysaccharides and the Genus Sphingomonas,”
Journal of General Microbiology
, vol. 139, pp. 1939-1945 (1993)). This culture is referred to as “Sphingomonas strain S7.”
S-7 is the subject of expired U.S. Pat. No. 3,894,976 issued to Kang et al. on Jul. 15, 1975 which discloses the use of S-7 in water based paints and U.S. Pat. No. 3,979,303 issued to Kang et al. on Sep. 7, 1976 which discloses the use of S-7 in oil well drilling. In addition, U.S. Pat. No. 5,772,912 issued to Lockyer et al. on Jun. 30, 1998 discloses the use of S-7 in anti-icing formulations and U.S. Pat. No. 4,462,836 issued to Baker et al. on Jul. 31, 1984 discloses the use of S-7 in cement.
Furthermore, published literature concerning this polysaccharide includes a 1977 review by the inventors of the Kang et al. patents which is based on the information in their published patents (See Kang, K. S. and W. H. McNeely, “A New Bacterial Heteropolysaccharide, In Extracellular Microbial Polysaccharides,”
American Chemical Society
, pp. 220-230 (1977)), and two brief studies by others concerning culture conditions for growing the bacterium ATCC21423 (See Lee, J. W., W. G. Yeomans, A. L. Allen, R. A. Gross, and D. L. Kaplan, “Compositional Consistency of a Heteropolysaccharide-7 Produced by
Beijerinckia indica,” Biotechnology Letters,
19 (1997); and Naumov, G. N., I. G. Multykh, and T. P. Shamrina, “Optimal Nitrogen and Phosphorous Concentrations in the Growth Medium for Exopolysaccharide Biosynthesis by
Beijerinckia indica,” Mikrobiologiya
, pp. 856-857 (1985)).
All of the above mentioned patents and references disclose methods for making S-7 which is contaminated with cell debris and protein from the culture that produced the polymer. Since the S-7 polymer is secreted from the cell and remains attached to the producing cell, precipitation with any of a variety of alcohols leads to the co-precipitation of the S-7 polymer with cells, cell debris, and proteins which are present in the culture broth. After precipitation and removal of the precipitate from the solution, the aqueous solvent is transparent since all of the cells co-precipitate with the S-7 polymer. After the contaminated precipitate is recovered, dried, milled, and resuspended in liquid, the resulting viscous solution is contaminated with cells, cell debris and proteins, and has an opaque or non-transparent appearance. For certain applications, a clear viscous solution is required which lacks the particulate contaminants.
The prior art referred to above describes useful properties of the non-purified polysaccharide S-7. In particular, S-7 is soluble in hot or cold water giving a homogenous viscous mixture. The viscosity of an aqueous suspension of S-7 is highly pseudoplastic and becomes increasingly viscous at low rates of shear or at rest. The high viscosity at low shear rates makes S-7 an effective suspension agent for solids. The low viscosity at high shear rates allows the S-7 polymer solution to be transported as by pumping. The magnitude of pseudoplasticity exceeds that of xanthan gum. For shear rates between 1-10 sec
−1
, a solution of S-7 is about 3-5 times more viscous than a comparable solution of xanthan gum. The aqueous viscosity is relatively constant over a wide range of pH, temperature, and salt concentration. The viscosity is compatible with commonly encountered mono-, di- and tri-valent metal ions. However, an aqueous solution of S-7 will form a gel in the presence of di- and tri-valent metal ions if the pH is raised to 9.5-10.5, and the gel is maintained when the pH is returned to neutrality.
Polysaccharides such as S-7 have several applications, for example, as a thickener, suspending agent and stabilizer. In addition, S-7 can be used to modify the viscosity of aqueous solutions. As polysaccharides such as S-7 have several applications, it is one of the purposes of the present invention to provide polysaccharides free from contamination with cell debris and protein which could be even further effective.
SUMMARY OF THE INVENTION
Accordingly, we have discovered a novel method for obtaining a polysaccharide containing reduced amounts of contaminating cellular debris and protein. Specifically, the present invention provides for a method for obtaining a polysaccharide substantially free from whole bacterial cells and bacterial cell debris including:
(a) fermenting a Sphingomonas bacterium, which produces the polysaccharide, to produce an aqueous fermentation having the polysaccharide dissolved therein;
(b) diluting the fermentation broth obtained with an equal volume of deionized water;
(c) partially hydrolyzing the polysaccharide by exposing the diluted fermentation broth to a temperature in excess of 100° C. for a time period from about ten minutes to about one hour;
(d) removing bacterial cells from the partially hydrolyzed polysaccharide by centrifugation of the heated fermentation broth and recovering a supernatant aqueous liquid;
(e) precipitating the polysaccharide from the supernatant aqueous liquid by adding a second liquid thereto, the second liquid being miscible with water, non-reactive with the polysaccharide, and a non-solvent for the polysaccharide; and
(f) harvesting the precipitated polysaccharide by separating it from the supernatant aqueous liquid.
The bacterium is preferably Sphingomonas strain S7 or a Sphingomonas bacterium modified with a S7c6 gene cluster or segment including at least the spsB and rhsACBD genes. Also, the fermentation broth preferably has a pH of about 6.
Furthermore, the method preferably includes a step of digesting contaminating cellular material by incubating the fermentation broth resulting from step (b) with at least one protease enzyme. The protease enzyme can be selected from the group consisting of Bioprase, MULTIFECT®, Protex 6L and proteinase K.
In addition, step (c) of the above method preferably includes autoclaving the diluted fermentation broth resulting from step (b) at a temperature in excess of 110° C. for about 10 to about 30 minutes. More preferably, step (c) includes autoclaving the diluted fermentation broth resulting from step (b) at a temperature of about 121° C. for about 15 minutes.
Also, it is preferable in step (d) of the above method that the bacterial cells are removed from the partially hydrolyzed polysaccharide in the fermentation broth by at least two centrifugation steps and the supernatant aqueous liquid recovered from each centrifugation step is used in the subsequent centrifugation step.
Furthermore, step (e) in the above method is preferably carried out at room temperature. In addition, the second liquid in step (e) can be selected from the group consisting of branched chain lower alkanols and lower alkyl ketones. Alternatively, the second liquid can be selected from the group consisting of methanol, ethanol, isopropanol, butanol, t-butanol, isobutanol, amyl alcohol and acetone.
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