Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom

Kern, Roger G.

Petersen, Gene R.

Richards, Gil F.

NASA Technical Reports Server

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United States Patent 
Kern et al. 
[54] METHOD FOR PRODUCING CAPSULAR 
[75] Inventors: Roger G. Kern, Pasadena; Gene R. 
Petersen, Pomona; Gil F. Richards, 
Duarte, all of Calif. 
(JPL), Pasadena, Calif. 
POLYSACCHARIDES 
[73] Assignee: Wornia Institute of Technology 
[21] Appl. No.: 799,419 
[22] Filed: Nor. 27,1991 
[51] Int. C l . 5  .............................................. C12P 19/04 
[52] U.S. Cl. .................................................... 435/101 
[58] Field of Scuch ......................................... 435/101 
1561 References Cited 
Tang et al., BiologicuZAbstructs, vol. 90(8): #86261, Oct. 
15, 1990. 
Dutton et al., Curbohydrute Research, vol. 103 (1982), 
“Industrial Applications of Some New Microbial Poly- 
saccharides” By J. K. Baird, et al. 1983 pp. 778-783. 
“Degradation of Bacterial Surface Carbohydrates By 
Virus-Associated Enzymes” By Hildegard Geyer, et al. 
“Turbulent-Flow Properties of Polysaccharide Solu- 
tions’’ By J. W. Hoyt, 1968, pp. 207-215. 
“Drag-Reduction Effectiveness of Polymer Solutions 
PUBLICATIONS 
pp. 107-128. 
1983-pp. 637-653. 
US005330903A 
pi] Patent Number: 5,330,903 
[45] Date of Patent: Jul. 19, 1994 
in the Turbulent-Flow Rheometer: A Catalog” By J. 
“Drag Reduction in Polysaccharide Solutions” By: J. 
“Yeasts Producing Exopolysaccharides with Drag-Re- 
ducing Activity” By G. R. Petersen, et al. 1990, pp. 
“Rheologically Interesting Polysaccharides From 
Yeasts” By Gene R. Petersen, et al. 1989 pp. 845-867. 
“Structural Modification of Polysaccharides, A Bio- 
chemical-Genetic Approach” By: Roger G. Kern and 
Gene R. Petersen, Dec. 1991 pp. 73-78. 
“Experiments in Molecular Genetics” By Jeffrey H. 
Miller, 1972, pp. 121-129. 
Primary Examiner-Ronald W. Griffin 
Attorney, Agent, or Firm-Graham & James 
Structurally altered capsular polysaccharides are pro- 
duced by mutant bacteria. These polysaccharides are 
isolated by selecting a wild type bacterial strain and a 
phage producing degradative enzymes that have sub- 
strate specificity for the capsular polysaccharides pro- 
duced by the wild type bacteria. Phage-resistant mu- 
tants producing capsular polysaccharides are selected 
and the structurally altered capsular polysaccharide is 
isolated therefrom. 
W. HOP, 1971, pp. 851-862. 
W. Hoyt, 1985, pp. 17-21. 
255-259. 
t571 ABSTRACT 
9 Claims, No Drawings 
https://ntrs.nasa.gov/search.jsp?R=20080012416 2019-08-30T04:01:34+00:00Z
5,330,903 
1 2 
polysaccharides because of the purity and uniformity of 
the product. 
Bacteriophage are viruses that infect bacteria. The 
viruses that infect bacteria encapsulated by a glycoca- 
The invention described herein was made in the per- 5 lyx are specially adapted to penetrate the polysaccha- 
formance of work under a NASA contract, and is sub- ride barrier. The tail fibers or spikes by which these 
ject to the provisions of Public Law 96-517 (35 U.S.C. viruses are seen to attach to bacteria contain en- 
§ 202) in which the Contractor has elected to retain zymes-generally referred to as endoglycanases or en- 
title. dopolysaccharases-that degrade the glycocalyx. 
This invention relates to novel and useful complex 10 These enzymes exhibit a high degree of substrate speci- 
polysaccharides and methods for producing them. ficity- GeYer et al, Supra at PP. 38-39. 
More particularly, this invention relates to structurally According to the Present invention, a Strain of encap- 
altered capsular polysaccharides produced by bacterial sulated bacteria and the specific phage which infects it 
mutants. are employed. The selected bacteria are optionally ex- 
15 posed to a mutagenic agent. Examples of useful muta- 
genic agents include radiation and chemical mutagens, 
METHOD FOR PRODUCING CAPSULAR 
POLY SACCHARIDES 
BACKGROUND OF THE INVENTION 
applications. Generally these molecules alter the physi- 
d properties-including viscosity and rheo1ogy-f 
complex polysaccharides me used as thickenen in the 
Complex polywc&ds have a variety of industrial such Bs nitrosoguanidine. The mUtageXlhd bacterial 
are grown to a desired density* and then 
for resistance to phage that specifically degrade the 
are mely to have a glycocalyx with altered Chemical 
fluid media in which they me suspended. Accordingly, 20 g l y ~ a l y x  of the wild type strain. surviving bacteria 
food industry, for waste fragments in d d -  
cants. Baird et al., “Industrial Applications Of Some 
vember 1983, pp. 778-83. For similar reasons, complex 
polysaccharides are also used to reduce turbulence of 
. fluid flow in pipelines and other containers. J. W. Hoyt, 
structure. The CPS of the selected bacterial strains is 
lated CPS is purified by known techniques and de- 
enzymes. 
In one embodiment of the present invention, we em- 
ployed a strain Klebsiella pneumoniae that produces a 
capsular known as K63, and a phage 
5845/52 from the World Health Organization. 
strain produces the K63 capsular protein. The K63 cps 
ling or cutting operations, and as Water soluble lubri- 
New Microbial Polysaccharides,” Biotechnology, No- 25 graded to Oligomer by further treatment with phage 
harvested with enzymes derived from phage* The 
Drag Reduction In Polysaccharide Solutions, Trends In 3o (“K63 that specifically degrades the Same poly- Bio techno lo~~  37 17-20 (1985)‘ The physico-chemical saccharide. We obtained Klebsiella pneumoni~e strain basis for these effects is not understood, id. although 
correlations between drag reduction and the extensional 
viscosity Of plysaccharides have been G. R. is a straight chain heteropolymer of fucose, galacuronic 
35 acid and galactose, linked, in that order by a 1-3 glyco- et al., “Rheologically Interesting 
rides From Yeasts”, Appl. Biochem. Biotech., 20/21, sidic linkages. The ~ 6 3  phage enzymatically hydro- 
845-67 (1989). lyzes the a 1-3 linkage between the galactose and fu- 
cose monomers. Both the nature of the CPS polymer 
plex polysaccharides is not understood, the identifica- and the mode of action of the K63 phage enzyme are 
tion of useful complex polysaccharides requires screen- 40 representative, and support the general applicability of 
ing of candidate polymers. Accordingly, it is desirable our method. 
to develop methods for Producing new candidate corn- We selected mutants for further study that were ei- 
plex polysaccharides for evaluation. ther spontaneously generated or were induced by expo- 
sure to the mutagenic agents nitrosoguanidine or ultra- 
45 violet radiation. J. H. Miller, Experiments In Molecular SUMMARY OF THE INVENTION 
Structurally altered capsular polysaccharides are Genetics (1 972). Following treatment to induce muta- 
Produced by mutant bacteria. These PolYsaccharideS tion, cells were grown on DIFCO Nutrient Broth, sup- 
are isolated by selecting a wild type bacterial Strain and plemented with 1% glucose, at 30’ C., while rotated at 
a phage producing degradative enzymes that have sub- 150 rpm. Cells were grown to early stationary phase 
strate specificity for the capsular polysaccharides pro- 50 (OD590 of approximately 3.5). Calcium chloride (0.1 M) 
duced by the wild type bacteria. Phage-resistant mu- was added to stabilize the cps matrix. 
tants producing capsular polysaccharides are selected The fresh stationary phase culture, less than hours 
and the structurally altered capsular polysaccharide is old, contained approximately 2.5 X 109 viable cells/ml. 
isolated therefrom. Phage 63 was added at a concentration sufficient to 
55 achieve a multiplicity of infection (“MOI”) of between 
one and five. The culture was incubated at room tem- DETAILED DESCRIPTION OF THE 
perature for about 10 minutes to permit phage to attach INVENTION 
The cells of many bacterial species-specifically, to the bacterial cells. The infected culture was then 
gram positive bacteria-are surrounded by a gl ycocalyx 
also referred to as a capsular polysaccharide (“CPS’) 60 
several hundred nanometers in thickness. The glycoca- 
lyx consists of a complex polysaccharide polymer that 
appears to be species specific. H. Geyer et al. “Degrada- 
tion Of Bacterial Surface Carbohydrates By Virus- 
Associated Enzymes”, Pure And Applied Chemistq 55, 65 
pp. 637-53 (1983). Hence bacteria have proved to be an 
important source of complex polysaccharides. Baird et 
al. (1983). Bacteria are an especially desirable source of 
Because the basis for these properties Of 
diluted 10 fold into fresh glucose supplemented nutrient 
broth and grown overnight to stationary phase. 
Phage resistant mutants were isolated as single colo- 
nies on petri dishes after streaking with the overnight 
culture of infected bacteria. Resistant colonies that did 
not form plaques were selected. Phage resistance of the 
selected colonies was confirmed by administration of a 
drop of phage/enzyme concentrate. The zone of lysis 
produced by the diffusion of the phage/enzyme concen- 
trate was dramatically reduced for those strains with 
5,330,903 
3 4 
phage resistance when compared with wild type bac- 100 ml and all measurements were made with water as 
teria. the solvent. Sample observations were normalized 
Mutants whose phage re&ance was due to altered (wt/ppm or mg/l) typically 100-500 wt/ppm. Our 
cps were selected from among those Phage resistant observations were reproducible to within 1-2%. We 
mutants already identified. The phage resistant colonies 5 considered measurements that differed by less than 5% 
were grown for 5 to 7 days so that mucoid (CPS pro- to be 
tinguished. The colonies were retested for phage resis- wild type and mutant cps (from 8 mutant strains) are 
tance as before. Mucoid mutants that remained phage in Table 1. Mutant strains 1, and were resistant were assumed to have altered CPS. Non- io spontaneous while strains 3, 4, 5, 6, and 8 were mucoid phage resistant mutants were assumed to be induced by nitrosoguanidine. All mutants showed a resistant for other reasons, for example, lacking virus 
receptor sites. Approximately 90 % of the 5845/52 decrease in ppvulation while altered NMR spectra 
rion. 15 Our results show a 10% improvement in drag reduction 
In order to test the capacity of the altered CPS from in the cps from mutant bacteria. as also 
mutant cells to affect rheological properties of aqueous shown in Table 1, cps from mutant bacteria is more 
media, we partially purified the cps. The cultwed resistant to mechanical shearing BS determined by re- 
mutant bacterial cells, like those produced by large- peated observations of drag reduction with a rheome- 
scale industrial fermentations, is resistant to high speed 20 ter. 
TABLE 1 centrifugation. We succeeded in achieving substantial 
purification of the altered CPS using a concentrated 
Characterization of K63 CPS Mutants: extract of phage enzymes. 
Drag Reduction Raults A concentrated mixture of CPS specific enzyme and 
phage fragments was prepared by preparing a phage 25 
lysate from stationary phase cultures, and concentrating 
the lysate 80 fold with an AMICON hollow fiber filter 
ducing) and non-mucoid mutant C&nies could be dis- Our observations of drag reduction coefficients for 
phage resistant mutants have altered CPS by this trite- have been detected only in the spontaneous mutants. 
% SHEAR % DRAG 
K 63 wt 55.9 84.9 
K63-JPL1 67.7 95.6 
(HPIIO), followed by dialysis to remove low molecular K63-JPL2 66.7 95.6 
39. This material showed a 24-fold enhancement in 30 K63-JPL4 67.6 98.2 
K63-JPL6 67.6 95.7 
K63-JPL8 67.2 99.7 
REDUCTION STABILITY 
weight substances. The final concentration factor was K63-JPL3 67.0 99.4 
enzymatic activity as determined by a reducing end K63-JPLS 67.0 99.4 
sugar assay, as compared with crude phage lysate. 
Phage/enzyme concentrate was added in small quan- 
tities-1 mlfliter-to cultures of mutant bacterial cells. 
This results in a rapid and dramatic reduction (> 100 35 While we have presented a number of embodiments 
fold) in the viscosity of the culture media. Altered CPS of this invention by way of examples, it is apparent that 
was thereafter separated from the bacterial cells by low our basic construction can be altered to provide other 
speed centrifugation at 20" C. Immediately after centrif- embodiments that also employ our invention. The scope 
ugation, the supernatant was precipitated by the addi- of our invention is defmed by the scope of the claims, 
tion of 4 volumes of isopropanol at -20" C. We have 40 rather than by the examples, which are only intended to 
also used cold ethanol in place of cold isopropanol. The be illustrative. 
resulting precipitate was rehydrated and extensively We claim: 
dialyzed against deionized water. 1. A method for producing an isolated capsular poly- 
The CPS polymer obtained from wild-type K63 bac- 
(a) providing a wild type bacterial strain that pro- 
daltons. Digestion of this material with phage 63 en- duces a capsular polysaccharide and a bacterio- 
zyme reproducibly yielded a homogeneous mono-dis- phage that degrades at least one capsular polysac- 
charide produced by the wild type bacterial strain; perse polysaccharide with a molecular weight of 
(b) selecting phage resistant mutants of the bacterial 9.Ox 105 daltons. 
gested CPS harvested from wild type and mutant K63 
bacteria using a turbulent flow rheometer fabricated (') propagating the mutants to Obtain a culture 
according to the design of Hoyt [J. W. Hop, Polymer 
erties Of natural Polymers", The Chemical Society Spe- 55 
cia1 Publication, No. 23, (London) pp. 86-91 (1968)] 
modified by altering pipe diameter to operate at a fmed 
Reynolds number of 16,500. The drag reduction was 
calculated: 
saccharide comprising the steps of: 
had and apparent molecular weight of 3.5 x 106 45 
We measured the drag reduction produced by undi- 50 strain that produce a 
thereof; and; 
Of the mutants. 
Letts, 9, 851-62 (1971); J. W. Hop, "The solution Prop- (d) harvesting the altering polysaccharides 
2. The method for producing an capsular 
polysaccharide of claim 1, wherein the wild type back- 
rid Strain is Klebsiella PneUmOniUe 5845/82. 
3. The method for producing an isolated capsular 
polysaccharide of claim 1 wherein the capsular polysac- 
chaiide is K63. 
4. The method for producing an isolated capsular 
polysaccharide of claim 1 wherein the bacteriophage is 
phage K63. 
5. The method for producing an isolated capsular 
polysaccharide of claim 1 wherein the step of selecting 
mutants comprises the step of treating the selected wild 
type bacterial strain with a mutagenic agent. 
Drag Reduction (%) = 100 X (PDw/wnr - PDSo/urion)/P&/wnr 
Where PDsoivenr and PDmiu!jon are the pressure differ- 
ences in the solvent and solution, respectively, mea- 
sured from a strip chart recording of pressures taken at 65 
two points, 12.7 cm apart, along a 1 mm pipe. Between 
sample runs, the rheometer was rinsed with tap water 
until the baseline returned to normal. Sample size was 
5 
5,330,903 
6 
6. The method for producing an isolated capsular 
polysaccharide of claim 5 wherein the mutagenic agent 
is radiation of nitrosoguanidine. 
polysaccharide of claim 1 wherein the step of selecting 
phage resistant mutants further comprises the steps of: 
(a) exposing bacterial cultures to degradative phage 
enzymes with substrate specificity for the capsular 10 
polyaa&de of the wild type bacterid strain, 
and 
(b) selecting those cultures surviving step (a) above 
that produce a capsular polysaccharide. 
8. The method for producing an isolated capsular 
polysaccharide of claim 1 wherein the step of harvest- 
ing further comprises the steps of: 
(a) treating the mutant bacterial cultures with phage 
(b) separating the capsular polysaccharide from other 
components of the treated mutant bacteria by cen- 
trifugation; and 
(c) precipitation of the supmatant obtained in step 
(b) above with cold isopropanol or cold ethanol. 
9. The method for producing an isolated capsular 
polysaccharide of claim 8 wherein the step of separating 
further comprises the step of digestion of the precipitate 
with concentrated phage enzyme. 
7. The method for producing an isolated capsular 5 enzyme; 
* * * * *  15 
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Method for producing capsular polysaccharides

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