GB2241705A - Preparing glucans - Google Patents

Abstract

A process for preparing a cyclosophoran (a cyclic glucan) comprises cultivating a suitable mutant strain of a microorganism that has been prepared by introducing a transposon into the parent strain, the parent strain being capable of producing both cyclosophoran and extracellular polysaccharide while the mutant strain is incapable of producing extracellular polysaccharide (EPS). The parent stratin may be any suitable strain that produces EPS and cyclosophorans, e.g. of Agrobacterium or Rhizobium.

Description

PREPARING GLUCANS This invention relates to a process for preparing glucans and, in particular, cyclosophorans.

Cyclosophorans are cyclic ss(1+2)D glucans secreted by several strains of Agrobacterium and Rhizobium.

Xanthomonas bacteria can produce an unusual branched cyclosophoran. The culture fluids normally contain cyclosophorans of varying molecular weight, or degree of polymerisation (dp). Typical values lie in the range dp = 17 - 24. The distribution of dp values has been found to be characteristic of the particular bacterial strain of Agrobacterium or Rhizobium, as reported by Koizumi et al, J. Chromatography 299 (198t) 21'.-2.zA, Ut 218.

These cyclic compounds are of interest for at least two reasons. Firstly, genetic mutants of Rhizobium meliloti which are unable to export cyclosophorans do not fix nitrogen in their plant host. Cyclic glucans may therefore play an essential role in the plant-bacterium symbiosis. Secondly, there is evidence that cyclosophorans may reserMle, and perhaps surpass, cyclodextrins in their complex-forming properties.

Cyclosophorans are normally secreted into the culture medium together with large amounts of highly viscous extracellular polysaccharide (EPS). The EPS may itself be of importance, and has been the subject of investigation. Production of the highly viscous EPS l 1keE aeration and agitation of the culture broth di-ficu31, nd slso hinders removal of bacterial cells from the broth. Separation of the cyclosophorans requires fractionation with alcohol followed by column chromatographic separation using ion-exchange and Sephadex resins. These procedures are time-consuming and wasteful in the use of solvents.

Higashuira et al, Agric. Biol. Chem. 49 (1985) 1865, have described the production and isolation of cyclosophorans from EPS mutants of R. leguminosarum bv.

phaseoli AHU133, obtained by chemical mutagenesis.

EP-A-0106311 and EP-A-0108355 describe processes for preparing cyclosophorans by cultivation of a strain of Agrobacterium or Rhizobium phaseoli, respectively, which does not produce EPS. Such EPS strains were obtained by conventional mutagenic techniques.

Known EPS mutants can produce reasonable levels of cyclosophorans without EPS. However, their stability is limited.

According to a first aspect of the present invention, a cyclosophoran is prepared by cultivation of a suitable EPS strain that has been prepared by introducing a transposon into the parent EPS-producing strain. The resulting strain is relatively stable.

According to a second aspect of the present invention, a cyclosophoran is prepared by cultivation of a strain of Rhizobium leguminosarum having the characteristics of the strain that was deposited on 22nd February 1990 at the National Collection of Industrial and Marine Bacteria and having the accession number NCIMB 40260.

The parent strain is any suitable strain producing EPS and cyclosophorans, e.g. of Agrobacterium or Rhizobium. An EPS mutant is derived from the parent strain by introducing a transposon, by methods generally known per se. Whereas if point mutation occurs (a single base change on DNA), this can revert. By the transposon technique, however, a deletion occurs (i.e. some DNA is lost), this is not easily repaired and so is stable.

For example, by subjecting the parent strain to suitable conditions in the presence of a transposon, preferably a transposon having a characteristic marker such as kanamycin-resistance, stable EPS mutants can be readily obtained by simple screening of relatively few colonies by comparison with the covnentional mutagenic procedures previously described, e.g. chemical or by irradiation The introduction of transposon involves mixing, on plates, the wild-type parent strain with, say, E. coli containing a plasmid in which the transposon resides. The whole of the plasmid then mates into the parent strain and selection is carried out.

Once obtained, the EPS mutant can be cultivated under conditions known per se, in a suitable nutrient medium, e.g. under conditions described in the prior art described above. Depending on the cultivated strain, the cyclosophoran or, usually, mixture of cyclosophorans that is produced can be used, say, as a complexing agent, to give inclusion complexes of cyclosophorans with, for example, steroids, prostaglandins, ubiquinone, vitamins, indomethacins, amphotericin B or aspartame. Inclusion complexes can be used for the purposes of, for example, solubilisation, stabilisation, catalysis, separation, chiral resolution and slow release.

The following Example illustrates the invention.

Example The deposited EPS mutant R. leguminosarum was grown in shake flask culture, at pH 6.8 and 290C, in the following medium: Component g/l Sodium glutamate 22.4 Mannitol 80.0 MgSO4.7H2O 1.25 CaCl2. 6H2O 0,22 K2HPO4 1.25 FeCl3. 6H 20 0.06 Biotin 0.0072 Thiamine 0.0072 Pantothenic acid 0.0072 Trace elements 3 ml The results of a typical laboratory batch production fermentation are shown in the accompanying Figure 2.

-1 -l Some 15 g/l 1 cyclosophoran are produced with 15 g/l cells after 150 hours. Mannitol has been used as the carbon source, in order to facilitate identification of the product by glucose analysis. Other carbon sources, e.g. glucose, can be used, and that will obviously be more economic. In those circumstances, cyclosophorans can be detected by HPLC.

Cyclosophorans are very water-soluble. They may be precipitated with excess isopropanol. Because the fermentation broth has a low viscosity, the bacteria may be removed by centrifugation.

In a second, pilot plant run, the cells were removed by cross-flow microfiltration, and the product was concentrated by ultrafiltration to 113 girl , and dried by rotary evaporation. Using 1000 mol wt cut-off membranes, the ultrafiltration flux rate was 1 to 7 litrelm2 /h at 1.4 bar.

3.

The 0.05 m pilot plant run produced 5 kg of final product. Figure 1 charts the fermentation. The long lag phase is the result of the low (2%) inoculum size used for convenience in this run. Because the broth is not viscous, the oxygen transfer problems associated with high biopolymer concentrations are absent. It may be desirable to increase the product/cell ratio. For example, this might be achieved through fed-batch operation.

Cyclosophorans may be isolated, free from pigment, by filtration through a charcoal column. The cyclic glucans bind to the column and can be eluted separately with 30% ethanol. The collected material was evaporated to dryness and stored for analysis.

Neutral sugars present in the powdered material were released by a Saeman hydrolysis and analysed as alditol acetates by glc. Glucose was the only sugar detected.

Glycosidic linkages were determined as methylated alditol acetates by glc mass spectrometry. The only linkage detected was (1+2) linked glucose, indicating pure cyclosophoran. The distribution of ring sizes was investigated by fast atom bombardment mass spectrometry (FAB-MS): the spectrum indicated the presence of major components with ring sizes corresponding to dp = 17,18,19,20 and 21. This spectrum of ring sizes is characteristic of wild-type R. leguminosarum and has been dubbed a type II pattern (see Koizumi et al, supra).

Claims (7)

1. A process for preparing a cyclosophoran, which comprises cultivating a suitable mutant strain that has been prepared by introducing a transposon into the parent strain, the parent strain being capable of producing both cyclosophoran and extracellular polysaccharide while the mutant strain is incapable of producing extracellular polysaccharide.

2. A process according to claim 1, which comprises cultivating a microorganism having the characteristics of Rhizobium leguminosarum NCIMB 40260.

3. A microorganism capable of producing cyclosophoran but incapable of producing extracellular polysaccharide, being a mutant strain that has been prepared by introducing a transposon into a parent strain, the parent strain being capable of producing both cyclosophoran and extracellular polysaccharide.

4. A microorganism according to claim 3, being a mutant of a parent strain of the genus Agrobacterium or the genus Rhizobium.

5. Rhizobium leguminosarum NCIMB 40260.

6. A process according to claim 1, substantially as hereinbefore described with reference to the Example and the accompanying figures.

7. A cyclosophoran when prepared by a process according to any of claims 1, 2 or