AU603339B2 - Process for the production of sorbitol by enzymatic reaction, and microorganisms suitable for the purpose - Google Patents

Description

1AL COMMONWEALTH OF AUSTRALIA PATENT ACT 1952 COMPLETE SPECIFICATION, 0 (Original) T359 FOR OFFICE U3E Class Int. Class Application Number: Lodged: 6/ os s/ Complete Specification Lodged: Accepted: Published: Priority: A, :l Related Art: f .4 4 4, 4 4, 4 4 9, ft6 ,Name of Applicant: 4* Address of Applicant: 9 44

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'4' KERNFORSCHUNGSANLAGE JULICH GESELLSCHAFT MIT BESCHRANKTER HAFTUNG Postfach 1913, D-5170 Julich, Federal Republic of Germany.

Stephanie BRINGER-MEYER Hermann SAHM DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Actual Inventor(s) Address for Service: 14 4 14 41 4gia&4 Complete Specification for the invention entitled: "PROCESS FOR THE PRODUCTION OF SORBITOL BY ENZYMATIC REACTION, AND MICROORGANISMS SUITABLE FOR THE PURPOSE" The following stbatement is a full description of this invention, including the best, method of performing it known to us -1- T 2-- -2- Process for the production of sorbitol by enzymatic reaction, and microorganisms suitable for the purpose The invention relates to a' method of recovering sorbitol by enzymatic reaction starting from aqueous mixtures containing glucose and fructose and to mutants of Zymomonas mobilis suitable for this purpose.

Sorbitol has about 50 60% of the sweetening power of sucrose and is used to a considerable extent in cosmetics, pharmacy, the food industry and in the industrial sector on account of its favourable physiological and its technological application properties. Since sorbitol is utilized in the organism in an insulin-saving manner, it is used in particular as a sugar substitute for diabetics.

In the method of preparing sorbitol which is at present commonly used, aqueous glucose solutions are hydrogenated at 120 150°C and H12 pressures of 70 180 bars in the presence of heavy-metal catalysts (e.g.

Raney nickel) and the product solutions purified by a multi-stage process comprising filtration, Ion-exchange and activated-charcoal treatment. Since the purity of the sorbitol depends on that of the initial material, high-purity glucose solutions are used for sorbitol production. -ligh substrate and process costs, and especially energy costs for high pressures and temperatures make these chemical methods of sorbitol production relatively complicated and costly.

Attempts had therefore already been made to prepare sorbitol at normal pressure and low temperatures by means of biological systems.

For Instance, German Offenlegungsschrift 33 26 546 discloses a method for the continuous enzymatic production of gluconic acid as well as sorbitol and mannitol from glucose and fructose or from disaccharldes containing glucose and/or fructose. in this case glucose is oxidized to gluconic acid in a membrane reactor by the enzyme glucose dehydrogenase. The fructose contained in the substrate mixture may be optionally reduced in a parallel reaction to mannitol with the enzyme mannitol dehydrogenase or to sorbitol with the enzyme sorbitol dehydrogenase. For glucose oxidation and fructose reduction the coenzyme NAD(-l), which in reaction is the acceptor of a hydride ion and in reaction is the donor of a hydride ion, is also absolutely necessary. Since NAD(I-), on account of its low molecular weight, cannot be retained in the membrane reactor used in the process, the coenzyme, which is itself already costly, must also be immobilized with a water-soluble polymer. This method thus necessitates the purification of a plurality of enzymes and also the synthesis of polymerbonded cofactors. Technically speaking, the method of German Offenlegungsschrift 33 26 546 appears to be too complicated for the preparation of sorbitol.

Japanese patent Al 82/19 00 53 discloses a method in which sorbitol is recovered from glucose in the presence of NADPH by cell-free extracts of the yeasts Pichia guercuum or Pichia xylosa. In this method considerable disadvantages can be seen in the preliminary cultivation and processing of the yeast cells and in the costly use of NADPH, so that the expenditure especially in comparison with the very low yields of 0.006 and 0.020 moles sorbitol/mole glucose is disproportionately high.

The use of purified or unpurified enzyme preparations and the cofactors NAD(H) or NADPH thus also makes the biological methods of sorbitol preparation of the prior artcomplicated and so costly that they are so far hardly used in practice.

i *The object of the invention is therefore a method of preparing sorbitol S in a relatively simple and less costly manner.

SThis object is achieved according to the invention in the method Sinitially mentioned by the aqueous mixture being directly fermented by stable mutants of Zymomonas mobllis which do not ferment fructose, with the formaation of an aqueous mixture containing fructose, sorbitol, glucose, ethanol, CO 2 and gluconic acid, from which the sorbitol is isolated as required.

From L.Viikari (Appl.Microbiol.and Biotechn. 20 (1984) 118-123) it is known that sorbitol can be obtained fron aqueous mixtures of glucose and fruceose in ethanol fermentation by a wild-type strain of Zymononas mobilis, but preferentially the fructose is fermented to ethanol and C02 whereas the yield of sorbitol remains low. Only when using a stable mutant of Zynnmonas mobilis, as no developed, a successful continuous production of sorbitol is achieved.

4 It was, in fact, surprisingly found that sorbitol may be obtained in a relatively high yield from aqueous mixtures which contain both glucose and fructose, if the fermentation of glucose to ethanol is effected with stable fructose-negative mutants of Zymomonas mobilis which retain their fructose-negativity for at least days, and preferably for at least 100 days.

This favourable procedure has, in addition to simple and economical application, the particular advantage that S* no addition of NAD cofactors is necessary.

t St The reaction preferably takes place with aqueous mixtures which contain at least 100 g/l, preferably 100 S 15 to 200 g/l and in particular 140 to 180 g/l each of Ott glucose anf fructose, the operation being conveniently carried out either batchwise or also continuously with the retention or recycling of biomass.

20 Suitable for the application of the method according to the invention are special new, stable mutants of Zymomonas mobilis, which can be obtained by physically and/or chemically mutagenic means, isolation of the sorbitol-forming, fructose-negative mutants and selection 25 of the stable mutants by continuous culture over at least S. 10 days and have the characteristic that in glucose/fructose mixtures they form sorbitol from fructose and produce ethanol, CO 2 and gluconic acid only from glucose, but not from fructose.

The Zymomonas mobilis mutants of this invention are morphologically indistinguishable from wild-type Zymomonas mobilis. The only biochemical difference is the inability of the mutants to decompose fructose to ethanol and carbon dioxide.

The mutant registered in the German collection of M 2O.;PASDAT.43,61085-8S.rsp,4

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4amicroorganisms in Gottingen under the registration number DSM 3126 has been found in practice to be particularly useful.

The sorbitol may be isolated from the reaction mixture obtained by known methods, e.g. by chromatography or fructionated precipitations.

By the method according to the invention, especially in the case of fairly high concentrations in the initial solution, the reduction product sorbitol is formed from fructose. Prolonged reaction times, which are caused by retarded growth of Zymomonas mobilis in the presence of such high sugar concentrations may be avoided in accordance with the invention by the use of continuous fermentation methods with the retention or recycling of biomass.

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Here the flow rate of the substrate solution through the fermentation reactor may preferably be adjusted so that sorbitol is obtained in high yields When there are high cell densities in the fermenter, high sugar concentrations and flow rates at which glucose residues remain in the effluent are favourable.

The unreduced initial product fructose in many applications does not have to be separated. Any separation of the unreacted sugars which may be required may be effected by known methods. A particular advantage of the method according to the invention is the possibility of the deliberate setting of different sorbitol/fructose ratios by means of the concentrations selected for the initial solutions.

Suitable methods of biomass retention are immobilizations with alginates or carrageenins and for biomass retention by-pass filtrations of the fermenter effluent by bacteria-proof filters or hollow-fibre modules or else sedimentation or centrifuging.

4 4, 1 The induction of the mutants according to the invention in wild strains of Zymomonas mobilis may be effected by irradiating the cells with ultraviolet light, or preferably by chemical mutagenesis with non-alkylating agents such as nitrite, bisulphite, hydroxylamine, or with alkylating agents such as alkylalkane sulphonates (e.g.ethylmthane sulphonate, ethylethane sulphonate, diethyl sulphate) and N-nitroso compounds (e.g.dialkyl-nitrosamines, N-nitrosoureas, N-alkyl-N'nitro-N-nitrosoguanadine), or particularly preferably, by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.

14. 0 *r 9 Media which contain the following components (quantities in g/l H 2 0): KH PO H2 O4 0.5-1.0; (NH 4 2

SO

4 0.5-1.0; MgSO 4 .7H 2 0: 0.5-0.75; yeast extract: 5-10; fermentable sugar (glucose): 10-130 are suitable for the culture of the mutants according to the invention. The pH of the medium should lie between 4 and 7 at the beginning of and during fermentation and the temperature between 250 and 37 0 C, and preferably 28 to 32 C.

Further characteristics can be seen from the following description of the invention by means of examples.

I_ i r__-lll Illi~__--ii~l__tl-li.W~lr~Y IIUII~~ _I- 6 1) Induction and selection of fructose-negative stable mutants A cellular suspension of the wild strain of Zymomonas mobilis ATCC 29191 was suspended with a cell density of 2.8 x 108 living cells/ml, corresponding to an optical density (OD 550 nm) of 7.0, in 0.1 M potassium I phosphate buffer, pH 6.0, with 1.9 x 10- 4 M N-methyl-N'nitro-N-nitroso-guanidine (NTG). After incubation for minutes at ambient temperature, the cells were washed twice with 0.9% NaCl solution. This was followed by incubation for 20 hours, for mutation development, in a liquid medium containing (g/l H 2

KH

2

PO

4

(NH

4 2 S0 4 1.0; MgSO 4 .7H 2 0, 0.5; yeast extract, 5; pH 5.0) with 2% glucose at 30°C. A subsequent incubation in S 15 a liquid medium with 2% fructose 0.3 mg/ml ampicillin was used for mutant concentration. These developmental and concentration phases were repeated three times.

Fructose-negative mutants were identified and isolated by replica plating on agar plates containing glucose or fructose (liquid medium 2% agar).

The stability of the fructose-negative mutants was verified by plating-out undiluted culture samples on fructose-containing agar plates (liquid medium 2% 25 agar). Cultures of the mutant DSM 3126 remained stable in liquid culture during and after a period of 6 months, 4 with transfer intervals of 2 7 days. The fructosenegativity of this mutant was also preserved unchanged during growth in continuous culture over 5 days.

The mutant DSM 3126 is morphologically identical with wild-type Zymomonas mobilis. Furthermore, the Zymomonas mobilis mutant is biochemically identical to wild-type Zymomonas mobilis except that it is unable to decompose fructose to ethanol and carbon dioxide.

Mutation of wild-type Zymomonas mobilis cells P *0020 bAT.G3,61085-85.rsp,6

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6aaccording to the above methods routinely gave fructosenegative mutants having the same characteristics as DSM 3126.

2) Recovery of sorbitol Example 1 A medium comprising 148.0 g/1 fructose and 151.0 g/1 glucose dissolved in the medium described in 1) was inoculated in a fermenter with regulated pH and temperature with cells of a preliminary culture (5 g cell i r moist mass/1) of the mutant Zymomonas mobilis DSM 3126.

From the time of inoculation to the end of the fermentation process the pH was kept at 5.0 and the S temperature at 30 0 C. The results are shown in Table 1.

o o Example 2 The fermentation was carried out as in example 1, but with higher sugar concentrations. The results are shown in Table 1.

t- 900820,PASDAT.043,61085-85.rsp,6 -7- Table 1 Example 1 Example 2 Initial concentrations: Fructose 148.0 176.4 Glucose 151.0 175.5 Final concentrations: Sorbitol 65.3 93.3 Fructose 76.9 75.5 Glucose 14.7 23.3 Ethanol 54.6 54.0 Fermentatior. time (hours) 75 110 As can be seen, sorbitol formation is increased at higher sugar concentrations and with longer fermentation times.

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Claims (5)

1. Method of recovering sorbitol by enzymatic reaction from aqueous mixtures containing glucose and fructose, characterized in that the aqueous mixture is directly fermented by means of stable mutants of Zymomonas mobilis which do not ferment fructose, with the formation of an aqueous mixture containing fructose, sorbitol, glucose, ethanol, CO 2 and gluconic acid, from which the sorbitol is isolated if necessary.

2. Method as in claim 1, characterized in that the sugar concentrations of the aqueous mixtures are at least 100 g/l each, preferably 120 to 200 g/1 each, and particularly preferably 140 to 180 g/l each of glucose and Sfructose.

3. Method as in claim 1 or 2, characterized in that the fermentation is effected batchwise or in particular continuously with the retention or recycling of biomass.

4. Sorbitol-forming stable fructose-negative mutants of Zymomonas mobilis obtainable by mutation of a wild strain of Zymomonas mobilis by physical and/or chemical mutagenic agents, isolation of sorbitol-forming fructose- negative mutants and selection of the stable mutants by continuous culture over at least 10 days. S

5. Mutant of Zymomonas mobilis as in claim 4, characterized by the registration S" number DSM 3126 -9- The stp. fe-ature5jOM ositions and compot is ef rr to or indicated in the specification and/or claims Of this application, inA4.vdually or collectively, and any andal cmb, ions of any two or more of said steps or a n l c oiC JA~ I Dated this 12th day of AUGUST, 1986 KERNFORSCHUNGSANLAGE JULICH GESELLSCHAFT MIT BESCHRANKTER HAFTUNG By Its Patent Attorneys DAVIES COLLISON I I I I II I V V I 4 1 4 V V