Process for the production of fructose transferase enzyme

An improved process for obtaining fructosyl transferase enzyme from the yeast Aureobasidium pullulans. Prior purification process steps are eliminated by use of conditions which do not produce black pigments and viscous polysaccharides.

FIELD OF THE INVENTION 
This invention relates to an improved process for the production of 
fructosyl transferase enzyme by the black yeast Aureobasidium pullulans. 
BACKGROUND OF THE INVENTION 
Because fructose is sweeter than either glucose or sucrose, much effort has 
gone into developing processes for producing syrups in which more than 50% 
of the carbohydrate is fructose. Recently, a novel way to obtain fructose 
syrup of greater than 50% fructose content was disclosed in British Pat. 
No. 2,000,144. According to that procedure, a sucrose substrate is 
subjected to the action of a fructosyl transferase enzyme to convert the 
sucrose to an intermediate syrup containing predominantly fructose 
polymers and glucose. This syrup, in which the fructose is in polymeric 
form, can be further treated to produce fructose syrups in which more than 
50% of the carbohydrate is fructose. An economical source of fructosyl 
transferase enzyme is essential to the successful operation of this 
process. 
British Pat. No. 2,000,144 describes a process for the production and 
isolation of a fructosyl transferase enzyme from the fermentation broth of 
Aureobasidium pullulans. However, the fermentation carried out according 
to the process of that disclosure produces a black, viscous broth which 
contains large amounts of the polysaccharide, pullulan. Extensive 
processing is necessary to remove the color and the pullulan before the 
fructosyl transferase enzyme can be isolated. Furthermore, the amount of 
enzyme obtained from the fermentation broth is comparatively low. There, 
therefore, exists a need for an improved process for the production of a 
fructosyl transferase enzyme preparation. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, there is provided an improved 
process for the production of fructosyl transferase enzyme. This process 
involves inoculating a culture medium with cells from a strain of 
Aureobasidium pullulans. The culture medium contains about 16 to about 24% 
weight by volume (w/v of sucrose, about 1 to about 2.4% (w/v) yeast 
extract or its nutrient equivalent, and about 1% (w/v) of an inorganic 
nitrate salt. The mixture is cultured at a pH of between 6 to 8 at a 
temperature of from 28.degree. to 32.degree. C. until a yield of fructosyl 
transferase enzyme is obtained. The enzyme preparation is then recovered 
from the culture medium. 
DETAILED DESCRIPTION OF THE INVENTION 
For the purposes of this specification, the following definitions are 
provided for the various terms used herein: 
1. Enzyme Preparation 
The term "enzyme preparation" is used herein to refer to any composition of 
matter which exhibits the desired enzymatic activity. The term is used to 
refer, for example, to cell extracts, refined and concentrated 
preparations derived from the cells and from culture liquors. The enzyme 
preparation may also include a composition in which the enzyme is bound to 
or absorbed on an inert carrier. 
2. Fructosyl Transferase Enzyme 
As used herein, this term refers to any enzyme that catalyzes the transfer 
of a fructosyl group from a donor, e.g., sucrose, to an acceptor. It 
includes the enzyme preparation derived from Aureobasidium pullulans, ATCC 
No. 9348 (synonymous with Pullularia pullulans). 
3. l Fructosyl Transferase Unit 
As used herein, one fructosyl transferase unit is defined as the amount of 
enzyme activity required to produce one micromole or reducing sugar, 
calculated as flucose, per minute under the following conditions: (a) pH 
5.5, (b) temperature 55.degree. C., and (c) substrate concentration at 60 
g food-grade sucrose per 100 ml of an aqueous reaction mixture. 
Reducing sugar (calculated as glucose) can be determined using a "Technicon 
Autoanalyzer II" (Technicon, Inc., Tarrytown, New York). Analysis is 
carried out by a conventional alkaline ferricyanide method, Analytical 
Biochemistry 45, No. 2, pp. 517-524 (1972), adapted for use in the 
"Autoanalyzer II". Unless otherwise designated, enzyme activity 
determinations are performed by continual monitoring of a reaction mixture 
consisting of the following composition: 
7.5 ml of 80% (w/v) aqueous food-grade sucrose solution. 
2.3 ml 0.1 M citrate buffer pH 5.5. 
0.2 ml enzyme sample containing that amount of fructosyl transferase enzyme 
which will produce from 5-25 micrograms of reducing sugar (calculated as 
glucose) per minute per ml of reaction mixture. 
Any strain of Aureobasidium pullulans capable of producing a fructosyl 
transferase enzyme can be employed in the process of this invention. 
Suitable strains of this yeast include NRRL No. 3937, ATCC No. 12535, NRRL 
No. 1673, NRRL No. Y 2311, NRRL No. YB 3892, ATCC No. 15223, and NRRL No. 
YB 3861. A particularly suitable strain is ATCC No. 9348. 
The process of this invention employs conventional fermentation equipment 
and procedures. Generally, the yeast culture is maintained or preserved on 
agar slants with periodic transfer to maintain viability. In the inoculum 
development, the culture is transferred from the slant to a liquid culture 
medium in order to provide an active culture in sufficient volume to 
inoculate the final production medium. The inoculum development stage may 
consist of a single transfer to a liquid medium or may include several 
successive transfers as required to provide for activation of the culture 
or to build sufficient volume for inoculation of a final production 
medium. It is in the final production medium where the desired enzyme is 
produced for use, either directly or after further processing or 
purification. 
The improved fructosyl transferase enzyme yields obtained by this process 
are due to part to the use of a more concentrated surcrose solution as a 
carbohydrate source for the growth of the microorganism. Increased yields 
of fructosyl transferase enzyme are obtained by the use of culture medium 
containing from about 16% to about 24% (w/v) sucrose in the medium. A 
preferred range of sucrose concentration is from about 20% to about 24% 
(w/v). Higher concentrations of sucrose fail to give appreciably enhanced 
yields of the enzyme. 
The improved yields of fructosyl transferase enzyme obtained by the process 
of this invention are also due in part to the use of increased 
concentrations of yeast extract in the medium. Suitable concentrations of 
yeast extract are from about 1% to about 2% (w/v). A preferred 
concentration of yeast extract is about 2.0% (w/v). A suitable yeast 
extract is that available from Difco Laboratories, Inc., Detroit, 
Michigan. Other preparations which are the nutrient equivalent of yeast 
extract may be substituted for this ingredient. For example, Amber BYF 
50X, a pure autolyzed brewers' yeast fraction available from the Amber 
Laboratories, Juneau, Wisconsin, may be used at a concentration of from 
about 5% to about 10% (w/v) in place of the yeast extract. 
For the most successful preparation of fructosyl transferase enzyme by the 
process of this invention, a water-soluble, inorganic nitrate salt is also 
added to the culture medium in which the cells of Aureobasidium pullulans 
are grown. Any of the common nitrate salts, such as sodium nitrate, are 
suitable. A preferred concentration of the salt is about 1% (w/v). 
The pH of the culture medium used in the process of this invention is 
adjusted to between 6 and 7 and the medium is sterilized by heating for 
from 1/2 to 2 hours at 120.degree. C. before it is inoculated with the 
cell culture. The pH of the culture medium is maintained between 6.0 and 
8.0 during the course of the fermentation by the addition of dilute acid 
or base as necessary. A preferred range for the fermentation is from about 
6.5 to about 7.5. When the fermentation medium of this invention was 
employed, the pH of the medium tended to increase or hold constant rather 
than decrease as had been the case with the fermentations of the prior art 
process. 
Another unexpected result of the use of the fermentation medium of this 
invention was the discovery that the black pigment formed in the prior art 
fermentations was not obtained. This avoided the inconvenience and expense 
of a separate precipitation step to remove this pigment before the enzyme 
could be isolated. 
A further unexpeted benefit was derived from the process of this invention. 
It was discovered that almost none of the viscous polysaccharide, 
pullulan, was produced. In the prior art process, a costly and 
time-consuming step was required to remove this high molecular weight 
product before the enzyme could be separated . Since the removal of this 
product and the precipitation of the black pigment are no longer 
necessary, enzyme isolation is greatly simplified. 
The fermentation process of this invention is conveniently carried out at a 
temperature of from about 28.degree. C. to about 32.degree. C. A preferred 
temperature range is from about 30.degree. C. to about 31.degree. C. 
Although the fermentation can be carried out below 28.degree. C. and above 
32.degree. C., the yield of fructosyl transferase enzyme produced when the 
fermentation is carried out in those temperature ranges is substantially 
lower than that obtained when the fermentation is run at temperatures 
between 28.degree. C. and 32.degree. C. 
A satisfactory yield of fructosyl transferase enzyme is usually present 
after the fermentation has been carried out for approximately 100 hours. 
However, the optimum time for the fermentation varies somewhat depending 
on the exact temperature used as well as on the concentration of the 
nutrient materials employed. 
In this new process, over 80% of the furctosyl transferase enzyme is 
present in the broth. This contrasts with the prior art fermentation 
wherein nearly half of the enzyme was cell-bound. Because the bulk of the 
enzyme is now present in the broth, a simple enzyme recovery process is 
feasible. 
A fructosyl transferase enzyme preparation suitable for many applications 
can be obtained by simply removing the yeast cells from the fermentation 
mixture. This separation can be accomplished by any of the conventional 
means, such as centrifugation or filtration. The enzyme solution can be 
concentrated, if desired, by evaporation at temperatures below those which 
cause inactivation of the enzyme. This is conveniently carried out in a 
rotary vacuum evaporator at temperatures below 50.degree. C. 
If a solid enzyme preparation is desired, the enzyme can be adsorbed on 
diatomaceous earth. This is accomplished by simply adding the diatomaceous 
earth to the cell-free fermentation broth. Addition of a water-soluble 
organic solvent, such as 2-propanol or acetone, causes the enzyme to 
precipitate and be adsorbed on the diatomaceous earth. The composite of 
diatomaceous earth and fructosyl transferase enzyme is then isolated by 
filtration or centrifugation.

The procedure of this invention is further illustrated by the following 
examples in which all parts are by weight and all percentages are weight 
by volume (w/v) unless expressly stated to be otherwise. 
EXAMPLE 1 
The medium used for inoculum development is as follows: 
0.5% Dibasic Potassium Phosphate 
0.1% Sodium Chloride 
0.02% Magnesium Sulfate.Heptahydrate 
0.06% Ammonium Nitrate 
0.3% Yeast Extract (Difco Laboratories) 
6% Sucrose 
pH of medium adjusted to 6.8 
The seed flasks, 500-ml Erlenmeyer's containing 100 ml of sterile medium, 
are inoculated from a slant culture of the black yeast, Aureobasidium 
pullulans. The particular strain of the yeast employed was designated in 
the catalogue of the American Type Culture Collection (Rockville, 
Maryland) as ATCC No. 9348. The seed flasks, after development on a 
reciprocal shaker for 48 hours at 30.degree. C., were used to inoculate 
second-stage seed flasks. Five milliliters of the inoculum from the first 
seed flask was added to 100 ml of fresh sterile medium in a second 500-ml 
Erlenmeyer seed flask. The second seed flasks were developed on a 
reciprocal shaker for 24 hours at 30.degree. C. before the entire contents 
of one flask was used to inoculate 4 liters of sterile medium in a 
7.5-liter fermentor. The media used for the various runs is given in the 
following table: 
TABLE I 
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FERMENTATION MEDIA 
Composition, % Weight/Volume 
Run No. 
Component 1 2 3 4 5 
______________________________________ 
K.sub.2 HPO.sub.4 
0.5 0.5 0.5 0.5 0.5 
MgSO.sub.4 . 7H.sub.2 O 
0.05 0.05 0.05 0.05 0.05 
NaNO.sub.3 1.0 1.0 1.0 1.0 1.0 
Yeast Extract 
2.0 2.4 2.4 2.0 -- 
(Difco Labs.) 
Amber BYF 50X 
-- -- -- -- 8.0 
(Amber Labs.) 
Sucrose 20 24 20 20 20 
Antifoam.sup.a 
0.025 0.025 0.025 0.025 0.025 
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.sup.a The antifoam agent was polypropylene glycol, mol. wt. 2000. 
In each run, the pH was adjusted to 6.5 before inoculation from the seed 
flasks. Runs Nos. 1, 2 and 3 were made at 30.degree.-31.degree. C. Runs 
Nos. 4 and 5 were made at 33.degree.-34.degree. C. The fermentations were 
carried out with an agitator speed of 580 rpm and with 4 liters of air per 
minute passing through the mixture. It was necessary to add 75 ml of 2 N 
HCl to Run No. 4 after 27 hours to reduce the pH from 7.5 to 6.5. After 50 
hours, an additional 30 ml of 2 N HCl was added to Run No 4 to reduce the 
pH from 7.5 to 6.5. At periodic intervals, samples were removed from the 
fermentor, the broth was separated from the cells by centrifugation and 
analyzed for fructosyl transferase enzyme activity. The results of these 
analyses are given in Table II. 
TABLE II 
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FRUCTOSYL TRANSFERASE ENZYME PRODUCTION 
Units/ml in Broth 
Time Run No. 
(hrs) 1 2 3 4 5 
______________________________________ 
27 77 58 56 59 39 
42 145 126 123 79 88 
50 178 171 158 81 106 
66 238 234 221 104 144 
90 290 355 302 144.sup.a 
159.sup.a 
114 440 438 380 -- -- 
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.sup.a The fermentation was stopped after 90 hours when the medium became 
very black. 
These results show that fermentations run above 33.degree. C. (Runs Nos. 4 
and 5) are much less satisfactory for the production of fructosyl 
transferase enzyme than the runs at 30.degree.-31.degree. C. These results 
further show that neither increasing the sucrose concentration in the 
medium above 20% (Run No. 2) nor increasing the yeast extract 
concentration in the medium above 2% (Runs Nos. 2 and 3) increases the 
production of fructosyl transferase enzyme in the fermentation. A 
comparison of Runs Nos. 4 and 5 shows that an alternate nutrient supply 
can be substituted for yeast extract in the fermentation. 
COMATIVE TEST 1 
(Prior Art Process) 
Inoculum development was carried out as in Example 1. The entire contents 
of one second-stage seed flask was used to inoculate 4 liters of sterile 
medium in a 7.5-liter fermentor. The medium used had the following 
composition: 
0.5% Dibasic Potassium Phosphate 
0.1% Sodium Chloride 
0.02% Magnesium Sulfate.Heptahydrate 
0.06% Ammonium Nitrate 
0.3% Yeast Extract (Difco Laboratories) 
12% Sucrose 
0.025% Propylene Glucol, mol. wt. 2000 
The pH of the medium was adjusted to 6.8 before inoculation from the seed 
flasks. The fermentation was carried out at 30.degree. C. with an agitator 
speed of 500 rpm and with 4 liters of air per minute passing through the 
mixture. The pH of the mixture dropped to 4.2 after 32.5 hours and 
remained at 4.2.+-.0.1 throughout the rest of the reaction. At periodic 
intervals, samples were removed from the fermentor, the broth was 
separated from the cells by centrifugation and analyzed for fructosyl 
transferase enzyme activity. The results of these analyses are as follows: 
______________________________________ 
FRUCTOSYL TRANSFERASE ENZYME PRODUCTION 
Time 
(hrs) Units/ml in Broth 
______________________________________ 
19 19.5 
25.5 26.2 
43 31.7 
67 40.6 
72 47.6 
90 49.7 
150 53.4 
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The reaction mixture was very black. This comparative test illustrates the 
comparatively low enzyme yields and highly pigmented reaction mixtures 
that were obtained by the prior art process.