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Gluco-oligosaccharide and galacto-oligosaccharide production by Rhodotorula minuta IFO879
JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 82, No. 2, 124-127. 1996
Gluco-Oligosaccharide and Galacto-Oligosaccharide by Rhodotorula minuta IF0879 NORIMASA Central Research Laboratory,
Ajinomoto
AND
ONISHI*
Production
KENZO YOKOZEKI
Co. Inc., 1-I Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa 210, Japan
Received
21 March
1996/Accepted
7 May 1996
Three strains of yeast, Sterigmatomyces elviae CBS8119, Rhodotorula minuta IF0879 and Sirobasidium magnum CBS6803, which are known to produce galacto-oligosaccharide (Gal-OS) from lactose, were found to possess strong transglucosylation abilities and to produce gluco-oligosaccharide (Glc-OS) from cellobiose. Among them, R. minutu IF0 879 was selected as the best producer of Glc-OS. Efficient production conditions for Glc-OS from cellobiose and Gal-OS from lactose were investigated. Using toluene-treated resting cells of R. minutu IF0879, the maximal amounts of Glc-OS produced from 200 mg/ml cellobiose and of Gal-OS produced from 200 mg/ml lactose were 70 mg/ml (a yield by weight of 35%) and 76 mg/ml (a yield by weight of 38x), respectively. Since the by-product glucose was found to inhibit oligosaccharide production, it was removed from the reaction mixture by devising a suitable culture method such that the enzymatic reaction was accompanied by cell growth to consume the glucose. Under these conditions, the productivities were markedly improved: 201 mg/ml of Glc-OS was produced from 400 mg/ml cellobiose (a yield by weight of 51%) and 230 mg/ml of Gal-OS from 360 mg/ml of lactose (a yield by weight of 64%). The structures of the major components of Glc-OS and Gal-OS obtained by this method were identified as cellotriose and 0-bo-galactopyranosyl-(1--*4)-O-~o-galactopyranosyl-(l~4)-o-glucopyranose (4’-galactosyl-lactose), respectively. [Key words: galactosyl-lactose,
gluco-oligosaccharide, transglucosylation,
galacto-oligosaccharide,
Oral administration of galacto-oligosaccharide (GalOS), a transgalactosylation product of lactose, promotes proliferation of intestinal bifidobacteria (1, 2). Bifidobacteria in the human large intestine are helpful in the maintenance of good health (3, 4). Gal-OS, which is used as an energy source by bifidobacteria, is known to be a bifidus factor, and the development of an efficient GalOS production method is highly desirable. We previously found that 3 strains of yeast, Sterigmatomyces elviae CBS81 19, Rhodotorula minuta IF0879, and Sirobasidiurn magnum CBS6803, possess high levels of transgalactosylation activity suitable for Gal-OS production (5). Efficient Gal-OS production conditions for intact cells of S. elviae CBS8119 was also studied (5). In addition, we showed that the Gal-OS-producing reaction was catalyzed by a ;3-galactosidase with a high level of transgalactosylation activity (6). Many ,3-galactosidases are known to catalyze not only the hydrolysis of ,3-galactoside linkages but also ,3-glucoside linkages (7-11). We found that toluene-treated resting cells of S. elviae CBSSll9, R. minuta IF0879, and S. magnum CBS6803 produced gluco-oligosaccharide (Glc-OS) at high yields when cellobiose was used as the substrate. It may be that the Gal-OS-producing ;3-galactosidase of these yeasts also recognizes ,3-glucoside linkages and has strong transglucosylation activity. In this paper, we describe the optimal conditions for efficient Glc-OS production from cellobiose and Gal-OS production from lactose by a representative strain, R. minuta IF0879, and identify the main transglycosylation products.
* Corresponding
yeast,
Rhodotorula minuta,
cellotriose,
MATERIALS
METHODS
4-
transgalactosylation]
Microorganisms R. minuta IF0879,
AND
and cultivation and S. magnum
S. elviae CBS81 19, CBS6803, which were
selected as Gal-OS producers in our previous study (5), were used. A loopful of cells subcultured on a potatodextrose agar slant was inoculated into 5 ml of medium containing 20 g glucose, 10 g lactose, 10 g peptone, 10 g yeast extract, 5 g (NH&S04, 3 g K2HP04, 1 g KH2P04, 0.5 g MgS04. 7H20 in 1 I of distilled water (pH 7.0) in a test tube and cultivated aerobically at 30°C for 48 h. Then, 1 ml of the culture was transferred into 50ml of the same fresh medium in a 500-ml flask and cultivated aerobically at 30°C for 60 h on a reciprocal shaker. The cells were harvested by centrifugation at 10,000 x g at 5°C for 10 min, washed with 100 mM potassium phosphate buffer (pH 6.0), and resuspended in 20mM potassium phosphate buffer (pH 6.0) to provide a concentrated cell suspension (wet weight, 500 mg/ml). Glc-OS and Gal-OS production by cells One drop of toluene was added
toluene-treated
to 5 ml of cell suspension and stirred vigorously. The standard reaction mixture contained 200 mg/ml substrate (cellobiose or lactose) in 100 mM potassium phosphate buffer (pH 6.0), and 5 ml of toluene-treated cell suspension in a total volume of 50 ml, and was incubated at 60°C without shaking. The reaction was stopped by boiling for 10min and the amounts of Glc-OS or Gal-OS produced in the reaction mixture were determined, as described below under “Analytical Method”. The transglycosylation activity is expressed as the total amount of Glc-OS or Gal-OS produced from cellobiose or lactose under this standard reaction condition. Glc-OS and Gal-OS production with cell culture The cell suspension (5 ml) was transferred to 45 ml of Glc-OS- or Gal-OS- production medium in a 500-ml
author.
124
VOL. 82, 1996
GLUCO-OLIGOSACCHARIDE
flask. The Glc-OS production medium contained 200 or 400g cellobiose, 0.9 g yeast extract, 4.5 g (NH&S04, 2.7 g K2HP04, 0.9 g KH2P04, 0.45 g MgS04. 7Hz0, and 0.9 g CaCOr in 900 ml distilled water, pH 6.0. The GalOS production medium was the same as that used for Glc-OS except that it contained 360g lactose instead of cellobiose. The saccharides (cellobiose and lactose) and CaCOr were sterilized separately from the other media components and were added aseptically. The cultivation was carried out aerobically at 30°C on a reciprocal shaker, and was stopped by boiling for 10 min. Analytical method The amounts of Glc-OS, GalOS and other saccharides produced were assayed by high-performance liquid chromatography using an L6000 pump (Hitachi, Tokyo), a Shodex SE-50 refractive index detector (Showa Denko Co., Tokyo), and a CR1B data processor (Shimadzu, Kyoto) with a Shodex ION Pak S-801 column (4.6~ 5OOmm; Showa Denko Co.) under the following conditions: column temperature, 80°C; mobile phase, distilled water; flow rate, 0.6 ml/min. Isolation and identification of products After 24-h cultivation for Glc-OS production from 200mg/ml cellobiose, the culture broth (20ml) was centrifuged (lO,OOOxg, 10 min) to remove the cells. The resulting supernatant was applied to an active carbon column (25 x 500 mm) equilibrated with distilled water, and the column was rinsed with 1 I of 4% (v/v) ethanol to remove residual glucose and cellobiose. Trisaccharide, the main product of Glc-OS, was eluted with 11 of 8% (v/v) ethanol. The eluate was concentrated to 20ml in vucuo, and when freeze-dried yielded 824 mg trisaccharide. Trisaccharide from Gal-OS was also isolated from the culture broth as previously described (5) and 1,066 mg of trisaccharide was obtained. l3C nuclear magnetic resonance (13C-NMR) spectra of the products in D20 were recorded with a JNM-GX 400 spectrometer (Nihondenshi Co. Ltd., Tokyo) using 3(trimethylsilyl)-propionic acid -d4 sodium salt as an internal standard. Infrared spectra were recorded with a 1600 FT-IR spectrometer (Perkin-Elmer, Norwalk, Conn., USA) as a KBr disc. Authentic cellotriose was purchased from Seikagaku Co. (Tokyo) and 4’-galactosyl-lactose from Sigma Chemical Co. (St. Louis, MO, USA).
Transglycosylation activities of S. elviue CBS8119, R. minutu IF0879 and S. magnum CBS6803 Transglycosylation (transgalactosylation and transglucosylation) activities were investigated for three yeast strains with strong Gal-OS production activities. As shown in Transgalactosylation of Gal-OS-producing
Microorganism S. elviae CBS81 19 minuta IF0819 S. magnum CBS6803 R.
and transglucosylation microorganisms
Oligosaccharide
formed
activities
(mg/ml)
Gal-OS
GIc-OS
45.5 44.0 36.8
35.3 59.2 54.0
Transgalactosylation and transglycosylation activities are expressed as the total amounts of Gal-OS or Glc-OS formed in the reaction mixture. In each reaction, 200 mg/ml lactose or cellobiose was used as the substrate (Materials and Methods). The reactions were carried out at 60°C for 2 h in potassium phosphate buffer, pH 6.0.
PRODUCTION
125
Table 1, each strain exhibited strong transglucosylation activity, and produced Glc-OS from cellobiose in addition to its transgalactosylation activity. Though S. elviae CBS81 19 showed the highest Gal-OS-producing activity, it was not the best strain for Glc-OS production. R. minuta IF0879 had the highest Glc-OS-producing activity and was selected for further study. Reaction conditions for Glc-OS and Gal-OS production by toluene-treated resting cells of R. minufu IF0879 The effects of pH and temperature on the production of Glc-OS or Gal-OS were examined. As shown in Figs. 1A and lB, the optimal pH and temperature were almost the same for both Glc-OS and Gal-OS productionabout 4.0 to 6.0 and 7O”C, respectively. The activity at 60°C was 80% of the maximum. However the reaction mixture turned brown at 7O”C, so a reaction temperature of 60°C was used to avoid coloring. Profiles of the effects of temperature and pH on activity were almost same for the transgalactosylation and transglycosylation reactions. These results indicated that the transgalactosylation and transglycosylation reactions appeared to be catalyzed by the same P-glycosidase, with a broad specificity, which recognized both $-glucoside linkages and ,3-galactoside linkages. Production of Glc-OS and Gal-OS by toluene-treated resting cells of R. minutu IF0879 The time courses of Glc-OS and Gal-OS production are shown in Figs. 2A and 2B, respectively. Glc-OS production from 200mg/ ml cellobiose reached a maximum of 70 mg/ml (a yield by weight of 35x), after 8-h incubation at 60°C which was comprised of 52 mg/ml trisaccharide and 18 mg/ml tetrasaccharide. The amount of Glc-OS produced rapidly decreased as the level of by-product glucose increase (Fig. 2). Pentasaccharide or larger oligosaccharides were scarcely formed during the reaction. Gal-OS production was maximal at 76mg/ml and was comprised of 63 mg/ml trisaccharide and 13 mg/ml tetrasaccharide, which was produced from 200 mg/ml lactose after 24-h incubation at 60°C (Fig. 2B). Only low levels of Gal-OS I
7 e
100
.-f 3 g
1
I
100 -
I
s-s .1 80 .=
?360
RESULTS AND DISCUSSION
TABLE I.
AND GALACTO-OLIGOSACCHARIDE
B
80 60 -
-
40
40 20 -
20.
2
I
4
PH
I
6
\
13
8
’
10
030405080708090 Temperature (“Cl
FIG. 1. Effects of pH and temperature on Glc-OS and Gal-OS production. (A) pH: Mixtures containing 0.5 ml of a toluene-treated cell suspension of R. minuta IF0879, 200 mg/mI cellobiose (for GlcOS) or lactose (for Gal-OS) and 100 mM buffer at various pH values in a total volume of 5 ml were incubated at 60°C for 1 h. Buffers used were sodium acetate-HCl (pH 1.3 to 2.3), sodium acetate (pH 3.3 to 6.0). uotassium ohosnhate (PH 6.0 to 7.0), and Tris-HCl (PH 7.5 to 8.8): (B) Temperaturk Mixtures containing 0.5 ml of a toluene-treated cell suspension of R. minuta IF0879, 200 mg/mI cellobiose (for GlcOS) or lactose (for Gal-OS), and 100 mM potassium phosphate buffer (pH 6.0) in a total volume of 5 ml were incubated at various temperatures for 1 h. Symbols: 0, Glc-OS; 0, Gal-OS.
ONISHI AND YOKOZEKI
126
J.
200
B i
0
0 0
102030405060 Time
0
10
20
30
Time
(h)
40
50
60
(h)
FIG. 2. Time courses of Glc-OS (A) and Gal-OS (B) production by toluene-treated resting cells. A mixture containing 5 ml of a toluene-treated cell suspension of R. minuta IF0879, 2OOmg/ml cellobiose (for Glc-OS) or lactose (for Gal-OS) and 100mM potassium phosphate buffer (pH 6.0) in a total volume of 50 ml was incubated at 60°C. Symbols: A, cellobiose or lactose; 0, glucose; 0 , galactose; 0, trisaccharide; A, tetrasaccharide; n , total Glc-OS or Gal-OS. degradation and by-product glucose formation were observed during subsequent reaction for 24 h or longer. From these observations, Glc-OS- and Gal-OS-producing +glycosidase seems to possess high ,%glucoside linkage hydrolyzing activity but little ,3-galactoside linkage hydrolyzing activity. On the other hand, it appears that another +glycosidase (,3-glucosidase), catalyzing only the hydrolysis of ,%glucoside linkages, may be present in the cells of R. minuta IF0879. To determine whether or not the production of Glc-OS and Gal-OS is catalyzed by the same enzyme, as well as to clarify the mechanism of the rapid degradation of Glc-OS, purification and characterization of the enzyme is now in progress. Effect of glucose The effect of glucose on Glc-OS and Gal-OS production by R. minuta IF0879 was also examined. As shown in Fig. 3, glucose was inhibitory to both transglycosylation and transgalactosylation, the
FERMENT.
BIOENG.,
amounts of Glc-OS or Gal-OS formed decreasing as more glucose was added. This phenomenon has previously been observed in transgalactosylation reactions with toluene-treated resting S. elviae CBS8119 cells (5), and we expected that removal of by-product glucose from the reaction mixture would improve Glc-OS productivity. Glc-OS and Gal-OS production with cell culture In order to remove the by-product glucose from the reaction mixture, the enzymatic reaction was carried out accompanied by cell growth (the cell culture method). Just as Gal-OS production by S. elviae CBS8119 previously increased using the cell culture method (5), so the productivities of both Glc-OS and Gal-OS by R. minuta IF0879 were also improved by employing the same system. As a result, 117 mg/ml Glc-OS (a yield by weight of 59x), comprising 68 mg/ml trisaccharide and 49 mg/ml tetrasaccharide, was produced from 200 mg/ml cellobiose after 34-h cultivation. With a higher concentration of cellobiose of 400 mg/ml, 201 mg/ml Glc-OS (a yield by weight of 51x), comprising 130 mg/ml trisaccharide and 71 mg/ml tetrasaccharide, was produced after 64-h cultivation at 30°C (Fig. 4). In a quantitative study of Glc-OS formation from cellobiose by transglucosylation using j-glucosidase of Aspergillus niger, a maximal yield from 133 mg/ml cellobiose of 23.5% (by weight) was reported (12). Recently, Christakopoulos et al. (13) reported more efficient Glc-OS formation using ,3-glucosidase of Fusarium oxysporum: Glc-OS (cellotriose) was formed from 10% (v/w) cellobiose with a maximal yield of 37% (by weight). In the present study, the enzymatic reaction of R. minuta IF0879 accompanied by cell growth, gave a Glc-OS productivity considerably superior to that using the ,9glucosidase of F. oxysporum. As far as we know, this is the best method of Glc-OS production reported so far. In the case of Gal-OS production, the enzymatic reaction accompanied by maximized cell growth gave 230mg/ml Gal-OS (a yield by weight of 64?& com-
1.2
0.8 0.6
0 0’
I
I
0
100
lactose (for Gal-OS), 100mM potassium phosphate buffer (pH 6.0), and the indicated concentrations of glucose in a total volume of 50 ml Symbols:
0, Glc-OS;
60
80
0.0 100
Time(h)
FIG. 3. Effect of glucose concentration on Glc-OS and Gal-OS production. A mixture containing 5 ml of a toluene-treated cell suspension of R. minula IF0879, 200 mg/ml cellobiose (for Glc-OS) or at 60°C for 1 h.
40
_I
200
Glucose added (mglmi)
was incubated
20
0, Gal-OS.
FIG. 4. Time courses of Glc-OS production by a cell culture of R. minuta IF0 879. The cell suspension (5 ml) was inoculated into 45 ml of Glc-OS-producing medium containing 20 g cellobiose in a 500-ml flask, and was cultivated aerobically at 30°C on a reciprocal shaker. The cell concentration is expressed as the optical turbidity at 562 nm after 50-fold dilution with 0.1 N HCl. Symbols: A, cellobiose; 0, glucose; A, Glc-OS (tetrasaccharide); 0, Glc-OS (trisaccharide); n , total Glc-OS; x , cell concentration.
VOL. 82. 1996
GLUCO-OLIGOSACCHARIDE
AND GALACTO-OLIGOSACCHARIDE
PRODUCTION
127
Utagawa of our laboratory for their encouragement and helpful suggestions throughout the study. We also thank Dr. H. Takesada for her technical expertise in obtaining the 13C-NMR spectra.
1.2
REFERENCES
0.0 0
20
40
60
80
100
Time(h) FIG. 5. Time courses of Gal-OS production by a cell culture of R. minuta IF0 879. The cell suspension (5 ml) was inoculated into 45 ml of Gal-OS-producing medium containing 18 g lactose in a 5Ol-ml flask, and was cultivated aerobically at 30°C on a reciprocal shaker. The cell concentration is expressed as the optical turbidity at 562 nm after 50-fold dilution with 0.1 N HCl. Symbols: A, lactose; 0, glucose; 0 , galactose; A, Gal-OS (tetrasaccharide); 0, Gal-OS (trisac-
charide);
n
, total Gal-OS; x , cell concentration.
prised of 185 mg/ml trisaccharide and 45 mg/ml tetrasaccharide, from 360mg/ml lactose after cultivation for 60 h (Fig. 5). This Gal-OS productivity level was almost equal to that achieved by the enzymatic reaction of S. elviae CBS8119 accompanied by cell growth, which is the best Gal-OS production method reported to date (5). Identification of Glc-OS and Gal-OS The structures of the trisaccharide products of Glc-OS and GalOS, were identified by 13C-NMR and infrared spectra, which were compared with authentic cellotriose and 4’galactosyl-lactose spectra. All the spectra of the trisaccharides of Glc-OS and Gal-OS agreed well with those of cellotriose and 4’-galactosyl-lactose, respectively (data not shown). From these results, it was clear that a ;3 1,4 linkage was formed during transglycosylation, and that cellotriose and 4’-galactosyl-lactose were the main products of Glc-OS and Gal-OS, respectively. ACKNOWLEDGMENTS We thank
Drs.
K. Kubota,
T. Tanaka,
A. Yamashiro,
and
T.
1. Tanaka, R., Takayama, H., Morotomi, M., Kuroshima, T., Ueyama, S., Matsumoto, K., Kuroda, A., and Mutai, M.: Effects of administration of TOS and Bifidobncterium breve 4006 on the human fecal flora. Bifidobacteria Microflora, 2, 17-24 (1983). 2. Ohtsuka, K., Benno, Y., Endo, K., Ozawa, O., Ueda, H., Uchida, T., and Mitsuoka, T.: Effects of 4’ galactosyllactose intake on fecal microflora. Bifidus, 2, 143-149 (1989). (in Japanese) 3. Mitsuoka, T.: Bifidobacteria and their role in human health. J. Ind. Microbial., 6, 263-268 (1990). 4. Hughes, D. B. and Hoover, D. G.: Bifidobacteria: their potential for use in American dairy products. Food. Technol., 45, 64-83 (1991). 5. Ooishi, N., Yamashiro, A., and Yokozeki, K.: Production of gala&o-oligosaccharide from lactose by Sterigmatomyces elviae CBS8119. Appl. Environ. Microbial., 61, 4022-4025 (1995). T.: Purification and properties of a 6. Onishi, N. and Tanaka, novel thermostable galacto-oligosaccharide-producing ,9-galactosidase from Sterigmatomyces elviae CBS8119. Appl. Environ. Microbial., 61, 4026-4030 (1995). 7. Got, R. and Marnay, A.: Isolement, purification et quelques caracteristiques physicochimiques de deux ,3-hexosidases du sue diaestif d’Helix aomatia. Eur. J. Biochem., 4. 240-246 (1968). M., Perez, N., and Cabezas, J. A:: $-Galac;osidase 8. Lianillo, and $-glycosidase activities of the same enzyme from rabbit liver. Int. J. Biochem., 8, 557-564 (1977). 9. Takase, M. and Horikoshi, K.: Purification and properties of a f-glycosidase from Thermus sp. Z-1. Agric. Biol. Chem., 53, 559-560 (1989). 10. Gorgan, D. W.: Evidence that ,%galactosidase of Sulfolobus solfataricus is only one of several activities of a thermostable ;?-D-glycosidase. AppI. Environ. Microbial., 57, 1644-1649 (1991). 11. Gabelsberger, J., Liebl, W., and Schleifer, K.: Purification and properties of recombinant ,3-glycosidase of the hyperthermophilic bacterium Thermotoga maritima. Appl. Microbial. Biotechnol., 40, 44-52 (1993). 12. Watanabe, T., Sato, T., Yosbioka, S., and Kuwahara, M.: Purification and properties of Aspergillus niger @-glucosidase. Eur. J. Biochem., 209, 651-659 (1992). 13. Christakopoulos, P., Bhat, M. K., Kekos, D., and Macris, B. J.: Enzymatic synthesis of trisaccharides and alkyl j!-D-glucosides by the transglycosylation reaction of Fusarium oxysporum. Int. J. Biol. Macromol., 16, 331-334 (1994).
Gluco-Oligosaccharide and Galacto-Oligosaccharide by Rhodotorula minuta IF0879 NORIMASA Central Research Laboratory,
Ajinomoto
AND
ONISHI*
Production
KENZO YOKOZEKI
Co. Inc., 1-I Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa 210, Japan
Received
21 March
1996/Accepted
7 May 1996
Three strains of yeast, Sterigmatomyces elviae CBS8119, Rhodotorula minuta IF0879 and Sirobasidium magnum CBS6803, which are known to produce galacto-oligosaccharide (Gal-OS) from lactose, were found to possess strong transglucosylation abilities and to produce gluco-oligosaccharide (Glc-OS) from cellobiose. Among them, R. minutu IF0 879 was selected as the best producer of Glc-OS. Efficient production conditions for Glc-OS from cellobiose and Gal-OS from lactose were investigated. Using toluene-treated resting cells of R. minutu IF0879, the maximal amounts of Glc-OS produced from 200 mg/ml cellobiose and of Gal-OS produced from 200 mg/ml lactose were 70 mg/ml (a yield by weight of 35%) and 76 mg/ml (a yield by weight of 38x), respectively. Since the by-product glucose was found to inhibit oligosaccharide production, it was removed from the reaction mixture by devising a suitable culture method such that the enzymatic reaction was accompanied by cell growth to consume the glucose. Under these conditions, the productivities were markedly improved: 201 mg/ml of Glc-OS was produced from 400 mg/ml cellobiose (a yield by weight of 51%) and 230 mg/ml of Gal-OS from 360 mg/ml of lactose (a yield by weight of 64%). The structures of the major components of Glc-OS and Gal-OS obtained by this method were identified as cellotriose and 0-bo-galactopyranosyl-(1--*4)-O-~o-galactopyranosyl-(l~4)-o-glucopyranose (4’-galactosyl-lactose), respectively. [Key words: galactosyl-lactose,
gluco-oligosaccharide, transglucosylation,
galacto-oligosaccharide,
Oral administration of galacto-oligosaccharide (GalOS), a transgalactosylation product of lactose, promotes proliferation of intestinal bifidobacteria (1, 2). Bifidobacteria in the human large intestine are helpful in the maintenance of good health (3, 4). Gal-OS, which is used as an energy source by bifidobacteria, is known to be a bifidus factor, and the development of an efficient GalOS production method is highly desirable. We previously found that 3 strains of yeast, Sterigmatomyces elviae CBS81 19, Rhodotorula minuta IF0879, and Sirobasidiurn magnum CBS6803, possess high levels of transgalactosylation activity suitable for Gal-OS production (5). Efficient Gal-OS production conditions for intact cells of S. elviae CBS8119 was also studied (5). In addition, we showed that the Gal-OS-producing reaction was catalyzed by a ;3-galactosidase with a high level of transgalactosylation activity (6). Many ,3-galactosidases are known to catalyze not only the hydrolysis of ,3-galactoside linkages but also ,3-glucoside linkages (7-11). We found that toluene-treated resting cells of S. elviae CBSSll9, R. minuta IF0879, and S. magnum CBS6803 produced gluco-oligosaccharide (Glc-OS) at high yields when cellobiose was used as the substrate. It may be that the Gal-OS-producing ;3-galactosidase of these yeasts also recognizes ,3-glucoside linkages and has strong transglucosylation activity. In this paper, we describe the optimal conditions for efficient Glc-OS production from cellobiose and Gal-OS production from lactose by a representative strain, R. minuta IF0879, and identify the main transglycosylation products.
* Corresponding
yeast,
Rhodotorula minuta,
cellotriose,
MATERIALS
METHODS
4-
transgalactosylation]
Microorganisms R. minuta IF0879,
AND
and cultivation and S. magnum
S. elviae CBS81 19, CBS6803, which were
selected as Gal-OS producers in our previous study (5), were used. A loopful of cells subcultured on a potatodextrose agar slant was inoculated into 5 ml of medium containing 20 g glucose, 10 g lactose, 10 g peptone, 10 g yeast extract, 5 g (NH&S04, 3 g K2HP04, 1 g KH2P04, 0.5 g MgS04. 7H20 in 1 I of distilled water (pH 7.0) in a test tube and cultivated aerobically at 30°C for 48 h. Then, 1 ml of the culture was transferred into 50ml of the same fresh medium in a 500-ml flask and cultivated aerobically at 30°C for 60 h on a reciprocal shaker. The cells were harvested by centrifugation at 10,000 x g at 5°C for 10 min, washed with 100 mM potassium phosphate buffer (pH 6.0), and resuspended in 20mM potassium phosphate buffer (pH 6.0) to provide a concentrated cell suspension (wet weight, 500 mg/ml). Glc-OS and Gal-OS production by cells One drop of toluene was added
toluene-treated
to 5 ml of cell suspension and stirred vigorously. The standard reaction mixture contained 200 mg/ml substrate (cellobiose or lactose) in 100 mM potassium phosphate buffer (pH 6.0), and 5 ml of toluene-treated cell suspension in a total volume of 50 ml, and was incubated at 60°C without shaking. The reaction was stopped by boiling for 10min and the amounts of Glc-OS or Gal-OS produced in the reaction mixture were determined, as described below under “Analytical Method”. The transglycosylation activity is expressed as the total amount of Glc-OS or Gal-OS produced from cellobiose or lactose under this standard reaction condition. Glc-OS and Gal-OS production with cell culture The cell suspension (5 ml) was transferred to 45 ml of Glc-OS- or Gal-OS- production medium in a 500-ml
author.
124
VOL. 82, 1996
GLUCO-OLIGOSACCHARIDE
flask. The Glc-OS production medium contained 200 or 400g cellobiose, 0.9 g yeast extract, 4.5 g (NH&S04, 2.7 g K2HP04, 0.9 g KH2P04, 0.45 g MgS04. 7Hz0, and 0.9 g CaCOr in 900 ml distilled water, pH 6.0. The GalOS production medium was the same as that used for Glc-OS except that it contained 360g lactose instead of cellobiose. The saccharides (cellobiose and lactose) and CaCOr were sterilized separately from the other media components and were added aseptically. The cultivation was carried out aerobically at 30°C on a reciprocal shaker, and was stopped by boiling for 10 min. Analytical method The amounts of Glc-OS, GalOS and other saccharides produced were assayed by high-performance liquid chromatography using an L6000 pump (Hitachi, Tokyo), a Shodex SE-50 refractive index detector (Showa Denko Co., Tokyo), and a CR1B data processor (Shimadzu, Kyoto) with a Shodex ION Pak S-801 column (4.6~ 5OOmm; Showa Denko Co.) under the following conditions: column temperature, 80°C; mobile phase, distilled water; flow rate, 0.6 ml/min. Isolation and identification of products After 24-h cultivation for Glc-OS production from 200mg/ml cellobiose, the culture broth (20ml) was centrifuged (lO,OOOxg, 10 min) to remove the cells. The resulting supernatant was applied to an active carbon column (25 x 500 mm) equilibrated with distilled water, and the column was rinsed with 1 I of 4% (v/v) ethanol to remove residual glucose and cellobiose. Trisaccharide, the main product of Glc-OS, was eluted with 11 of 8% (v/v) ethanol. The eluate was concentrated to 20ml in vucuo, and when freeze-dried yielded 824 mg trisaccharide. Trisaccharide from Gal-OS was also isolated from the culture broth as previously described (5) and 1,066 mg of trisaccharide was obtained. l3C nuclear magnetic resonance (13C-NMR) spectra of the products in D20 were recorded with a JNM-GX 400 spectrometer (Nihondenshi Co. Ltd., Tokyo) using 3(trimethylsilyl)-propionic acid -d4 sodium salt as an internal standard. Infrared spectra were recorded with a 1600 FT-IR spectrometer (Perkin-Elmer, Norwalk, Conn., USA) as a KBr disc. Authentic cellotriose was purchased from Seikagaku Co. (Tokyo) and 4’-galactosyl-lactose from Sigma Chemical Co. (St. Louis, MO, USA).
Transglycosylation activities of S. elviue CBS8119, R. minutu IF0879 and S. magnum CBS6803 Transglycosylation (transgalactosylation and transglucosylation) activities were investigated for three yeast strains with strong Gal-OS production activities. As shown in Transgalactosylation of Gal-OS-producing
Microorganism S. elviae CBS81 19 minuta IF0819 S. magnum CBS6803 R.
and transglucosylation microorganisms
Oligosaccharide
formed
activities
(mg/ml)
Gal-OS
GIc-OS
45.5 44.0 36.8
35.3 59.2 54.0
Transgalactosylation and transglycosylation activities are expressed as the total amounts of Gal-OS or Glc-OS formed in the reaction mixture. In each reaction, 200 mg/ml lactose or cellobiose was used as the substrate (Materials and Methods). The reactions were carried out at 60°C for 2 h in potassium phosphate buffer, pH 6.0.
PRODUCTION
125
Table 1, each strain exhibited strong transglucosylation activity, and produced Glc-OS from cellobiose in addition to its transgalactosylation activity. Though S. elviae CBS81 19 showed the highest Gal-OS-producing activity, it was not the best strain for Glc-OS production. R. minuta IF0879 had the highest Glc-OS-producing activity and was selected for further study. Reaction conditions for Glc-OS and Gal-OS production by toluene-treated resting cells of R. minufu IF0879 The effects of pH and temperature on the production of Glc-OS or Gal-OS were examined. As shown in Figs. 1A and lB, the optimal pH and temperature were almost the same for both Glc-OS and Gal-OS productionabout 4.0 to 6.0 and 7O”C, respectively. The activity at 60°C was 80% of the maximum. However the reaction mixture turned brown at 7O”C, so a reaction temperature of 60°C was used to avoid coloring. Profiles of the effects of temperature and pH on activity were almost same for the transgalactosylation and transglycosylation reactions. These results indicated that the transgalactosylation and transglycosylation reactions appeared to be catalyzed by the same P-glycosidase, with a broad specificity, which recognized both $-glucoside linkages and ,3-galactoside linkages. Production of Glc-OS and Gal-OS by toluene-treated resting cells of R. minutu IF0879 The time courses of Glc-OS and Gal-OS production are shown in Figs. 2A and 2B, respectively. Glc-OS production from 200mg/ ml cellobiose reached a maximum of 70 mg/ml (a yield by weight of 35x), after 8-h incubation at 60°C which was comprised of 52 mg/ml trisaccharide and 18 mg/ml tetrasaccharide. The amount of Glc-OS produced rapidly decreased as the level of by-product glucose increase (Fig. 2). Pentasaccharide or larger oligosaccharides were scarcely formed during the reaction. Gal-OS production was maximal at 76mg/ml and was comprised of 63 mg/ml trisaccharide and 13 mg/ml tetrasaccharide, which was produced from 200 mg/ml lactose after 24-h incubation at 60°C (Fig. 2B). Only low levels of Gal-OS I
7 e
100
.-f 3 g
1
I
100 -
I
s-s .1 80 .=
?360
RESULTS AND DISCUSSION
TABLE I.
AND GALACTO-OLIGOSACCHARIDE
B
80 60 -
-
40
40 20 -
20.
2
I
4
PH
I
6
\
13
8
’
10
030405080708090 Temperature (“Cl
FIG. 1. Effects of pH and temperature on Glc-OS and Gal-OS production. (A) pH: Mixtures containing 0.5 ml of a toluene-treated cell suspension of R. minuta IF0879, 200 mg/mI cellobiose (for GlcOS) or lactose (for Gal-OS) and 100 mM buffer at various pH values in a total volume of 5 ml were incubated at 60°C for 1 h. Buffers used were sodium acetate-HCl (pH 1.3 to 2.3), sodium acetate (pH 3.3 to 6.0). uotassium ohosnhate (PH 6.0 to 7.0), and Tris-HCl (PH 7.5 to 8.8): (B) Temperaturk Mixtures containing 0.5 ml of a toluene-treated cell suspension of R. minuta IF0879, 200 mg/mI cellobiose (for GlcOS) or lactose (for Gal-OS), and 100 mM potassium phosphate buffer (pH 6.0) in a total volume of 5 ml were incubated at various temperatures for 1 h. Symbols: 0, Glc-OS; 0, Gal-OS.
ONISHI AND YOKOZEKI
126
J.
200
B i
0
0 0
102030405060 Time
0
10
20
30
Time
(h)
40
50
60
(h)
FIG. 2. Time courses of Glc-OS (A) and Gal-OS (B) production by toluene-treated resting cells. A mixture containing 5 ml of a toluene-treated cell suspension of R. minuta IF0879, 2OOmg/ml cellobiose (for Glc-OS) or lactose (for Gal-OS) and 100mM potassium phosphate buffer (pH 6.0) in a total volume of 50 ml was incubated at 60°C. Symbols: A, cellobiose or lactose; 0, glucose; 0 , galactose; 0, trisaccharide; A, tetrasaccharide; n , total Glc-OS or Gal-OS. degradation and by-product glucose formation were observed during subsequent reaction for 24 h or longer. From these observations, Glc-OS- and Gal-OS-producing +glycosidase seems to possess high ,%glucoside linkage hydrolyzing activity but little ,3-galactoside linkage hydrolyzing activity. On the other hand, it appears that another +glycosidase (,3-glucosidase), catalyzing only the hydrolysis of ,%glucoside linkages, may be present in the cells of R. minuta IF0879. To determine whether or not the production of Glc-OS and Gal-OS is catalyzed by the same enzyme, as well as to clarify the mechanism of the rapid degradation of Glc-OS, purification and characterization of the enzyme is now in progress. Effect of glucose The effect of glucose on Glc-OS and Gal-OS production by R. minuta IF0879 was also examined. As shown in Fig. 3, glucose was inhibitory to both transglycosylation and transgalactosylation, the
FERMENT.
BIOENG.,
amounts of Glc-OS or Gal-OS formed decreasing as more glucose was added. This phenomenon has previously been observed in transgalactosylation reactions with toluene-treated resting S. elviae CBS8119 cells (5), and we expected that removal of by-product glucose from the reaction mixture would improve Glc-OS productivity. Glc-OS and Gal-OS production with cell culture In order to remove the by-product glucose from the reaction mixture, the enzymatic reaction was carried out accompanied by cell growth (the cell culture method). Just as Gal-OS production by S. elviae CBS8119 previously increased using the cell culture method (5), so the productivities of both Glc-OS and Gal-OS by R. minuta IF0879 were also improved by employing the same system. As a result, 117 mg/ml Glc-OS (a yield by weight of 59x), comprising 68 mg/ml trisaccharide and 49 mg/ml tetrasaccharide, was produced from 200 mg/ml cellobiose after 34-h cultivation. With a higher concentration of cellobiose of 400 mg/ml, 201 mg/ml Glc-OS (a yield by weight of 51x), comprising 130 mg/ml trisaccharide and 71 mg/ml tetrasaccharide, was produced after 64-h cultivation at 30°C (Fig. 4). In a quantitative study of Glc-OS formation from cellobiose by transglucosylation using j-glucosidase of Aspergillus niger, a maximal yield from 133 mg/ml cellobiose of 23.5% (by weight) was reported (12). Recently, Christakopoulos et al. (13) reported more efficient Glc-OS formation using ,3-glucosidase of Fusarium oxysporum: Glc-OS (cellotriose) was formed from 10% (v/w) cellobiose with a maximal yield of 37% (by weight). In the present study, the enzymatic reaction of R. minuta IF0879 accompanied by cell growth, gave a Glc-OS productivity considerably superior to that using the ,9glucosidase of F. oxysporum. As far as we know, this is the best method of Glc-OS production reported so far. In the case of Gal-OS production, the enzymatic reaction accompanied by maximized cell growth gave 230mg/ml Gal-OS (a yield by weight of 64?& com-
1.2
0.8 0.6
0 0’
I
I
0
100
lactose (for Gal-OS), 100mM potassium phosphate buffer (pH 6.0), and the indicated concentrations of glucose in a total volume of 50 ml Symbols:
0, Glc-OS;
60
80
0.0 100
Time(h)
FIG. 3. Effect of glucose concentration on Glc-OS and Gal-OS production. A mixture containing 5 ml of a toluene-treated cell suspension of R. minula IF0879, 200 mg/ml cellobiose (for Glc-OS) or at 60°C for 1 h.
40
_I
200
Glucose added (mglmi)
was incubated
20
0, Gal-OS.
FIG. 4. Time courses of Glc-OS production by a cell culture of R. minuta IF0 879. The cell suspension (5 ml) was inoculated into 45 ml of Glc-OS-producing medium containing 20 g cellobiose in a 500-ml flask, and was cultivated aerobically at 30°C on a reciprocal shaker. The cell concentration is expressed as the optical turbidity at 562 nm after 50-fold dilution with 0.1 N HCl. Symbols: A, cellobiose; 0, glucose; A, Glc-OS (tetrasaccharide); 0, Glc-OS (trisaccharide); n , total Glc-OS; x , cell concentration.
VOL. 82. 1996
GLUCO-OLIGOSACCHARIDE
AND GALACTO-OLIGOSACCHARIDE
PRODUCTION
127
Utagawa of our laboratory for their encouragement and helpful suggestions throughout the study. We also thank Dr. H. Takesada for her technical expertise in obtaining the 13C-NMR spectra.
1.2
REFERENCES
0.0 0
20
40
60
80
100
Time(h) FIG. 5. Time courses of Gal-OS production by a cell culture of R. minuta IF0 879. The cell suspension (5 ml) was inoculated into 45 ml of Gal-OS-producing medium containing 18 g lactose in a 5Ol-ml flask, and was cultivated aerobically at 30°C on a reciprocal shaker. The cell concentration is expressed as the optical turbidity at 562 nm after 50-fold dilution with 0.1 N HCl. Symbols: A, lactose; 0, glucose; 0 , galactose; A, Gal-OS (tetrasaccharide); 0, Gal-OS (trisac-
charide);
n
, total Gal-OS; x , cell concentration.
prised of 185 mg/ml trisaccharide and 45 mg/ml tetrasaccharide, from 360mg/ml lactose after cultivation for 60 h (Fig. 5). This Gal-OS productivity level was almost equal to that achieved by the enzymatic reaction of S. elviae CBS8119 accompanied by cell growth, which is the best Gal-OS production method reported to date (5). Identification of Glc-OS and Gal-OS The structures of the trisaccharide products of Glc-OS and GalOS, were identified by 13C-NMR and infrared spectra, which were compared with authentic cellotriose and 4’galactosyl-lactose spectra. All the spectra of the trisaccharides of Glc-OS and Gal-OS agreed well with those of cellotriose and 4’-galactosyl-lactose, respectively (data not shown). From these results, it was clear that a ;3 1,4 linkage was formed during transglycosylation, and that cellotriose and 4’-galactosyl-lactose were the main products of Glc-OS and Gal-OS, respectively. ACKNOWLEDGMENTS We thank
Drs.
K. Kubota,
T. Tanaka,
A. Yamashiro,
and
T.
1. Tanaka, R., Takayama, H., Morotomi, M., Kuroshima, T., Ueyama, S., Matsumoto, K., Kuroda, A., and Mutai, M.: Effects of administration of TOS and Bifidobncterium breve 4006 on the human fecal flora. Bifidobacteria Microflora, 2, 17-24 (1983). 2. Ohtsuka, K., Benno, Y., Endo, K., Ozawa, O., Ueda, H., Uchida, T., and Mitsuoka, T.: Effects of 4’ galactosyllactose intake on fecal microflora. Bifidus, 2, 143-149 (1989). (in Japanese) 3. Mitsuoka, T.: Bifidobacteria and their role in human health. J. Ind. Microbial., 6, 263-268 (1990). 4. Hughes, D. B. and Hoover, D. G.: Bifidobacteria: their potential for use in American dairy products. Food. Technol., 45, 64-83 (1991). 5. Ooishi, N., Yamashiro, A., and Yokozeki, K.: Production of gala&o-oligosaccharide from lactose by Sterigmatomyces elviae CBS8119. Appl. Environ. Microbial., 61, 4022-4025 (1995). T.: Purification and properties of a 6. Onishi, N. and Tanaka, novel thermostable galacto-oligosaccharide-producing ,9-galactosidase from Sterigmatomyces elviae CBS8119. Appl. Environ. Microbial., 61, 4026-4030 (1995). 7. Got, R. and Marnay, A.: Isolement, purification et quelques caracteristiques physicochimiques de deux ,3-hexosidases du sue diaestif d’Helix aomatia. Eur. J. Biochem., 4. 240-246 (1968). M., Perez, N., and Cabezas, J. A:: $-Galac;osidase 8. Lianillo, and $-glycosidase activities of the same enzyme from rabbit liver. Int. J. Biochem., 8, 557-564 (1977). 9. Takase, M. and Horikoshi, K.: Purification and properties of a f-glycosidase from Thermus sp. Z-1. Agric. Biol. Chem., 53, 559-560 (1989). 10. Gorgan, D. W.: Evidence that ,%galactosidase of Sulfolobus solfataricus is only one of several activities of a thermostable ;?-D-glycosidase. AppI. Environ. Microbial., 57, 1644-1649 (1991). 11. Gabelsberger, J., Liebl, W., and Schleifer, K.: Purification and properties of recombinant ,3-glycosidase of the hyperthermophilic bacterium Thermotoga maritima. Appl. Microbial. Biotechnol., 40, 44-52 (1993). 12. Watanabe, T., Sato, T., Yosbioka, S., and Kuwahara, M.: Purification and properties of Aspergillus niger @-glucosidase. Eur. J. Biochem., 209, 651-659 (1992). 13. Christakopoulos, P., Bhat, M. K., Kekos, D., and Macris, B. J.: Enzymatic synthesis of trisaccharides and alkyl j!-D-glucosides by the transglycosylation reaction of Fusarium oxysporum. Int. J. Biol. Macromol., 16, 331-334 (1994).