Hydrolysis of Lactose by Microbial β -Galactosidases. Formation of Oligosaccharides with Special Reference to 2-0-β-D-galactopyranosyI-D-glucose

Hydrolysis of Lactose by Microbial -Galactosidases. Formation of Oligosaccharides with Special Reference to 2-0--D-galactopyranosyl-D-glucoseTAKAHIRO TOBA and SUSUMU ADACHI Laboratory of Animal Products Technology
College of Agriculture Tohoku University
Sendai, Japan
These disaccharides were isolated by preparative paper chromatography and their structures were explored by gas- liquid chromatography of their methyl- ated methanolyzates. Comparisons of mo- bility and color of the spots on paper electrophoretograms were also carried out. The disaccharides proved to be 2-0-/3-D-galact o pyr an osyl- D-glucose, 3-0- /3-D-galactopyran osyl-D-glucose, 6-0- /3-D-galactopyran osyl-D-glucose, 3-0-/3-D-galactopyran osyl-D-galactose, and 6-0-/3-D-galactopyranosyl-D-galactose.
INTRODUCTION
/3-Galactosidases which hydrolyze lactose to glucose and galactose also catalyze transgalac- tosylation reactions (7). The number of oligo- saccharides formed from lactose via transfer reactions varies from three to eleven (1, 9, 10, 13, 14, 24). Pazur has reported that/3-galactosi- dase from S. fragilis synthesizes the disaccha- r ides 3-0-/3-D-galactopyranosyl-D-glucose, 6- 0-/3- D-g al actopyranosyl-D-glucose (allolac- tose), and 6-0-/3-D-galactopyranosyl-D-galactose (9, 10). These disaccharides also have been reported as the transfer products of/3-galactosi-
Received August 28, 1977.
dases from Escbericbia coil and Helix pomatia (1, 20). Allolactose and 6-0-/3-D-galactopyrano- syl-D-galactose were produced by a mold en- zyme (19, 20), and 3-0-/3-D-galactopyranosyl- D-glucose and allolactose by calf-intestine en- zyme (20, 22).
The principal conclusion of most of these studies has been that only these three disaccha- rides occur during the hydrolysis of lactose by /3-galactosidases, although seven aldodisaccha- rides may be formed theoretically from glucose and galactose by this reaction. In recent years methods have been developed for the enzymat- ic synthesis of such disaccharides from sub- stances other than lactose, and the formation of 3-0-/3-D-galactopyranosyl-D-galactose (11, 16) and 4-0-/3-D-galactopyranosyl-D-galactose (6) by ~-galactosidases has been elucidated. These re- sults suggest that other disaccharides which have not been reported previously could be detected in lactose hydrolyzates.
In this paper, we report the detection of other transfer products from lactose with /3- galactosidases from S. fragilis and A. niger. The results confirm and extend previous knowledge of the disaccharides' transfer products.
MATERIALS AND METHODS
Materials
Two kinds of microbial /3-galactosidases, "Lactase Y" (/3-galactosidase from S. fragilis) and "Lactase A" (/3-galactosidase from A. ni- ger), were obtained from a commercial source (Kyowa Hakko Kogyo Co., Ltd.). Reagent grade sugars were obtained commercially: lac- tose, raffinose, and stachyose (Wako Pure Chemical Industries Ltd.), and melibiose (Difco Laboratories). Crystalline kojibiose, nigerose, and 2,4,6-tri-0-methyl-D-galactose were kindly given by Prof. K. Matsuda, and Prof. S. Hirase, respectively.
1978 J Dairy Sci 61:33--38 33
34 TOBA AND ADACHI
Hydrolysis Procedure of Lactose
For Lactase Y, 2.5 ml of reaction mixture containing .75 g of lactose, enzyme (about 350 ONPG units) (4), and 1.0 ml of Mcllvaine buffer (pH 7.0) were incubated at 40 C for several hours. Toluene was added to prevent growth of microorganisms. For Lactase A the above conditions were used except the pH was 4.0 and the temperature 55 C to stop the reaction. The reaction mixture was boiled for 5 to 10 rain to inactivate the enzyme.
Paper Chromatography of Sugars
Samples were evaporated under reduced pressure at 35 to 45 C. The samples were chromatographed on Toyo Roshi No. 514 filter paper (40 × 40 cm) with multiple ascents (three or four times). The solvent system was n-butanol-pyridine-water (6:4:3), and sugars were detected with aniline hydrogen phthalate (AHP) reagent (8). Rgal values, distance moved relative to D-galactose, were measured.
Two Dimensional Analysis of Sugars by Paper Chro mato-elactrophoresis
The lactose hydrolyzate was applied to the paper (40 x 40 cm) 16 cm from one side edge and 4 cm from bottom, and the same sample as a marker was spotted 1 cm from the two adjoining edges. After drying, a paper strip (36 × 14.5 cm) loaded with disaccharides was cut out of the paper with the guidance of the markers.
Paper electrophoresis was in .05 M-sodium borate at pH 9.8 at right angles to the chroma- tographic development. The applied potential was 400 V for 3 h which gave a final current of 21 to 35 mamps. The apparatus and technique of electrophoresis were essentially those of Aso and Hamada (2). Diphenylamine aniline phos- phoric acid (DAAP) (15), triphenyl tetrazolium chloride (TTC) (18) and AHP were used for detection and characterization of the disaccha- rides on paper electrophoretograms. M G values, the mobility of a compound relative to D-glu- cose, were calculated.
Gas-liquid Chromatography of Sugars
Gas-liquid chromatography of sugar tri- methylsilyl (TMS) ethers was carried out with a Hitachi model 063 gas chromatograph fitted a hydrogen flame ionization detector and a stain-
less steel column (2 m × 3 mm ID). Nitrogen was used as a carrier gas at a flow rate of 30 ml/min.
The sugars obtained from the transfer reac- tions were converted into their trimethylsilyl (TMS)-derivatives by shaking with reagent ( p y r i d i n e-hexamethyldisilazane-trifluoroace tic acid, 10:9:1, vol/vol) and separated on a column of Chromosorb W (Shimazu Seisakusho Ltd.) coated with 1.5% SE-52. The temperature was programmed from 150 to 300 C at 3 C/min.
Classification of sugars was on a comparison of retention times with those of D-glucose, D-galactose, maltose, cellobiose, melibiose, su- crose, trehalose, and raffinose, and in some cases by co-injection of the sample with stan- dards.
Sugar oximes were silylated by the method of (17) to form TMS-ethers which could be determined quantitatively by gas-liquid chroma- tography. For this analysis a column of 1.5% OV-17 on Shimalite W (Shimazu Seisakusho Ltd.) was programmed from 110 to 275 C at 3 C/min.
Gas-liquid chromatography of methyl glyco- sides of methylated sugars was on a Japan Electron Opticus Laboratory JGC-20KFP gas chromatograph. The column was stainless steel (2 m x 3 mm ID) and packed with 8% diethyl- ene glycol succinate on Diasolid L (Nihon Chro- mato Kogyo Kaisha Ltd.). It was operated at 200 C with nitrogen as a carrier gas at 20 ml/min.
Methylation of Disaccharides
Disaccharides were methylated by a pub- lished method (12) with the following quanti- ties: .5 to 2.0 mg of disaccharides was shaken with .2 ml methyl iodide, .2 ml N,N-dimethyl- formamide, and .2 g silver oxide at room temperature in the dark for 18 h. The mixture was filtered, the residue washed with chloro- form, and the filtrate evaporated to dryness. The products were boiled with 5% methanolic hydrogen chloride for 8 h. The resulting methyl glycosides were identified by comparing their retention times with those of authentic samples run under the same conditions. Authentic samples of methyl tri-0-methyl-D-glucoside were prepared from kojibiose, nigerose, lactose, and melibiose. Methyl 2,4,6-tri-0-methyl-D- gatactoside was prepared from its methyl ether.
Journal of Dairy Science Vol. 61, No. 1, 1978
FORMATION OF OLIGOSACCHARIDES FROM LACTOSE 35
Methyl 2,3,4-tri-0-methyl-D-galactoside was identified by comparing the gas chromatograms of methanolyzates of methylated raffinose and stachyose.
RESULTS
Formation of Oligosaccharides During/~-Galactosidase Action. S. fragilis ~-galactosidase
Samples were analyzed by paper and gas chromatography at hourly intervals during incu- bation. Paper chromatography revealed twelve oligosaccharides formed from lactose during hydrolysis (spot A to L in Fig. 1). The lactose spot is included in this count, because one oligosaccharide product overlapped the lactose spot as will be seen later. Maximum oligosac- charide formation occurred in 10 h under the conditions used. None of the oligosaccharides except lactose was found at zero time.
Figure 2 shows two chromatograms of sac- charides formed from lactose at 10 h hydrolysis with S. fragilis/3-galactosidase and 24 h with A. niger. Peaks indicating the presence of mono-, di-, and trisaccharides were obtained in these hydrolyzates. The retention times of peaks 2 to 10 corresponded roughly to those of the standard disaccharides and peak 11 to trisaccha- rides.
Hydrolyzate prepared with S. fragilis t3-galac- tosidase was developed by preparative paper chromatography. Bands corresponding to spots
I I I I I I I t I ~ l Standard 2 4 12 32 48 2 4 12 32 48hr
S .fragilis A .niger ~gatactosidase ~-galactosidase
FIG. 1. Paper chromatogram of oligosaccharides formed from lactose during hydrolysis by two mi- crobial 13-galactosidases.
S.fracj i l is 13-galactosida se
910
2 A niger ~-galactosidase
i i - - t i I 0 10 20 30 4 0 50 6 0 70 8 0 90min
,aetent~o. t~me
Temperature
FIG. 2. Representative gas chromatogram of TMS- ethers of transfer products from lactose produced with microbial /3-galactosidases. Peak identification: 1-glu- cose plus galactose; 2-lactose; 5-oligosaccharide A; 6- lactose plus B; 7-A plus C; 9, IO-D plus E; and l l - F~L.
A, B, C, D, E, and F through L (Fig. 1), as detected by guide spots, were extracted with water and analyzed by gas-liquid chromatogra- phy. The retention times revealed that A, B, C, D, and E were disaccharides, while F~L were probably trisaccharides. Disaccharide(s) corre- sponding to peaks 3, 4, and 8 (Fig. 2) were not found in the fractions obtained from the paper strips. Oligosaccharides A, D, E, G, H, I, and J were produced in the large amounts.
A. niger t3-galactosidase
Paper chromatographic examination of lac- tose hydrolyzates produced with A. niger fJ- galactosidase revealed that ten oligosaccharides were produced by the enzyme (spot A'~J in Fig. 1). Figure 1 shows that the disaccharides were the same as those produced by S. fragilis /3-galactosidase but the trisaccharides were not. The oligosaccharide and especially trisaccharide production was relatively small in comparison with S. fragilis /3-galactosidase. Oligosaccharide A, D, E, G, and H were produced in the large amounts.
Determination of the Transfer Oligosaccharides During Hydrolysis
During hydrolysis of 30% lactose solution, portions of the hydrolyzate were analyzed quantitatively as sugar oxime TMS-ethers by gas-liquid chromatography. The amounts of each sugar was determined by peak area. As shown in Fig. 3, the course of lactose deple-
Journal of DairY Science Vol. 61, No. 1, 1978
36 TOBA AND ADACHI
jected to permethylation and methanolysis, and the resulting products were analyzed by gas-
i ~ O [ liquid chromatography. The data are summa- rized in Table 1. The three disaccharides, A, B, and D gave peaks corresponding to methyl
:80~ S 2,3,4,6-tetra-0-methyl-D-galactoside, A also gave 1~. / , 6 , 6 methyl 2,4,6-tri-0-methyl-D-glucoside, and B
_ methyl 3,4,6-tri-O-methyl-D-glucoside, and D ! 6 0 F methyl 2,3,4-tri-0-methyl-D-glucoside. There-
fore, disaccharides, A, B, and D were 3-0-3-D- galactopyranosyl-D glucose. 24)-3-D-galactopy-
4 0 ~ ranosyl-D-glucose, and 6-0-/3-D-galactopyrano- I I 1 % syl-D-glucose, respectively. Gas-liquid chroma-
20 ~ tographic analysis of disaccharides C and E ~ showed that these methanolyzates contained
methyl 2,3,4,6-tetra-0-methyl-D-galactoside, to O l ~ h r hr and C also contained methyl 2,4,6-tri-0-methyl-
0 10 20 30 40 50 0 10 20 30 40 50 Time Of incubation
S .fragili s" J3-gala ctosidase p-galactosida se
FIG. 3. Time course of lactose hydrolysis with microbial 3-galactosidases. ~ monosaccharides, --<>--o-- disaccharides, • • disaccharides except lactose, ~ 3-1,2- and ~-l,3-1inked disaccharides,
A A fl-l,6-1inked disaccharides, - - lactose, trisaccharides.
tion was consequently known. The amounts of transfer products with S. fragilis 3-galactosidase were larger than with A. niger. The amounts of 1,6-1inked disaccharides and trisaccharides was especially remarkable.
Characterization of Transfer Disaccharides
Each of the disaccharides isolated by the preparative paper chromatography was sub-
D-galactoside, and E contained methyl 2,3,4- tri-0-methyl-D-galactoside. Thus, disaccharides, C and D were 3-0-~-D-galactopyranosyl-D- galactose and 6-0-3-D-galactopyranosyl-D-galac- tose, respectively. These identities were sup- ported by the other chromatographic and elec- trophoretic evidences, as shown in Table 2.
Peaks of relative retention time 2.36 in the methanolyzate of methylated disaccharide frac- tion C and 2.40 in E could not be positively assigned to any methyl tri-0-methyl glycoside listed on Table 1.
DISCUSSION
Glycosidases catalyze transfer as well as hydrolytic reactions; that is, the sugar residue forming the glycone part of the substrate molecule may be transferred to water or to some other hydroxylic acceptor such as another
TABLE 1. Gas chromatographic identification of methanolyzates of the methylated transfer disaccharides.
Relative retention times (T) a Methyl glycoside Authentic Samples b
2,3,4,6-Tetra-O-methyl-D-galactose 3,4,6-Tri-O-methyl-D-glucose 2,4,6-Tri-O-methyl-D-glucose 2, 3,4-Tri-O-methyl-D-glucose 2, 3,6-Tri-O-methyl-D-glucose 2,4,6-Tri-O-methyl-D-galactose 2,3,4-Tfi-O-methyl-D-galactose
.83, 1.00 1.75, 2.07 B 1.84, 2.61 A 1.51, 2.06 D 1.91, 2.51 Lactose 2.25, 2.49 C 3.73 E
.83, 1.00 1.76, 2.08 1.85, 2.61 1.51, 2.07 1.91, 2.50 2.25, 2.51 3.72
avalues relative to methyl 2, 3,4,6-tetra-O-methyl~-D-galactopyranoside ( 5.90 min). bcorresponding to the signs in Fig. 1 and Table 2.
Journal of Dairy Science Vol. 61, No. 1, 1978
FORMATION OF OLIGOSACCHARIDES FROM LACTOSE
TABLE 2. Properties of the transfer disaecharides.
37
Color on paper chromatogram or paper electrophoretogram Sign Rgal M G AHP DAAP TTC
A .87 .73 Dark brown Dark green Red B .82 .46 Yellowish brown or orange Orange Negative C .74 .72 Dark brown Dark green Red Lactose .74 .40 Brown Blue Red D .65 .76 Chocolate brown Dark green Red E .57 .78 Chocol ate brown Dark green Red
sugar or alcohol (7). One also may regard the transfer reaction as competition between water and sugar molecules for the "substrate-enzyme" complex (1). The fact that increases in lactose concentration increase the amount of transfer oligosaccharides, agrees with this idea (14). With/3-galactosidases from various sources, this phenomenon was studied by Aronson (1), Pazur (9, 10), Wallenfels (19, 20, 22) and their co-workers.
Aronson (1) and Wallenfels and co-workers (20) demonstrated the intermediate formation of several oligosaccharides by transgalactosyla- tion from lactose with crude enzyme prepara- tions of S. fragilis, E. coli, mold, snail (Helix pomatia), and calf-intestine. Pazur (9, 10) reported the enzymatic synthesis of five 0-/3-D-galactosyl oligosaccharides by lactase from yeasts. With these enzymes,/3-D-galactosyl transfer occurred preferentially at the primary alcohol group of D-glucose and formed allolac- rose (6-O-fl-D-galactopyranosyl-D-glucose). With D-galactose as a receptor, the product was galactobiose (6-0-/3-D-galactopyranosyl-D-galac- tose); with the non-reducing D-galactosyl resi- dues of lactose or galactobiose as acceptors, the products were lactotriose or galactotriose. In general, 6-O-/3-D-galactopyranosyl-D-glucose and lactotriose were predominant products.
It has been further reported that/3-galacto- sidases from various sources differ in substrate specificity. Calf-intestinal /3-galactosidase of high purity apparently differed in substrate specificity from that of E. coi l The hydrolysis of different galactosylglucoses showed different rates with enzyme preparations from E. coli and from calf-intestine, namely, 1,6>1, 4>1, 3>1, 2, and 1,251, 351, 451,6, respectively (3). With E. coli /3-galactosidase the order for synthesis was 1,6>1, 4>/1,3 (21, 23). Judging
from our results /3-galactosidases from both S. fragilis and A. niger seemed to resemble the E. coli enzyme more than the calf enzyme. The order of velocity of synthesis of galactodisac- charide with /3-galactosidase from E. coli (23) and yeast (11) was 1,6>1,3. The results with /3-galactosidases of S. fragilis and A. niger also agree with this order.
The results show that all kinds of 0-/3-D-ga- lactosyl-D-glucose except the /3-1,1 linkage are in the lactose hydrolyzates. Consequently, it seems that the activated galactosyl group re- leased from lactose was transferred to C-2,3, or 6 of D-glucose which had been produced by the hydrolysis of lactose. Beck and Wallenfels reported that 2-0-/3-D-galactopyranosyl-D-glu- cose could be synthesized chemically from 4,6-benzyliden-~-methyl glucoside and s-ace- tobromogalactose (3). Gakhokidze synthesized this disaccharide by the zinc chloride catalyzed condensation of 2,3,4,6-tetra-0-acetyl-D-galac- tose and 1,3,4,6-tetra-O-acetyl-D-glucose (5). Enzymatic synthesis of 2-0-/3-D-galactopyrano- syl-D-glucose has not been reported.
It is possible that seven disaccharides other than lactose can be synthesized from lactose, if the activated galactosyl group also is transferred to the C-2,3,4, or 6 position of D-galactose. In this experiment, the presence of five of these disaccharides was confirmed, including two galactodisaccharides identified as 3-0-/3-D-galac- topyranosyl-D-galactose and 6-0-fi-D-galacto- pyranosyl-D-galactose. 3-0-/3-D-Galactopyrano- syl-D-galactose has been synthesized enzymat- ically by the transferase action of yeast/3-galac- tosidase on o-nitrophenyl /3-D-galactoside and D-galactose (11) and by the enzymatic rever- sion action of emulsin on D-galactose (16). But the synthesis of this galactodisaccharide from lactose by fl-galactosidases of S. fragilis and A.
Journal of Dairy Science Voi. 61, No. 1, 1978
3 8 TOBA AND ADACHI
niger has n o t been repor ted . An add i t iona l d isacchar ide spo t was pro-
duced by A. niger 3-galactosidase and resolved on paper c h r o m a t o - e l e c t r o p h o r e t o g r a m s . The s t ruc tu re of this d isacchar ide will be discussed in a s u b s e q u e n t paper .
ACKNOWLEDGMENTS
We would like to acknowledge K. Matsuda ( T o h o k u Univers i ty) for the gifts of koj ib iose and nigerose, and S. Hirase ( K y o t o Univers i ty of Indust r ia l Ar ts and Text i le Fibers) for 2 ,4 ,6- t r i -0-methyl-D-galactose .
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