APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1977, p. 137-146 Copyright © 1977 American Society for Microbiology
Vol. 33, No. 1 Printed in U.S.A.
Hydrolysis of Lactose by Immobilized Microorganisms KUNIO OHMIYA,* HIROSHI OHASHI, TAKESHI KOBAYASHI, AND SHOICHI SHIMIZU Department of Food Science and Technology, Nagoya University, Nagoya, 464 Japan Received for publication 1 June 1976
Cells ofLactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the /3-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial f3-galactosidases were negligibly affected by the immobilization. K, values of /3-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of /3-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free /8-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.
The use of immobilized whole cells as an enzyme source will generally eliminate the need for release of intracellular enzymes and for the succeeding purification steps; also, in some cases the enzymes are more stable if immobilized within their natural environment, the cell. These benefits have led to studies on immobilized microbial cells. Corynebacterium glutamicum (17) was entrapped in polyacrylamide gel to produce glutamic acid in successive batches of fresh medium. Streptomyces phaeochromogenes cells were heated at 80°C for 1.5 h and successfully immobilized in a collagen sheet by the method of aggregation of soluble collagen as a result of pH changes (19). Chibata et al. (3) immobilized Escherichia coli and other bacteria containing aspartase by various methods, and enzymatically active immobilized cells were obtained. Very recently, Martin and Perlman (9) reported the immobilization of Gluconobacter melanogenus cells capable of converting L-sorbose to L-sorbosone in polyacrylamide gel. Entrapping of microorganisms having ,-galactosidase activity, however, has not been reported. B-Galactosidase catalyzes the splitting of lactose to glucose and galactose and is fairly important for the treatment of milk for milkintolerant people who cannot digest lactose. We have already described the immobilization
of 3-galactosidase extracted from Aspergillus oryzae in polyacrylamide gel beads (15). In the present paper, we deal with the immobilization of microbial cells having 8-galactosidase activity and the enzymatic properties of the immobilized cells. MATERIALS AND METHODS Materials. Lactose was the product of BDH Chemicals Ltd. Glucose, galactose, and the other reagents were used in guaranteed grade. Achromycin (tetracycline hydrochloride) used as a growth inhibitor was purchased from Takeda Chemical Industries, Ltd. Cultivation of microorganisms. Lactobacillus bulgaricus (B-1), L. acidophilus (Ack), Streptococcus cremoris (H-61), and S. thermophilus (510) were kind gifts from the Institute of Animal Industry, Ministry of Agriculture and Forestry, Chiba, Japan. All these bacteria were propagated in a medium containing 2% peptone, 1% yeast extract, 0.5% meat extract, 1% glucose, 10% tomato juice, and 0.01% CaCl2, which was adjusted to pH 7.0 with concentrated NH4OH and sterilized at 120°C for 15 min (14). For batch growth, lactobacilli were cultivated at 37°C and streptococci at 30°C for 48 h under anaerobic conditions. E. coli (E-106) was given by the Laboratory of Fermentation Chemistry, Nagoya University. This strain has /8-galactosidase constitutively and was cultivated in peptone-glycerol medium (0.5% peptone, 0.1% glycerol, 2.65% Na2HPO4, 0.45% KH2PO4, 0.03% MgSO4, 0.1% NH4Cl, 0.0033% 137
138
OHMIYA ET AL.
CaCl2, pH 7.0) at 37°C for 18 h under aerobic conditions. Kluyveromyces (Saccharomyces) lactis (from Tokyo Tanabe Co., Ltd.), K. (S.) fragilis (H-2-3, from the Laboratory of Fermentation Chemistry of Nagoya University), and Candida pseudotropicalis var. lactosa (IFO-0616, from the Institute of Fermentation Organization, Osaka) were grown in medium containing 30% corn steep liquor and 1% glycerol at pH 6.0, 30°C, for 48 h under aerobic conditions. Neurospora crassa (IFO-96) was propagated in bouillon medium (1% meat extract, 1% peptone, 5% NaCl, pH 7.0) at 30°C for 7 days. The media described above were sterilized at 120°C for 15 min. All of the microbial cells were harvested by centrifugation at 10,000 x g for 15 min and washed in phosphate buffer (0.7 M, pH 6.5). These cell suspensions were used for cell immobilization as soon as possible. Isolation of free enzymes from microorganisms. Cell pellets of each microorganism obtained by centrifugation were disrupted in a mortar with alumina powder and a small volume of phosphate buffer under chilled conditions (5WC). Then the clear supernatant was separated from the cell debris by centrifugation at 10,000 x g for 15 min. The precipitate was resuspended in the same buffer and sonicated to liberate the enzyme remaining in the cell. The supernatant containing f8-galactosidase was dialyzed against a large volume of phosphate buffer overnight. The dialysate was lyophilized and stored as crude f8-galactosidase in a desiccator at low temperature until used. The crude 8-galactosidase from L. bulgaricus, E. coli, or K. lactis was solubilized in distilled water and is referred to as free enzyme of L. bulgaricus, E. coli, or K. lactis, respectively. Immobilization of microorganisms. These microorganisms and the free enzymes were immobilized in acrylamide gel beads according to the method described previously (15); these preparations are referred to as the immobilized cells and immobilized enzymes, respectively. The concentration of the cell suspensions was usually lower than 3 g of wet cell per 10 ml of phosphate buffer. Evaluation of effectiveness factor. The effectiveness factor, Ef, was evaluated as the ratio of the apparent rate of enzymatic reaction with diffusional effect to the rate under conditions without any diffusional effect. Such conditions were created by grinding immobilized microorganisms in a chilled mortar. Reuse of immobilized microorganisms. To use immobilized cells repeatedly, 1 g of immobilized cells was wrapped with nylon cloth that does not leak any beads. The enzyme reactor was pretreated before the first run as follows; it was immersed into 50 ml of 4.5% lactose solution (phosphate buffer, pH 6.5) at 30°C, shaken at 120 cycles/min for 10 min, and centrifuged at 1,000 x g for 1 min in the centrifugal tube with a rack at the middle to remove lactose solution in the reactor. Then every run was carried out under the same conditions for 30 min. After each run, the reactor was washed twice with 200 ml of the same buffer. The residual solution in the reactor was removed by centrifugation at 1,000
APPL. ENVIRON. MICROBIOL. x g for 1 min at room temperature. One cycle of reaction and washing took about 1 h. Standard assay of ,B-galactosidase activity. (i) ,Bgalactosidase activity in cell suspensions. One milliliter of the suspension containing 2 g of wet cells in 10 ml of phosphate buffer was mixed with 4 ml of the same buffer and 0.15 ml of toluene. This mixture was shaken for 1 h at 320 rpm, 30°C, to increase lactose penetration into the cells by toluene extraction of the lipid contained in the cell membrane. One milliliter of this toluene-treated cell suspension was combined with 5 ml of lactose solution (11.25%) and 6.5 ml of the same buffer and incubated at 30°C. After 15 min, a 3-ml aliquot of the reaction mixture was put immediately into a slender test tube preheated in boiling water and held for 3 min in order to stop further hydrolysis before inactivation of the enzyme. Then this aliquot was centrifuged to remove cells at 10,000 x g for 10 min after chilling in ice water. The amounts of glucose and galactose were determined by glucose oxidase (Glucostat special reagent kit, Worthington Biochemicals Co.) and by galactose dehydrogenase (galactose ultraviolet test, Boehringer Mannheim Co. Ltd.), respectively. (ii) Activity of crude j8-galactosidase. The solution of crude /8-galactosidase (0.5 ml) was mixed with phosphate buffer (pH 6.5, 7.0 ml) and lactose solution (11.25%, 5.0 ml) and incubated at 30°C for 30 min. The same procedure as described above, except for cell removal, was used for this reaction mixture to evaluate the amount of glucose released. (iii) ,B-Galactosidase activities of immobilized cells and immobilized enzymes. /8-Galactosidase activity in the polyacrylamide gel beads containing the microbial cells or extracted enzyme was assayed with 4.5% lactose in phosphate buffer (pH 6.5, 0.7 M) at 30°C for 30 min under reciprocal shaking at 100 cycles/min. The details have been described (15). TLC. Hydrolysis of lactose in skim milk was carried out with the immobilized cells of these microorganisms as follows: 30 ml of the solution prepared with skim milk powder was incubated with immobilized cells (10 g) at 30°C under reciprocal shaking for 5 h. Then the skim milk was deproteinized with 1% solutions of Ba(OH)2 and ZnSO4 and filtered through filter paper (Toyo, no. 5C). The filtrates were spotted on precoated thin-layer chromatography (TLC) plates (silica gel) purchased from Merck & Co. The chromatography was developed by a solvent (22) containing n-butanol-methanol-boric acid solution (0.03 M) (5:3:1) at room temperature for about 1 h. Sugars were detected by the anthrone-H2SO4 reagent (12). Properties of skim milk treated with immobilized cells. Intensity of sweetness, flavor quality, and degree of lactose hydrolysis were determined on both the milk treated with active immobilized cells and the control treated with inactive immobilized cells.
RESULTS
,1-Galactosidase activity of microorganisms. Nine species or strains of bacteria, yeast, and mold were selected and evaluated for
HYDROLYSIS OF LACTOSE
VOL. 33, 1977
inherent 83-galactosidase activity. The amounts of glucose (in milligrams) released from lactose by 100 mg of dry cells of each microorganism in 4.5% lactose solution during 15 min at 30°C, pH 6.5, and percentage of lactose hydrolysis are listed in Table 1. E. coli and K. lactis revealed very strong f3-galactosidase activity. The activity of L. bulgaricus was lower than those of the former but the highest among the lactic acid bacteria. Immobilization of microbial cells. Three species of microbial cells with high f3-galactosidase activity were entrapped in polyacrylamide gel beads. The cell suspensions used for the immobilization contained 2 g of cell pellet per 10 ml of phosphate buffer (pH 6.5, 0.7 M) because beads of normal size could not be obtained when larger amounts of cell pellet were used. Typical beads containing K. lactis cells are shown in Fig. 1. It can be clearly seen that the cells were dispersed homogeneously and entrapped in the acrylamide gel beads. Immobilization of free enzymes from these microorganisms was also done by this procedure. The activity yields of these immobilized cells and immobilized enzymes were in the range of 27 to 61% (Table 2). Properties of immobilized microorganisms. (i) Optimum pH. The pH range studied was 4.5 to 9.0. Figure 2 shows the pH profile for free enzymes, immobilized enzymes, and immobilized cells with enzymatic activity (L. bulgaricus, E. coli, and K. lactis). pH optima for activity of these immobilized cells were 5.5, 8.0, and 6.3, respectively. Each value was very close to that of free enzyme. (ii) Optimum temperature and thermostability. Immobilized cells of L. bulgaricus and E. coli showed the highest activity at near 55°C (Fig. 3A, B). The optimum temperature of immobilized K. lactis cells was 370 (Fig. 3C). Temperature-activity profiles of these immobilized cells were comparable to those of free enzymes. Thermostability of immobilized cells of the three microorganisms was determined by the residual activities after 24 h of incubation in the buffer at a given temperature. The residual activities evaluated according to the standard assay are depicted in Fig. 4. E. coli was the most stable among the three. The stability was slightly increased by the immobilization because the initial activity was maintained even after storage of immobilized E. coli cells at 45°C for 24 h. (iii) Ef value. The values for immobilized cells of L. bulgaricus, E. coli, and K. lactis were 1.1, 0.9, and 1.1, respectively. These values were almost equal to unity within the range of
139
TABLE 1. f&Galactosidase activity of microorganisms Glucose
Microorganisms (100 mg of dry cells)
pro- a
duced
Lactose
hydrolY-
hydroly
(mg) Lactobacillus bulgaricus (B-1) L. acidophilus (Ack) Streptococcus thermophilus (510) S. cremoris (H-61) Escherichia coli (E-106) Kluyveromyces (Sacchromyces) lactis K. (S.) fragilis (H-2-3) Candida pseudotropicalis var. lactosa (IFO-0616) Neurospora crassa (IFO-96)
21.1 3.8 3.1 3.0 174.0 266.4
3.8 0.7 0.5 0.5 31.0 47.4
4.7 2.0
0.8 0.4
9.1
1.6
Reaction was carried out at 30°C, pH 6.5, for 15 min in 4.5% lactose. a
experimental error. This suggests that the diffusional effects within the beads used in this experiment were not significant. (iv) K,,, value. The effect of substrate concentration on the hydrolysis of lactose by immobilized cells was investigated. The K,m values were evaluated by plotting the reciprocal of the substrate concentration against the reciprocal of the reaction velocity. The data for immobilized cells and for free enzymes at pH 6.5 are summarized in Table 3. The values for E. coli and L. bulgaricus were six- to sevenfold lower than that for K. lactis. All of these values were negligibly affected by the treatment for immobilization. (v) Ki value. Galactose and glucose effects on ,f-galactosidase activity were studied for the free enzymes and immobilized cells. The initial rates of lactose hydrolysis by immobilized cells of L. bulgaricus and E. coli were almost constant, although the concentration of galactose was increased. The same result was observed with the free enzymes from these bacteria. Since these results revealed clearly that these enzymes were not obviously inhibited by galactose, we estimated that the K, values of the enzymes were higher than the highest inhibitor levels tested. The activity from K. lactis, however, gradually decreased with increased amounts of galactose added. The inhibition effect with increasing concentrations of galactose on the activity of immobi1 zed K. lactis cells was examined at different concentrations of lactose; i.e., the reaction rate, v, was measured with varying inhibition concentrations, [I], and 1/v was plotted against [I]. This convenient method is described by Dixon (5). A straight line was obtained for each substrate concentration (Fig. 5). This result suggests that the inhibition type is either competitive or mixed com-
140
APPL. ENVIRON. MICROBIOL.
OHMIYA ET AL.
A .
4~~~~~IV
.F O
}e "
-
*
FIG. 1. Photomicrographs of immobilized cells of K. lactis in polyacrylamide gel beads in wet state (upper: 1 scale, 10 ,um; lower: 1 scale, 2.5 ,um).
TABLE 2. Activity yield of immobilized f&galactosidase Immobilized enzyme
Immobilized cell
Microorganism
Absolute
tya
CalcuClu
..Extracactivi-. Yield" Absolute activ- Yield tion ra- lated acx
%
ity
tioc
yieldy
21 42 1:2 27 5.0X 10-4 Lactobacillus bulgaricus 3.5 x 10-3 10 40 1:4 38 1.3 x 10-2 Escherichia coli 3.9 x 10-2 15 1:2 29 61 6.3 x 1O-3 Kluyueromyces lactis 4.8 x 10-2 a Expressed as micromoles of glucose per milliliter per minute per milligram of immobilized cells or enzymes. bRatio of activity of immobilized cells or enzymes to that of toluene-treated cells or free enzymes. c Ratio of activity of f8-galactosidase extracted from a given amount of cells to that of the toluene-treated cells.
HYDROLYSIS OF LACTOSE
VOL. 33, 1977
141
pH
FIG. 2. Effect ofpH on the f-galactosidase activity of L. bulgaricus (A), E. coli (B), and K. lactis (C) at 30°C in 0.7 M phosphate buffer. Symbols: (0) Immobilized microorganisms; (A) immobilized enzymes; (X) free enzymes.
Temperature ( °C ) FIG. 3. Effect of temperature on the f3galactosidase activity of L. bulgaricus (A), E. coli (B), and K. lactis (C) at pH 6.5 in 0.7 M phosphate buffer. Symbols are as in Fig. 2.
*-.,,
100-
?s0~~~~~~~~~ A~~~~A
60
*020
~
20A
20
30
40
0~~~~~~~~~A 0 B
50
30 40 50 Temperature ( OC )
20
30
40
50
FIG. 4. Thermostability of f3galactosidases of L. bulgaricus (A), E. coli (B), and K. lactis (C) at pH 6.5 in 0.7 M phosphate buffer. Symbols are as in Fig. 2.
142
APPL. ENVIRON. MICROBIOL.
OHMIYA ET AL.
TABLE 3. Kinetic parameters of immobilized ,3galactosidlases Microorganism
Vmaza
Ke (lacM) K, and K,' (galactose, mM) K, (glucose, mM)
Lactobacillus bulgaricus 00b 230 (non)c 4.2 2.9 x 10-4 Immobilized cell 00 230 (non) 1.3 x 10-2 4.2 Free enzyme Escherichia coli 76 (non) X 5.4 7.5 x 10-7 Immobilized cell 70 (non) X 5.4 1.1 x 10-3 Free enzyme Kluyveromyces lactis 380 (non) 30 47 and 103(mix)d 8.3 x 10-4 Immobilized cell 340 (non) 46 and 103(mix) 30 3.3 x 10-4 Free enzyme a Expressed as millimoles per minute per milligram of beads or free enzyme per liter of reaction mixture. b Ki and Ki' values were higher than the highest inhibitor levels tested. c non, Noncompetitive inhibition. d mix, Mixed competitive inhibition.
201
10 60 -40 -80 ?
-20
0
20
40
Galactose (mM )
20 0 40 60 -20 Ga I actose ( mM ) FIG. 5. Effect of galactose on the rate of lactose hydrolysis offree enzymes (dotted line) and immobilized cells (solid line) of K. lactis at various lactose concentrations. Lactose concentrations (millimoles per liter of reaction mixture): (I) 31.4; (II) 52.6; (III) 105.2; (1) 39.1; (2) 62.5; (3) 173.6.
-60
-40
petitive (4). For distinguishing between these inhibition types, [SI/v was plotted against [I] at different values of [SI (substrate concentration). Straight lines were drawn, and they intersected at a point where [I] = Ki' (Fig. 6). K, and K,' values of 3-galactosidase in K. lactis cells immobilized in acrylamide gel were 47 and 103 mM, respectively. These values were almost the same as those of the free enzyme. The effects of glucose at various concentrations on the activity of these microorganisms were also examined by determining the amounts of galactose released from lactose. The patterns of the Lineweaver-Burk plots appeared to be those of typical noncompetitive inhibition (Fig. 7 to 9). From these plots, K,
FIG. 6. Effect of galactose on the rate of lactose hydrolysis offree enzymes (dotted line) and immobilized cells (solid line) of K. lactis at various lactose concentrations. Lactose concentrations, [SI (millimoles per liter of reaction mixture): (I) 31.4; (II) 52.6; (III) 105.2; (1) 39.1; (2) 62.5; (3) 173.6. [S]/v was plotted against [I].
values of glucose were evaluated by plotting the reciprocal values of Vmax obtained at a given concentration of glucose against the concentration of glucose added. These K, values were close to the respective values for free enzymes (Table 3). K. and Ki values obtained in this experiment for f3-galactosidase are summarized in Table 3. Reusability of immobilized cells. The reusability of these immobilized microbial cells, which were wrapped with nylon cloth as a reactor, was tested repeatedly (Fig. 10). The reactor from K. lactis showed a negligible decrease in activity upon initial contact with lactose solution. Those from E. coli and L. bulgaricus showed 25 and 35% decreases, respectively. The decreasing profile of immobilized enzyme from E. coli was consistent with that of immobilized cells (Fig. 10). Properties of the skim milk solution treated with immobilized cells. Changes in lactose of the deproteinized filtrate of the skim milk solu-
VOL. 33, 1977
HYDROLYSIS OF LACTOSE
] -200
100
-100
1 /(Lactose)
(M-1 )
FIG. 7. Inhibition pattern for free f&galactosiclase (dotted line) and immobilized cells (solid line) of bulgaricus with lactose as the varied substrate and glucose as the varied inhibitor. Glucose concen trations (millimoles per liter of reaction mixture): I) 106.7; (II) 53.3; (III) 0; (1) 95.2; (2) 47.6; (3) 0
La
143
tions that were treated with these immobilized cells were detected by TLC (Fig. 11). The position of authentic lactose (A) followed that of authentic galactose (B) and glucose (C). Deproteinized filtrate of the skim milk solution without any enzymatic treatment (control) contained only lactose (D). Figure liE suggests that lactose in the skim milk was completely hydrolyzed to galactose and glucose by immobilized cells of E. coli. The enzymatic actions of immobilized cells of L. bulgaricus and K. lactis were consistently detected by the appearance of glucose and galactose spots on the TLC (F and G, respectively). In addition to these components, unknown spots faintly appeared near the starting points. The skim milk treated with immobilized cells seemed to weaken the typical milk flavor (Table 4). The sweetness of the skim milk was enhanced as lactose hydrolysis proceeded.
A
300 c
E 200
E >
C
100'
------ ... --..
0
50
100
200 0
150
50
100
150
200
1/ (Lactose) (M_1) FIG. 8. Inhibition patterns for immobilized cells (A) and free f3galactosidase (B) of E. coli with lactose as the varied substrate and glucose as the varied inhibitor. Glucose concentrations (millimoles per liter of reaction mixture): (1) 55.6; (2) 0; (I) 53.3; (II) 26.7; (III) 0. Dotted lines were symmetrical parts of the plots
against ordinates.
-40
-20
0
20
40
-40
-20
0
20
40
1/(Lactose) (M-1) FIG. 9. Inhibition patterns for immobilized cell (A) and free ,&galactosidase (B) of K. lactis with lactose as the varied substrate and glucose as the varied inhibitor. Glucose concentrations (millimoles per liter of reac-
tion mixture): (1) 76.2; (2) 38.1; (3) 0; (I) 55.3; (II) 26.7; (III) 0.
144
OHMIYA ET AL.
.
APPL. ENVIRON. MICROBIOL.
*.
..
0~~~~~~ ,60 a 40.
a
TABLE 4. Characteristics of skim milk treated with immobilized microorganisms Skim milk treated with: Control Characteristic (inactive L. bulgar- E. coli
0
Milk flavor Sweetness Lactose hydrolyzed (%)
K.
lactis
immobilized cells)
icus
+++
++ ++
++ +++
++
+
0
42
100
46
++
topolymerization was incomplete even after 15 min under saturated conditions of nitrogen gas. This phenomenon was also seen when more than 10% solutions of free enzymes were used. 0_' Such disadvantages could be overcome by low7 5 6 8 3 4 1 2 ering the concentration of cells or enzymes. In Number of repeat runs it is very important FIG. 10. Reusability of immobilized cells of L. the immobilization process, bulgaricus (0), E. coli (A), and K. lactis (A) and to decrease the polymerization time as noted immobilized f&galactosidase from E. coli (x). All of previously (15), since acrylamide monomer and these immobilized cells and enzyme were wrapped organic solvents lead to inactivation of the enwith nylon cloths and reacted with 4.5% lactose solu- zymes. tion at pH 6.5, 300C. Adequate contact of the microorganisms with the mixture of toluene and chloroform may be required to promote the penetrability of the substrate lactose through the cell membrane. This was estimated from the low 3-galactosidase activity of immobilized microorganisms that were not pretreated with toluene for 1 h at 37°C. The extraction ratio of the enzyme from L. bulgaricus and K. lactis was about 1:2, and that from E. coli was smaller than 1:4 (Table 2). These extraction ratios may have been due to inadequate extraction of the enzyme from the cells or enzyme denaturation during the extraction procedure. On the other hand, the activity yields, with immobilization, of the free f3-galactosidases extracted from these microorganisms were not very different from those of the immo. .. D C B A bilized microorganisms (Table 2). The activity yields of extracted enzymes were calculated FIG. 11. Thin-layer chromatogram. A, B, C, and based on the activities of toluene-treated cells. H are authentic lactose, galactose, glucose, and raffi- These values were in the range of 10 to 21% and nose, respectively. D. E, F, and G are deproteinized were lower than those of immobilized microor-
K 20
S**+*
S-
filtrates of native skim milk, of skim milk treated with immobilized E. coli, of skim milk treated with immobilized L. bulgaricus, and of skim milk treated with immobilized K. lactis, respectively. An arrow shows starting points.
DISCUSSION Immobilization of microbial cells. Desirable formation of the gel matrix was affected by the concentration of the cell suspension. When a cell suspension containing more than 3 g of cell pellet in 10 ml of phosphate buffer was mixed with a 50% acylamide solution, the pho-
ganisms. These results suggest that immobilization of the microorganisms is a potential method for utilization of the microbial enzymes without an enzyme extraction procedure. Properties of p8-galactosidase. One of the kinetic parameters, apparent K,,, is known to be affected by the conditions surrounding the enzyme. An increase in the K,, value was reported in the case of enzymes entrapped in acrylamide gel (11, 18) but Mori et al. (10) reported that the diffusional effect through the Nernst layer was negligible. These authors also
VOL. 33, 1977
saw negligible diffusional effects in a previous report (15), in which the immobilized enzyme activity determined in a stirred tank reactor with baffles at 1,000 rpm was the same as that measured in a conical flask at 100 cycles/min, and the effectiveness factor was almost unity. The same conditions as used in the previous paper were used in this experiment to determine the enzymatic activity of immobilized microorganisms. These results, including the finding that Ef values were almost unity, suggest that diffusional effects around the beads were also negligible. Therefore, it seems reasonable that the K,,, values of immobilized microorganisms are almost the same as those of free enzymes. In the present report, galactose, one of the hydrolysates of lactose, was found to inhibit competitively ,B-galactosidase only in the case ofK. lactis; this type of inhibition has also been seen with the enzyme from Aspergillus niger (6, 8, 21). The data described above reveal the noncompetitive inhibition of glucose on f3-galactosidases of L. bulgaricus, E. coli, and K. lactis. Such results have not been mentioned in papers on /3-galactosidases except for the report of Kuby and Lardy (7), who found that glucose and sucrose (glucoside) apparently act as noncompetitive inhibitors of the /3-galactosidase from E. coli K-12. All these properties of /3galactosidase were negligibly changed by the immobilization procedure. Carbohydrate compounds. Unknown carbohydrate compounds were detected by TLC in milk treated with immobilized cells of L. bulgaricus and K. lactis (Fig. 11). These compounds were different from each other judging from Rf values, but they occurred near the position for authentic raffinose. Aronson (1) observed an immediate formation of oligosaccharides having different Rf values during the hydrolysis of lactose by extracts of the microbial cells, and he revealed that these saccharides were synthesized as a result of transgalactosidation. Transgalactosidation is known to occur with monosaccharides, oligosaccharides, alkyl alcohols, and even phenols (2, 13, 16, 20). In the present report, we also justified the existence of this transgalactosidation reaction by the detecting components like raffinose (Fig. 11). Since this reaction also proceeded in extracts from L. bulgaricus and K. lactis, it does not arise only in immobilized microorganisms. It is interesting that unknown carbohydrate compounds were not detected in milk treated with immobilized cells of E. coli. We have shown that direct immobilization of intact microorganisms is superior to the previous method (15), in which extraction of intra-
HYDROLYSIS OF LACTOSE
145
cellular enzyme and its purification are necessary before immobilization. ACKNOWLEDGMENTS We wish to thank the Institute of Animal Industry of the Ministry of Agriculture and Forestry, Laboratory of Fermentation Chemistry of Nagoya University, Institute of Applied Microbiology of the University of Tokyo, Institute of Fermentation Organization, and Tokyo Tanabe Co., Ltd., for supplying the microorganisms used in this experiment. This investigation was partially supported by a grant from Morinaga Hoshikai (Tokyo). LITERATURE CITED 1. Aronson, M. 1952. Transgalactosidation during lactose hydrolysis. Arch. Biochem. Biophys. 39:370-378. 2. Burstein, C., M. Cohn, A. Kepes, and J. Monod. 1965. Role du lactose et de ses produits metaboliques dans l'induction de l'operon lactose chez Escherichia coli. Biochim. Biophys. Acta 95:634-639. 3. Chibata, I., T. Tosa, and T. Sato. 1974. Immobilized aspartase-containing microbial cells: preparation and enzymatic properties. Appl. Microbiol. 27:878-885. 4. Cornish-Bowden, A. 1974. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 137:143-144. 5. Dixon, M. 1953. The determination of enzyme inhibitor constants. Biochem. J. 55:170-171. 6. Kobayashi, T., K. Ohmiya, and S. Shimizu. 1975. Immobilization of ,3-galactosidase by polyacrylamide gel, p. 169-183. In H. H. Weetall and S. Suzuki (ed.), Immobilized enzyme technology, Plenum Press, New York and London. 7. Kuby, S. A., and H. A. Lardy. 1953. Purification and kinetics of 13-D-galactosidase from Escherichia coli, strain K-12. J. Am. Chem. Soc. 75:890-896. 8. Lee, Y. C., and V. Wacek. 1970. Galactosidases from Aspergillus niger. Arch. Biochem. Biophys. 138:264271. 9. Martin, C. K. A., and D. Perlman. 1976. Conversion of L-sorbosone by immobilized cells of Gluconobacter melanogenus IFO 3239. Biotechnol. Bioeng. 18:217237. 10. Mori, T., T. Tosa, and I. Chibata. 1972. Studies on immobilized enzymes. X. Preparation and properties of aminoacylase entrapped into acrylamide gel-lattice. Enzymologia 43:213-226. 11. Mori, T., T. Tosa, and I. Chibata. 1974. Preparation and properties of asparaginase entrapped in the lattice of polyacrylamide gel. Cancer Res. 34:30663068. 12. Morris, D. 1948. Quantative determination of carbohydrates with Dreywood's anthrone reagents. Science 107:254-255. 13. Nishisawa, K., and Y. Hasimoto. 1970. Glucoside hydrolases and glycosyl transferases, p. 241-300. In W. Pignan, D. Horton, and A. Herp (ed.), The carbohydrates, 2nd ed., vol. 2. Academic Press, Inc., New York. 14. Ohmiya, K., and Y. Sato. 1975. Purification and properties of intracellular protease from Streptococcus cremoris. Appl. Microbiol. 30:738-745. 15. Ohmiya, K., C. Terao, S. Shimizu, and T. Kobayashi. 1975. Immobilization of 83-galactosidase by polyacrylamide gel in the presence of protective agents. Agric. Biol. Chem. 32:491-498. 16. Pridham, J. B., and K. Wallenfels. 1964. Enzymatic galactosidation and fucosylation of phenols. Nature (London) 202:488-489. 17. Slowinski, W., and S. E. Charm. 1973. Glutamic acid production with gel-entrapped Corynebacterium glu-
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tamicum. Biotechnol. Bioeng. 15:973-979. 18. Srere, D. A., B. Mattiason, and K. Mosbach. 1973. An immobilized three-enzyme system: a model for microenvironmental compartmentation in mitochondria. Proc. Natl. Acad. Sci. U. S. A. 70:2534-2538. 19. Vieth, W. R., S. S. Wang, and R. Saini. 1973. Immobilization of whole cells in a membraneous form. Biotechnol. Bioeng. 15:565-569.
APPL. ENVIRON. MICROBIOL. 20. Wallenfels, K., and 0. P. Malhotra. 1961. Galactosidases: Hydrolytic, synthetic and transfer reactions. Adv. Carbohydr. Chem. 16:255-257. 21. Woychik, J. H., and M. V. Wondorowski. 1972. Covalent bonding of fungal /3-galactosidase to glass. Biochim. Biophys. Acta 289:347-351. 22. Zweig, C., and J. Sherma. 1972. Handbook of chromatography, vol. 1, p. 463. CRC Press, Cleveland.
Vol. 33, No. 1 Printed in U.S.A.
Hydrolysis of Lactose by Immobilized Microorganisms KUNIO OHMIYA,* HIROSHI OHASHI, TAKESHI KOBAYASHI, AND SHOICHI SHIMIZU Department of Food Science and Technology, Nagoya University, Nagoya, 464 Japan Received for publication 1 June 1976
Cells ofLactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the /3-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial f3-galactosidases were negligibly affected by the immobilization. K, values of /3-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of /3-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free /8-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.
The use of immobilized whole cells as an enzyme source will generally eliminate the need for release of intracellular enzymes and for the succeeding purification steps; also, in some cases the enzymes are more stable if immobilized within their natural environment, the cell. These benefits have led to studies on immobilized microbial cells. Corynebacterium glutamicum (17) was entrapped in polyacrylamide gel to produce glutamic acid in successive batches of fresh medium. Streptomyces phaeochromogenes cells were heated at 80°C for 1.5 h and successfully immobilized in a collagen sheet by the method of aggregation of soluble collagen as a result of pH changes (19). Chibata et al. (3) immobilized Escherichia coli and other bacteria containing aspartase by various methods, and enzymatically active immobilized cells were obtained. Very recently, Martin and Perlman (9) reported the immobilization of Gluconobacter melanogenus cells capable of converting L-sorbose to L-sorbosone in polyacrylamide gel. Entrapping of microorganisms having ,-galactosidase activity, however, has not been reported. B-Galactosidase catalyzes the splitting of lactose to glucose and galactose and is fairly important for the treatment of milk for milkintolerant people who cannot digest lactose. We have already described the immobilization
of 3-galactosidase extracted from Aspergillus oryzae in polyacrylamide gel beads (15). In the present paper, we deal with the immobilization of microbial cells having 8-galactosidase activity and the enzymatic properties of the immobilized cells. MATERIALS AND METHODS Materials. Lactose was the product of BDH Chemicals Ltd. Glucose, galactose, and the other reagents were used in guaranteed grade. Achromycin (tetracycline hydrochloride) used as a growth inhibitor was purchased from Takeda Chemical Industries, Ltd. Cultivation of microorganisms. Lactobacillus bulgaricus (B-1), L. acidophilus (Ack), Streptococcus cremoris (H-61), and S. thermophilus (510) were kind gifts from the Institute of Animal Industry, Ministry of Agriculture and Forestry, Chiba, Japan. All these bacteria were propagated in a medium containing 2% peptone, 1% yeast extract, 0.5% meat extract, 1% glucose, 10% tomato juice, and 0.01% CaCl2, which was adjusted to pH 7.0 with concentrated NH4OH and sterilized at 120°C for 15 min (14). For batch growth, lactobacilli were cultivated at 37°C and streptococci at 30°C for 48 h under anaerobic conditions. E. coli (E-106) was given by the Laboratory of Fermentation Chemistry, Nagoya University. This strain has /8-galactosidase constitutively and was cultivated in peptone-glycerol medium (0.5% peptone, 0.1% glycerol, 2.65% Na2HPO4, 0.45% KH2PO4, 0.03% MgSO4, 0.1% NH4Cl, 0.0033% 137
138
OHMIYA ET AL.
CaCl2, pH 7.0) at 37°C for 18 h under aerobic conditions. Kluyveromyces (Saccharomyces) lactis (from Tokyo Tanabe Co., Ltd.), K. (S.) fragilis (H-2-3, from the Laboratory of Fermentation Chemistry of Nagoya University), and Candida pseudotropicalis var. lactosa (IFO-0616, from the Institute of Fermentation Organization, Osaka) were grown in medium containing 30% corn steep liquor and 1% glycerol at pH 6.0, 30°C, for 48 h under aerobic conditions. Neurospora crassa (IFO-96) was propagated in bouillon medium (1% meat extract, 1% peptone, 5% NaCl, pH 7.0) at 30°C for 7 days. The media described above were sterilized at 120°C for 15 min. All of the microbial cells were harvested by centrifugation at 10,000 x g for 15 min and washed in phosphate buffer (0.7 M, pH 6.5). These cell suspensions were used for cell immobilization as soon as possible. Isolation of free enzymes from microorganisms. Cell pellets of each microorganism obtained by centrifugation were disrupted in a mortar with alumina powder and a small volume of phosphate buffer under chilled conditions (5WC). Then the clear supernatant was separated from the cell debris by centrifugation at 10,000 x g for 15 min. The precipitate was resuspended in the same buffer and sonicated to liberate the enzyme remaining in the cell. The supernatant containing f8-galactosidase was dialyzed against a large volume of phosphate buffer overnight. The dialysate was lyophilized and stored as crude f8-galactosidase in a desiccator at low temperature until used. The crude 8-galactosidase from L. bulgaricus, E. coli, or K. lactis was solubilized in distilled water and is referred to as free enzyme of L. bulgaricus, E. coli, or K. lactis, respectively. Immobilization of microorganisms. These microorganisms and the free enzymes were immobilized in acrylamide gel beads according to the method described previously (15); these preparations are referred to as the immobilized cells and immobilized enzymes, respectively. The concentration of the cell suspensions was usually lower than 3 g of wet cell per 10 ml of phosphate buffer. Evaluation of effectiveness factor. The effectiveness factor, Ef, was evaluated as the ratio of the apparent rate of enzymatic reaction with diffusional effect to the rate under conditions without any diffusional effect. Such conditions were created by grinding immobilized microorganisms in a chilled mortar. Reuse of immobilized microorganisms. To use immobilized cells repeatedly, 1 g of immobilized cells was wrapped with nylon cloth that does not leak any beads. The enzyme reactor was pretreated before the first run as follows; it was immersed into 50 ml of 4.5% lactose solution (phosphate buffer, pH 6.5) at 30°C, shaken at 120 cycles/min for 10 min, and centrifuged at 1,000 x g for 1 min in the centrifugal tube with a rack at the middle to remove lactose solution in the reactor. Then every run was carried out under the same conditions for 30 min. After each run, the reactor was washed twice with 200 ml of the same buffer. The residual solution in the reactor was removed by centrifugation at 1,000
APPL. ENVIRON. MICROBIOL. x g for 1 min at room temperature. One cycle of reaction and washing took about 1 h. Standard assay of ,B-galactosidase activity. (i) ,Bgalactosidase activity in cell suspensions. One milliliter of the suspension containing 2 g of wet cells in 10 ml of phosphate buffer was mixed with 4 ml of the same buffer and 0.15 ml of toluene. This mixture was shaken for 1 h at 320 rpm, 30°C, to increase lactose penetration into the cells by toluene extraction of the lipid contained in the cell membrane. One milliliter of this toluene-treated cell suspension was combined with 5 ml of lactose solution (11.25%) and 6.5 ml of the same buffer and incubated at 30°C. After 15 min, a 3-ml aliquot of the reaction mixture was put immediately into a slender test tube preheated in boiling water and held for 3 min in order to stop further hydrolysis before inactivation of the enzyme. Then this aliquot was centrifuged to remove cells at 10,000 x g for 10 min after chilling in ice water. The amounts of glucose and galactose were determined by glucose oxidase (Glucostat special reagent kit, Worthington Biochemicals Co.) and by galactose dehydrogenase (galactose ultraviolet test, Boehringer Mannheim Co. Ltd.), respectively. (ii) Activity of crude j8-galactosidase. The solution of crude /8-galactosidase (0.5 ml) was mixed with phosphate buffer (pH 6.5, 7.0 ml) and lactose solution (11.25%, 5.0 ml) and incubated at 30°C for 30 min. The same procedure as described above, except for cell removal, was used for this reaction mixture to evaluate the amount of glucose released. (iii) ,B-Galactosidase activities of immobilized cells and immobilized enzymes. /8-Galactosidase activity in the polyacrylamide gel beads containing the microbial cells or extracted enzyme was assayed with 4.5% lactose in phosphate buffer (pH 6.5, 0.7 M) at 30°C for 30 min under reciprocal shaking at 100 cycles/min. The details have been described (15). TLC. Hydrolysis of lactose in skim milk was carried out with the immobilized cells of these microorganisms as follows: 30 ml of the solution prepared with skim milk powder was incubated with immobilized cells (10 g) at 30°C under reciprocal shaking for 5 h. Then the skim milk was deproteinized with 1% solutions of Ba(OH)2 and ZnSO4 and filtered through filter paper (Toyo, no. 5C). The filtrates were spotted on precoated thin-layer chromatography (TLC) plates (silica gel) purchased from Merck & Co. The chromatography was developed by a solvent (22) containing n-butanol-methanol-boric acid solution (0.03 M) (5:3:1) at room temperature for about 1 h. Sugars were detected by the anthrone-H2SO4 reagent (12). Properties of skim milk treated with immobilized cells. Intensity of sweetness, flavor quality, and degree of lactose hydrolysis were determined on both the milk treated with active immobilized cells and the control treated with inactive immobilized cells.
RESULTS
,1-Galactosidase activity of microorganisms. Nine species or strains of bacteria, yeast, and mold were selected and evaluated for
HYDROLYSIS OF LACTOSE
VOL. 33, 1977
inherent 83-galactosidase activity. The amounts of glucose (in milligrams) released from lactose by 100 mg of dry cells of each microorganism in 4.5% lactose solution during 15 min at 30°C, pH 6.5, and percentage of lactose hydrolysis are listed in Table 1. E. coli and K. lactis revealed very strong f3-galactosidase activity. The activity of L. bulgaricus was lower than those of the former but the highest among the lactic acid bacteria. Immobilization of microbial cells. Three species of microbial cells with high f3-galactosidase activity were entrapped in polyacrylamide gel beads. The cell suspensions used for the immobilization contained 2 g of cell pellet per 10 ml of phosphate buffer (pH 6.5, 0.7 M) because beads of normal size could not be obtained when larger amounts of cell pellet were used. Typical beads containing K. lactis cells are shown in Fig. 1. It can be clearly seen that the cells were dispersed homogeneously and entrapped in the acrylamide gel beads. Immobilization of free enzymes from these microorganisms was also done by this procedure. The activity yields of these immobilized cells and immobilized enzymes were in the range of 27 to 61% (Table 2). Properties of immobilized microorganisms. (i) Optimum pH. The pH range studied was 4.5 to 9.0. Figure 2 shows the pH profile for free enzymes, immobilized enzymes, and immobilized cells with enzymatic activity (L. bulgaricus, E. coli, and K. lactis). pH optima for activity of these immobilized cells were 5.5, 8.0, and 6.3, respectively. Each value was very close to that of free enzyme. (ii) Optimum temperature and thermostability. Immobilized cells of L. bulgaricus and E. coli showed the highest activity at near 55°C (Fig. 3A, B). The optimum temperature of immobilized K. lactis cells was 370 (Fig. 3C). Temperature-activity profiles of these immobilized cells were comparable to those of free enzymes. Thermostability of immobilized cells of the three microorganisms was determined by the residual activities after 24 h of incubation in the buffer at a given temperature. The residual activities evaluated according to the standard assay are depicted in Fig. 4. E. coli was the most stable among the three. The stability was slightly increased by the immobilization because the initial activity was maintained even after storage of immobilized E. coli cells at 45°C for 24 h. (iii) Ef value. The values for immobilized cells of L. bulgaricus, E. coli, and K. lactis were 1.1, 0.9, and 1.1, respectively. These values were almost equal to unity within the range of
139
TABLE 1. f&Galactosidase activity of microorganisms Glucose
Microorganisms (100 mg of dry cells)
pro- a
duced
Lactose
hydrolY-
hydroly
(mg) Lactobacillus bulgaricus (B-1) L. acidophilus (Ack) Streptococcus thermophilus (510) S. cremoris (H-61) Escherichia coli (E-106) Kluyveromyces (Sacchromyces) lactis K. (S.) fragilis (H-2-3) Candida pseudotropicalis var. lactosa (IFO-0616) Neurospora crassa (IFO-96)
21.1 3.8 3.1 3.0 174.0 266.4
3.8 0.7 0.5 0.5 31.0 47.4
4.7 2.0
0.8 0.4
9.1
1.6
Reaction was carried out at 30°C, pH 6.5, for 15 min in 4.5% lactose. a
experimental error. This suggests that the diffusional effects within the beads used in this experiment were not significant. (iv) K,,, value. The effect of substrate concentration on the hydrolysis of lactose by immobilized cells was investigated. The K,m values were evaluated by plotting the reciprocal of the substrate concentration against the reciprocal of the reaction velocity. The data for immobilized cells and for free enzymes at pH 6.5 are summarized in Table 3. The values for E. coli and L. bulgaricus were six- to sevenfold lower than that for K. lactis. All of these values were negligibly affected by the treatment for immobilization. (v) Ki value. Galactose and glucose effects on ,f-galactosidase activity were studied for the free enzymes and immobilized cells. The initial rates of lactose hydrolysis by immobilized cells of L. bulgaricus and E. coli were almost constant, although the concentration of galactose was increased. The same result was observed with the free enzymes from these bacteria. Since these results revealed clearly that these enzymes were not obviously inhibited by galactose, we estimated that the K, values of the enzymes were higher than the highest inhibitor levels tested. The activity from K. lactis, however, gradually decreased with increased amounts of galactose added. The inhibition effect with increasing concentrations of galactose on the activity of immobi1 zed K. lactis cells was examined at different concentrations of lactose; i.e., the reaction rate, v, was measured with varying inhibition concentrations, [I], and 1/v was plotted against [I]. This convenient method is described by Dixon (5). A straight line was obtained for each substrate concentration (Fig. 5). This result suggests that the inhibition type is either competitive or mixed com-
140
APPL. ENVIRON. MICROBIOL.
OHMIYA ET AL.
A .
4~~~~~IV
.F O
}e "
-
*
FIG. 1. Photomicrographs of immobilized cells of K. lactis in polyacrylamide gel beads in wet state (upper: 1 scale, 10 ,um; lower: 1 scale, 2.5 ,um).
TABLE 2. Activity yield of immobilized f&galactosidase Immobilized enzyme
Immobilized cell
Microorganism
Absolute
tya
CalcuClu
..Extracactivi-. Yield" Absolute activ- Yield tion ra- lated acx
%
ity
tioc
yieldy
21 42 1:2 27 5.0X 10-4 Lactobacillus bulgaricus 3.5 x 10-3 10 40 1:4 38 1.3 x 10-2 Escherichia coli 3.9 x 10-2 15 1:2 29 61 6.3 x 1O-3 Kluyueromyces lactis 4.8 x 10-2 a Expressed as micromoles of glucose per milliliter per minute per milligram of immobilized cells or enzymes. bRatio of activity of immobilized cells or enzymes to that of toluene-treated cells or free enzymes. c Ratio of activity of f8-galactosidase extracted from a given amount of cells to that of the toluene-treated cells.
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VOL. 33, 1977
141
pH
FIG. 2. Effect ofpH on the f-galactosidase activity of L. bulgaricus (A), E. coli (B), and K. lactis (C) at 30°C in 0.7 M phosphate buffer. Symbols: (0) Immobilized microorganisms; (A) immobilized enzymes; (X) free enzymes.
Temperature ( °C ) FIG. 3. Effect of temperature on the f3galactosidase activity of L. bulgaricus (A), E. coli (B), and K. lactis (C) at pH 6.5 in 0.7 M phosphate buffer. Symbols are as in Fig. 2.
*-.,,
100-
?s0~~~~~~~~~ A~~~~A
60
*020
~
20A
20
30
40
0~~~~~~~~~A 0 B
50
30 40 50 Temperature ( OC )
20
30
40
50
FIG. 4. Thermostability of f3galactosidases of L. bulgaricus (A), E. coli (B), and K. lactis (C) at pH 6.5 in 0.7 M phosphate buffer. Symbols are as in Fig. 2.
142
APPL. ENVIRON. MICROBIOL.
OHMIYA ET AL.
TABLE 3. Kinetic parameters of immobilized ,3galactosidlases Microorganism
Vmaza
Ke (lacM) K, and K,' (galactose, mM) K, (glucose, mM)
Lactobacillus bulgaricus 00b 230 (non)c 4.2 2.9 x 10-4 Immobilized cell 00 230 (non) 1.3 x 10-2 4.2 Free enzyme Escherichia coli 76 (non) X 5.4 7.5 x 10-7 Immobilized cell 70 (non) X 5.4 1.1 x 10-3 Free enzyme Kluyveromyces lactis 380 (non) 30 47 and 103(mix)d 8.3 x 10-4 Immobilized cell 340 (non) 46 and 103(mix) 30 3.3 x 10-4 Free enzyme a Expressed as millimoles per minute per milligram of beads or free enzyme per liter of reaction mixture. b Ki and Ki' values were higher than the highest inhibitor levels tested. c non, Noncompetitive inhibition. d mix, Mixed competitive inhibition.
201
10 60 -40 -80 ?
-20
0
20
40
Galactose (mM )
20 0 40 60 -20 Ga I actose ( mM ) FIG. 5. Effect of galactose on the rate of lactose hydrolysis offree enzymes (dotted line) and immobilized cells (solid line) of K. lactis at various lactose concentrations. Lactose concentrations (millimoles per liter of reaction mixture): (I) 31.4; (II) 52.6; (III) 105.2; (1) 39.1; (2) 62.5; (3) 173.6.
-60
-40
petitive (4). For distinguishing between these inhibition types, [SI/v was plotted against [I] at different values of [SI (substrate concentration). Straight lines were drawn, and they intersected at a point where [I] = Ki' (Fig. 6). K, and K,' values of 3-galactosidase in K. lactis cells immobilized in acrylamide gel were 47 and 103 mM, respectively. These values were almost the same as those of the free enzyme. The effects of glucose at various concentrations on the activity of these microorganisms were also examined by determining the amounts of galactose released from lactose. The patterns of the Lineweaver-Burk plots appeared to be those of typical noncompetitive inhibition (Fig. 7 to 9). From these plots, K,
FIG. 6. Effect of galactose on the rate of lactose hydrolysis offree enzymes (dotted line) and immobilized cells (solid line) of K. lactis at various lactose concentrations. Lactose concentrations, [SI (millimoles per liter of reaction mixture): (I) 31.4; (II) 52.6; (III) 105.2; (1) 39.1; (2) 62.5; (3) 173.6. [S]/v was plotted against [I].
values of glucose were evaluated by plotting the reciprocal values of Vmax obtained at a given concentration of glucose against the concentration of glucose added. These K, values were close to the respective values for free enzymes (Table 3). K. and Ki values obtained in this experiment for f3-galactosidase are summarized in Table 3. Reusability of immobilized cells. The reusability of these immobilized microbial cells, which were wrapped with nylon cloth as a reactor, was tested repeatedly (Fig. 10). The reactor from K. lactis showed a negligible decrease in activity upon initial contact with lactose solution. Those from E. coli and L. bulgaricus showed 25 and 35% decreases, respectively. The decreasing profile of immobilized enzyme from E. coli was consistent with that of immobilized cells (Fig. 10). Properties of the skim milk solution treated with immobilized cells. Changes in lactose of the deproteinized filtrate of the skim milk solu-
VOL. 33, 1977
HYDROLYSIS OF LACTOSE
] -200
100
-100
1 /(Lactose)
(M-1 )
FIG. 7. Inhibition pattern for free f&galactosiclase (dotted line) and immobilized cells (solid line) of bulgaricus with lactose as the varied substrate and glucose as the varied inhibitor. Glucose concen trations (millimoles per liter of reaction mixture): I) 106.7; (II) 53.3; (III) 0; (1) 95.2; (2) 47.6; (3) 0
La
143
tions that were treated with these immobilized cells were detected by TLC (Fig. 11). The position of authentic lactose (A) followed that of authentic galactose (B) and glucose (C). Deproteinized filtrate of the skim milk solution without any enzymatic treatment (control) contained only lactose (D). Figure liE suggests that lactose in the skim milk was completely hydrolyzed to galactose and glucose by immobilized cells of E. coli. The enzymatic actions of immobilized cells of L. bulgaricus and K. lactis were consistently detected by the appearance of glucose and galactose spots on the TLC (F and G, respectively). In addition to these components, unknown spots faintly appeared near the starting points. The skim milk treated with immobilized cells seemed to weaken the typical milk flavor (Table 4). The sweetness of the skim milk was enhanced as lactose hydrolysis proceeded.
A
300 c
E 200
E >
C
100'
------ ... --..
0
50
100
200 0
150
50
100
150
200
1/ (Lactose) (M_1) FIG. 8. Inhibition patterns for immobilized cells (A) and free f3galactosidase (B) of E. coli with lactose as the varied substrate and glucose as the varied inhibitor. Glucose concentrations (millimoles per liter of reaction mixture): (1) 55.6; (2) 0; (I) 53.3; (II) 26.7; (III) 0. Dotted lines were symmetrical parts of the plots
against ordinates.
-40
-20
0
20
40
-40
-20
0
20
40
1/(Lactose) (M-1) FIG. 9. Inhibition patterns for immobilized cell (A) and free ,&galactosidase (B) of K. lactis with lactose as the varied substrate and glucose as the varied inhibitor. Glucose concentrations (millimoles per liter of reac-
tion mixture): (1) 76.2; (2) 38.1; (3) 0; (I) 55.3; (II) 26.7; (III) 0.
144
OHMIYA ET AL.
.
APPL. ENVIRON. MICROBIOL.
*.
..
0~~~~~~ ,60 a 40.
a
TABLE 4. Characteristics of skim milk treated with immobilized microorganisms Skim milk treated with: Control Characteristic (inactive L. bulgar- E. coli
0
Milk flavor Sweetness Lactose hydrolyzed (%)
K.
lactis
immobilized cells)
icus
+++
++ ++
++ +++
++
+
0
42
100
46
++
topolymerization was incomplete even after 15 min under saturated conditions of nitrogen gas. This phenomenon was also seen when more than 10% solutions of free enzymes were used. 0_' Such disadvantages could be overcome by low7 5 6 8 3 4 1 2 ering the concentration of cells or enzymes. In Number of repeat runs it is very important FIG. 10. Reusability of immobilized cells of L. the immobilization process, bulgaricus (0), E. coli (A), and K. lactis (A) and to decrease the polymerization time as noted immobilized f&galactosidase from E. coli (x). All of previously (15), since acrylamide monomer and these immobilized cells and enzyme were wrapped organic solvents lead to inactivation of the enwith nylon cloths and reacted with 4.5% lactose solu- zymes. tion at pH 6.5, 300C. Adequate contact of the microorganisms with the mixture of toluene and chloroform may be required to promote the penetrability of the substrate lactose through the cell membrane. This was estimated from the low 3-galactosidase activity of immobilized microorganisms that were not pretreated with toluene for 1 h at 37°C. The extraction ratio of the enzyme from L. bulgaricus and K. lactis was about 1:2, and that from E. coli was smaller than 1:4 (Table 2). These extraction ratios may have been due to inadequate extraction of the enzyme from the cells or enzyme denaturation during the extraction procedure. On the other hand, the activity yields, with immobilization, of the free f3-galactosidases extracted from these microorganisms were not very different from those of the immo. .. D C B A bilized microorganisms (Table 2). The activity yields of extracted enzymes were calculated FIG. 11. Thin-layer chromatogram. A, B, C, and based on the activities of toluene-treated cells. H are authentic lactose, galactose, glucose, and raffi- These values were in the range of 10 to 21% and nose, respectively. D. E, F, and G are deproteinized were lower than those of immobilized microor-
K 20
S**+*
S-
filtrates of native skim milk, of skim milk treated with immobilized E. coli, of skim milk treated with immobilized L. bulgaricus, and of skim milk treated with immobilized K. lactis, respectively. An arrow shows starting points.
DISCUSSION Immobilization of microbial cells. Desirable formation of the gel matrix was affected by the concentration of the cell suspension. When a cell suspension containing more than 3 g of cell pellet in 10 ml of phosphate buffer was mixed with a 50% acylamide solution, the pho-
ganisms. These results suggest that immobilization of the microorganisms is a potential method for utilization of the microbial enzymes without an enzyme extraction procedure. Properties of p8-galactosidase. One of the kinetic parameters, apparent K,,, is known to be affected by the conditions surrounding the enzyme. An increase in the K,, value was reported in the case of enzymes entrapped in acrylamide gel (11, 18) but Mori et al. (10) reported that the diffusional effect through the Nernst layer was negligible. These authors also
VOL. 33, 1977
saw negligible diffusional effects in a previous report (15), in which the immobilized enzyme activity determined in a stirred tank reactor with baffles at 1,000 rpm was the same as that measured in a conical flask at 100 cycles/min, and the effectiveness factor was almost unity. The same conditions as used in the previous paper were used in this experiment to determine the enzymatic activity of immobilized microorganisms. These results, including the finding that Ef values were almost unity, suggest that diffusional effects around the beads were also negligible. Therefore, it seems reasonable that the K,,, values of immobilized microorganisms are almost the same as those of free enzymes. In the present report, galactose, one of the hydrolysates of lactose, was found to inhibit competitively ,B-galactosidase only in the case ofK. lactis; this type of inhibition has also been seen with the enzyme from Aspergillus niger (6, 8, 21). The data described above reveal the noncompetitive inhibition of glucose on f3-galactosidases of L. bulgaricus, E. coli, and K. lactis. Such results have not been mentioned in papers on /3-galactosidases except for the report of Kuby and Lardy (7), who found that glucose and sucrose (glucoside) apparently act as noncompetitive inhibitors of the /3-galactosidase from E. coli K-12. All these properties of /3galactosidase were negligibly changed by the immobilization procedure. Carbohydrate compounds. Unknown carbohydrate compounds were detected by TLC in milk treated with immobilized cells of L. bulgaricus and K. lactis (Fig. 11). These compounds were different from each other judging from Rf values, but they occurred near the position for authentic raffinose. Aronson (1) observed an immediate formation of oligosaccharides having different Rf values during the hydrolysis of lactose by extracts of the microbial cells, and he revealed that these saccharides were synthesized as a result of transgalactosidation. Transgalactosidation is known to occur with monosaccharides, oligosaccharides, alkyl alcohols, and even phenols (2, 13, 16, 20). In the present report, we also justified the existence of this transgalactosidation reaction by the detecting components like raffinose (Fig. 11). Since this reaction also proceeded in extracts from L. bulgaricus and K. lactis, it does not arise only in immobilized microorganisms. It is interesting that unknown carbohydrate compounds were not detected in milk treated with immobilized cells of E. coli. We have shown that direct immobilization of intact microorganisms is superior to the previous method (15), in which extraction of intra-
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cellular enzyme and its purification are necessary before immobilization. ACKNOWLEDGMENTS We wish to thank the Institute of Animal Industry of the Ministry of Agriculture and Forestry, Laboratory of Fermentation Chemistry of Nagoya University, Institute of Applied Microbiology of the University of Tokyo, Institute of Fermentation Organization, and Tokyo Tanabe Co., Ltd., for supplying the microorganisms used in this experiment. This investigation was partially supported by a grant from Morinaga Hoshikai (Tokyo). LITERATURE CITED 1. Aronson, M. 1952. Transgalactosidation during lactose hydrolysis. Arch. Biochem. Biophys. 39:370-378. 2. Burstein, C., M. Cohn, A. Kepes, and J. Monod. 1965. Role du lactose et de ses produits metaboliques dans l'induction de l'operon lactose chez Escherichia coli. Biochim. Biophys. Acta 95:634-639. 3. Chibata, I., T. Tosa, and T. Sato. 1974. Immobilized aspartase-containing microbial cells: preparation and enzymatic properties. Appl. Microbiol. 27:878-885. 4. Cornish-Bowden, A. 1974. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 137:143-144. 5. Dixon, M. 1953. The determination of enzyme inhibitor constants. Biochem. J. 55:170-171. 6. Kobayashi, T., K. Ohmiya, and S. Shimizu. 1975. Immobilization of ,3-galactosidase by polyacrylamide gel, p. 169-183. In H. H. Weetall and S. Suzuki (ed.), Immobilized enzyme technology, Plenum Press, New York and London. 7. Kuby, S. A., and H. A. Lardy. 1953. Purification and kinetics of 13-D-galactosidase from Escherichia coli, strain K-12. J. Am. Chem. Soc. 75:890-896. 8. Lee, Y. C., and V. Wacek. 1970. Galactosidases from Aspergillus niger. Arch. Biochem. Biophys. 138:264271. 9. Martin, C. K. A., and D. Perlman. 1976. Conversion of L-sorbosone by immobilized cells of Gluconobacter melanogenus IFO 3239. Biotechnol. Bioeng. 18:217237. 10. Mori, T., T. Tosa, and I. Chibata. 1972. Studies on immobilized enzymes. X. Preparation and properties of aminoacylase entrapped into acrylamide gel-lattice. Enzymologia 43:213-226. 11. Mori, T., T. Tosa, and I. Chibata. 1974. Preparation and properties of asparaginase entrapped in the lattice of polyacrylamide gel. Cancer Res. 34:30663068. 12. Morris, D. 1948. Quantative determination of carbohydrates with Dreywood's anthrone reagents. Science 107:254-255. 13. Nishisawa, K., and Y. Hasimoto. 1970. Glucoside hydrolases and glycosyl transferases, p. 241-300. In W. Pignan, D. Horton, and A. Herp (ed.), The carbohydrates, 2nd ed., vol. 2. Academic Press, Inc., New York. 14. Ohmiya, K., and Y. Sato. 1975. Purification and properties of intracellular protease from Streptococcus cremoris. Appl. Microbiol. 30:738-745. 15. Ohmiya, K., C. Terao, S. Shimizu, and T. Kobayashi. 1975. Immobilization of 83-galactosidase by polyacrylamide gel in the presence of protective agents. Agric. Biol. Chem. 32:491-498. 16. Pridham, J. B., and K. Wallenfels. 1964. Enzymatic galactosidation and fucosylation of phenols. Nature (London) 202:488-489. 17. Slowinski, W., and S. E. Charm. 1973. Glutamic acid production with gel-entrapped Corynebacterium glu-
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tamicum. Biotechnol. Bioeng. 15:973-979. 18. Srere, D. A., B. Mattiason, and K. Mosbach. 1973. An immobilized three-enzyme system: a model for microenvironmental compartmentation in mitochondria. Proc. Natl. Acad. Sci. U. S. A. 70:2534-2538. 19. Vieth, W. R., S. S. Wang, and R. Saini. 1973. Immobilization of whole cells in a membraneous form. Biotechnol. Bioeng. 15:565-569.
APPL. ENVIRON. MICROBIOL. 20. Wallenfels, K., and 0. P. Malhotra. 1961. Galactosidases: Hydrolytic, synthetic and transfer reactions. Adv. Carbohydr. Chem. 16:255-257. 21. Woychik, J. H., and M. V. Wondorowski. 1972. Covalent bonding of fungal /3-galactosidase to glass. Biochim. Biophys. Acta 289:347-351. 22. Zweig, C., and J. Sherma. 1972. Handbook of chromatography, vol. 1, p. 463. CRC Press, Cleveland.
