J. Biol. Macromol., 3(3) 97-103 (2003)

ArticlePurification and molecular characterization of β-galactosidase from yeast

Kluyveromyces lactis
Yuko Fujimura, Souji Rokushika, and Masatake Ohnishi*

Research Field of Cellular Macromolecules, Science Department of Biological Function,
Graduate School of Agriculture Science, The Prefecture University of Kyoto(Shimogamo, Sakyo-ward, Kyoto City, Kyoto, 606-8522, Japan)
Received June 5, 2003; accepted September 16, 2003
β-Galactosidase from yeast Kluyveromyces lactis was purified to be pure using the
gel filtration column chromatography and was examined for molecular weight,
subunit composition, enzymatic properties (optimum and stable pH, optimum andstable temperature, effect of added metal ions), and steady-state kinetic parameters
for the two substrates; lactose and οNPG.
Keywords:
β-galactosidase, yeast Kluyveromyces lactis, purification, molecular properties
β-Galactosidase (EC 3.2.1.32) has
been purified from many kinds of organism and has been studied in the various
fields including basic science and practically useful industries [1-5]. Purificationof β-galactosidase from Kluyveromyces
lactis strain Y1140 was reported for thecharacterization of its molecular constitution by Dickson R.C. et al. [6]. They
have examined the induction of the
enzyme β-galactosidase in K. lactis,
characterized the physical and molec---------------------------------------------------* Corresponding author.
Presented in part at the Annual Meetingof the Japan Society for Agric. Biol.
Chem., Tokyo, Japan, 2000.

ular properties of the induced β-galactosidase [6].
β-Galactosidase-catalyzed reactions
are studied in the various experimental
conditions, such as pH, temperature,ionic strength, high concentration, andpresence of organic reagents, of which
environmental factors are essentially
important in the regulation of the
enzyme reactions [7-12]. In the intracellular organelles and cytoplasm, biological functions of the proteins andenzymes are of great interest in understanding their mechanisms. However,the function and mechanism of proteins/
enzymes in the membrane and cytoplasm
are further difficult for investigation in

98β-Galactosidase from yeast K. lactis

this stage [13-15].
Kinetic analysis is sometimes very
effective for the characterization and/or
understanding of the enzyme-catalyzed
reaction and substrate specificity. In
fact, the steady-state kinetic parameters,
the Michaelis constant Km, and themolar activity k0, have been determinedfor glucoamylase from Rhizopus niveus
using various substrates in the presenceof urea, polyethylene glycol, or acetonitrile, and applied to the evaluation of
the mechanism on the enzyme-catalyzed
reactions and the subsite structure[11,12,16,17].
Under the specified conditions, it is very
possible that a glycohydrolase catalyzes
a synthesis reaction (condensation,transglycosylation) to produce saccharides having higher degree of polymerization than that of substrate. We areinteresting in enzymatic properties of βgalactosidase, which is considered tolocate at membrane of the yeast K. lactis
cells.
In this paper, β-galactosidase waspurified from yeast K. lactis, and themolecular and enzymatic properties were
examined for the successive investigationon the reaction mechanism.
EXPERIMENTAL

Materials
A commercial material of the enzyme
β-galactosidase from yeast K. lactis waspurchased from Amano Enzymes Co. Ltd.,
Nagoya. Lactose, glucose, galactose, οnitorophenyl-β-D-galactopyranoside(οNPG), ο-nitro phenol (οNP), glycerol,
sodium-hydroxide, -carbonate, -chloride,-dihydrogen phosphate, and –hydrogen
phosphate, potassium-chloride, and-dihydrogen phosphate, and magnesium
sulphate, all in guaranteed grade wereobtained from Nacarai Tesque Inc., Kyoto,
and were used without further purification. Bovine serum albumin was used ofa product from Sigma Chemical Co. A
glucose determination-kit, Glucose C-ΙΙ
Test Wako, was a product of Wako Pure
Chemical Industries, Ltd., Osaka.
Superdex 200 for the column chromatography was purchased from Pharmacia
Biotech. Japan Co., Tokyo.

Enzyme assay
β-Galactosidase-catalyzed hydrolysis
reaction was carried out in a 20mM
phosphate buffer pH 7.0 at 25.0ºC withlactose as a substrate to obtain thereaction time-curves. In this experiment, phosphate buffer contains 10mM
KCl and 1 mM MgSO4 . Reactions
were initiated by addition of the enzyme
solution to the substrate solution, andaliquots (usually 100µl) were takenout at appropriate time intervals andmixed with a 50mM NaOH solution(usually 100µl) to stop the enzymecatalyzed reaction. Amount of glucose
released from a substrate lactose by thecatalyzed reaction was measured using
a routine procedure with glucose assaykit, Glucose C-ΙΙ Test Wako (GOD) [18],which was used after 4-fold dilution in
the 20 mM phosphate buffer pH 7.0.
The β-galactosidase-catalyzed hydr-

Y. Fujimura et al.

olysis for another substrate oNPG was
measured spectrophotometrically in
20mM phosphate buffer pH 7.0 at 25ºC.
Reactions were initiated by addition of
the enzyme solution to the substrateoNPG solution, and aliquots (100µl)
were removed at appropriate timeintervals and mixed with a 100mM
Na2CO3 solution (2ml) to stop thecatalyzed reaction. Based on the absorbance at 420nm, the amount of oNP
released from the substrate οNPG by the
enzyme-catalyzed reaction was measured
to obtain the time-curves.
One unit of enzyme activity wasdefined to release 1 µmol of οNP per min
for the substrate oNPG or 1µmol of
glucose per min for the substrate lactose.
Concentrations of the enzyme were
determined spectrophotometrically using
the Bradford method with the Coomassie
reagent, a product of PIERCE Co. andbovine serum albumin as a standard
protein sample.

Purification of the enzyme preparation
A commertial product of β-galactosidase from yeast K. lactis, of Amano
Enzymes Co. Nagoya, was purified using
a gel filtration chromatography with
Superdex 200. A 50cm long 1.5cm
diameter column (bed volume 85ml) was
equilibrated with a 20mM phosphatebuffer pH 7.0 before use. The sample
containing β-galactosidase was added on
to the column and eluted with 1 - 1.5 bed
volume of a 20mM phosphate buffer, of
which flow rate is 0.38ml per min andfractions of 2ml volume were collected in

99

fraction tubes. All purification procedures were conducted at 4ºC unless noted
otherwise.
Each fraction was estimated for theprotein concentration by measurement ofabsorbance at 280nm using a Shimadzu
UV-1600 spectrophotometer and for thelactose hydrolysis enzyme-assay according to the method described above andthe specific activity was evaluated for the
purified β-galactosidase. Glycerol wasadded to the purified enzyme preparation to keep the stability.
RESULTS AND DISCUSSION

Purification of β-galactosidase
The enzyme sample of β-galactosidase (6.2mg in 600µl), was added to
the Superdex column and eluted with a
20 mM phosphate buffer containing 10
mM KCl and 1 mM MgSO4. The resultis shown in Fig. 1, clearly indicating thatthe first peak in the two peaks has thelactose-hydrolysis enzymr-catalyzed
activity. Fractions containing the
enzymatic activity were pooled andconcentrated by ultrafiltration. Afterthe ultrafiltration, protein concentrations and hydrolysis enzymatic activity
were determined to evaluate the specificactivity as follows: The specific activityof the enzyme preparation showed about
2-fold increase and the yield of total
activity was 72%. The purified enzyme
was stored in 20 mM phosphate buffer
pH 7.0 containing 20% glycerol.

100

β-Galactosidase from yeast K. lactis

dehydrogenase (53,000). Molecularweight of a subunit protein in β-galactosidase was estimated to be 120,000;
thus, β-galactosidase obtained from K.
lactis was concluded to be a dimmer.

β-Galactosidase catalyzed reaction in thepresence of metal ionsFig. 1. Superdex chromatogram for
purification of β-galactosidase
Column φ1.5 X 50cm, 20mM phosphatebuffer pH 7.0 containing 10mM KCl and
1mM MgSO4, fraction 2ml, flow rate
0.38ml/min, 4℃, ●; protein, ○; enzyme
activity.

Physicochemical and molecularproperties of the purified β-galactosidase preparation
The molecular weight of thepurified β-galactosidase preparation wasdetermined by gel filtration technique:standard proteins used were: ferritinfrom horse spleen (MW440,000), catalase
from bovine liver (232,000), β-glucosidasefrom Aspergillus niger (137,000), andbovine serum albumin (66,000). Molecular weight of the purifiedβ-galactosidase preparation was analyzed to be
240,000.
Subunit composition of the purified
β-galactosidase preparation was examined by SDS-PAGE technique with a 10%
polyacrylamide gel where standard
proteins were: rabbit muscle myosin(MW212,000), bovine plasma α2-macroglobulin (170,000), β-galactosidase from
E. coli (116,000), human transferring(76,000), and bovine liver glutamic

The effect of metal ions in the
enzyme-catalyzed reaction as analyzed
by using 30mM lactose as a substrate
in 20mM phosphate buffer pH 7.0 at
25ºC. The enzyme-catalyzed activity inaddition of KCl at concentration of 50 200mM was found to be 1.5 fold larger
than that in the reference (no addition),whereas the activity in the addition of
0.3 - 5mM MgSO4 was 3.3 fold larger
than that in the reference. Based onthese results, both 10mM KCl and 1mM
MgSO4 were chosen to be presence in 20
mM phosphate buffer pH 7.0 when the βgalactosidase-catalyzed reaction was
examined.

pH optimum and pH stability for the βgalactosidase-catalyzed reactions
The β-galactosidase-catalyzed
reactions were observed with 30mM
lactose as the substrate in 20mM
phosphate buffer containing 10mM KCl
and 1mM MgSO4 at the pH range of 5.5
to 9.0 at 25ºC. The experimental results
are shown in Fig. 2, where the enzymatic
reactions dropped at below pH 6.5 and atabove pH 7.0, suggesting that the
optimum pH of the enzyme-catalyzed
reaction is 7.0. Thus, the enzyme-

Y. Fujimura et al.

catalyzed reactions were carried out at
pH 7.0 in this study.
The pH stability for the β-galactosidase-catalyzed reactions was examinedby measuring the remaining activity
after 30min incubation at 25ºC with 30
mM lactose as the substrate in 20mM
phosphate buffer pH 5.5 - 9.0. The
experimental results are illustrated inFig. 2, suggesting that the enzyme isstable in a pH range of 7.0 to 8.5.

101

activity after 30min incubation at 5 50ºC and pH 7.0 with 30mM lactose as
the substrate. The experimental results
are illustrated in Fig. 3, where the
enzyme activity dropped off rapidly
above 35ºC, suggesting that the stabletemperature range is 5 - 35ºC.

Fig. 3. Effect of temprature on the βgalactosidase-catalyzed reaction andstability of the enzyme[E]0; 3.2nM, 20mM phosphate buffer pH 7.0
containing 10mM KCl and 1mM MgSO4,
stability; pH 7.0 for 30min

Fig. 2. Effect of pH on the β-galactosidase-catalyzed reaction and stabilityof the enzyme｢E｣0; 3.2nM, 20mM phosphate buffer
containing 10mM KCl and 1mM MgSO4,25ºC,
stability; 25ºC for 30min

Temperature-optimum and -stability forthe β-galactosidase-catalyzed reactions
The effect of temperature on the βgalactosidase-catalyzed reactions wasanalyzed at 5 - 80ºC with 30mM lactose
as the substrate in 20mM phosphatebuffer pH 7 containing 10mM KCl and 1
mM MgSO4. The experimental results
are shown in Fig. 3, suggesting that thetemperature optimum is 50ºC.
The temperature stability of the βgalactosidase-catalyzed reactions wasanalyzed by measuring the remaining

Steady-state kinetic properties of the βgalactosidase-catalyzed reactions
Based on the initial velocity, kinetic
parameters including the Michaelis
constant Km and molar activity k0 were
evaluated for lactose or οNPG as a
substrate, where concentrations wereselected at the range from 2.4 to 57mM
for lactose and from 0.38 to 3.8mM forοNPG. Some typical examples of thereaction-time courses are shown in Figs.
4 and 5 for the substrate lactose andoNPG, respectively. The experimental
data were treated by a software, Biograph, Kyoto Soft Co., [19].
From slopes of the reaction timecourses, the initial velocity (v) was

β-Galactosidase from yeast K. lactis

102

Fig. 4. β-Galactosidase-catalyzed
reaction for a substrate lactose[E]0; 5.9nM, pH 7.0, 25ºC.

Fig. 5.

β-Galactosidase-catalyzed

reaction for a substrate oNPG
[E]0; 1.6nM, pH 7.1, 25ºC.
Table 1. Kinetic parameters on the βgalactosidase-catalyzed reaction for lactose
and oNPG[E]0; 1.6nM, 20mM phosphate buffer
pH 7.0, 25ºC.

obtained at different concentrations of
substrate [S]0. As shown, that the plots
are excellently fitted to the MichaelisMenten fashion. Thus, the kinetic parameters evaluated by using the steadystate methods are summarized in Table 1,
where the weighted mean values wereobtained on the basis of the experiments
carried out more than three times.
With kinetic parameters Km and k0,
k0/Km values were calculated as shownin Table 1, suggesting that οPNG is abetter substrate for the β-galactoseidase-catalyzed reaction than lactose.

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Communicated by Hiroshi Ueno.