65
b-Galactosidase
Raymond R. Mahoney
University of Massachusetts, Amherst, Massachusetts, U.S.A.
I. INTRODUCTION the enzyme is used in clinical settings to aid lactose
digestion and in commercial operations to produce
-Galactosidase ( -D-galactoside galactohydrolase, low-lactose diary products.
EC 3.2.1.23) is often known as lactase because it
catalyzes the hydrolysis of lactose into its constituent
II. IMPORTANCE TO FOOD QUALITY
sugars viz., lactoseþH2O!galactoseþglucose.
All lactases are -galactosidases but some -galactosi-
Enzymatic hydrolysis of lactose is of interest in both
dases, including those from plant cells and mammalian
food science and nutrition, as well as in waste manage-
organs other than the intestine, have little or no activ-
ment and byproduct utilization. The product sugars
ity on lactose because their function is to hydrolyze
are sweeter, more soluble, more easily fermented, and
other galactosyl moieties, including glycolipids, glyco-
more readily absorbed from the intestine.
proteins, and mucopolysaccharides (1).
Consequently, hydrolysis of lactose in milk and whey
-Galactosidase hydrolyzes O-glycosyl bonds and
provides new functional properties for these ingredi-
exhibits strict specificity for the glycone part of the
ents which can be exploited in a variety of products
substrate (Fig. 1). The only changes that allow activity
(see Sec. IV below).
(albeit reduced) are replacement of the hydroxylmethyl
The principal reason for hydrolyzing lactose in fluid
group on carbon 6 with a methyl group or a hydrogen
milk, however, is to overcome the problem of lactose
atom. Consequently, the enzyme shows some activity
on -L-arabinosides and -D-fucosides. Other changes
such as methylation of the hydroxyl on carbons 2, 3, 4,
or 6; loss of the pyranose ring structure; or conversion
to the -anomeric form, lead to loss of activity (1).
Replacement of the glycosidic oxygen with sulfur
causes loss of activity but not loss of binding affinity
(1). The enzyme shows wide tolerance for the structure
of the aglycone which may be another sugar, an alkyl
group or an aryl group. The nature of the aglycone,
however, strongly influences the kinetic parameters (1).
Activity of intestinal lactase in mammals decreases
after weaning, and this leads to some degree of lactose
intolerance in many population groups. Consequently,
Figure 1 Hydrolysis of lactose by -galactosidase.
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
intolerance, which is widespread among non-Caucasian IV. USE IN FOODS
adults and leads to reduced milk consumption. This is
of concern since milk is a good source of high-quality Reduction of the lactose content of milk or whey by
protein and an especially good source of calcium. hydrolysis with -galactosidase leads to several pro-
Whey is the principal byproduct of cheese manufac- ducts/applications, as shown in Table 2. These applica-
ture and contains>70% lactose on a dry-weight basis. tions have been reviewed extensively elsewhere (2).
Hydrolysis of the lactose improves whey utilization in
a variety of products and processes (see Sec. IV).
V. PROPERTIES AS A PROTEIN
III. SOURCES
The best-understood -galactosidase in terms of pro-
tein structure is the enzyme from Escherichia coli
-Galactosidases are widely distributed in nature
encoded by the lac Z gene. This enzyme has a molecu-
according to their various functions, which include
lar weight of 464 kDa and is composed of four iden-
digestion, lysosomal degradation, and catabolism.
tical subunits (each 116 kDa), each containing 1023
However, lactases are found essentially only in the
residues (3). X-ray crystallography confirms that the
mammalian intestinal tract and in various microorgan-
protein is a tetramer with 222 point symmetry (4).
isms, including many fungi, yeasts, and bacteria (2).
Each subunit contains a binding site for Mg2þ and
In the intestine, the enzyme is located in the micro-
has five essentially independent domains. The key
villi of the mucosal cells of the brush border mem-
active site residues are located in a depression formed
brane. In microorganisms, the enzyme is usually
from loops connecting the C-terminal ends of -
intracellular in bacteria and yeasts but may be intra-
strands to surrounding -helices in an = barrel.
or extracellular in fungi.
Subunits and dimers of subunits are inactive whereas
For commercial use lactase is extracted from a few
activity in the tetramer state is due to completion of
microbial sources, primarily yeasts and fungi, which
each active site by a loop donated from a neighboring
are considered safe as hosts for food grade enzymes
monomer (4).
(see Table 1). It may be possible to use -galactosidases
At the primary sequence level, each subunit con-
from other sources by cloning them into one of these
tains 16 half-cystine residues per 116-kDa monomer;
hosts. The commercial enzymes are usually classified as
virtually none of these residues appear to be involved
acid pH (pH optimum <5) for use in acid whey, or
in disulfide linkages (5).
neutral pH (pH optimum 5.5 7.0) for use in milk or
sweet whey. The enzyme is also produced from
Escherichia coli for biochemical and analytical uses.
Table 2 Applications of Lactose Hydrolysis
Product/process Advantages
Table 1 Commercial Sources of -Galactosidase for
Hydrolysis of Lactose in Foods
Low-lactose milk Overcomes the problem of
lactose intolerance
Molecular
Sweetened condensed milk, Reduce lactose crystallization
Organism pH Optimuma weight (kDa)
ice cream, dulce de leche in concentrated or frozen
Bacteria milk products
Bacillus spp. related to 5.5 6.5 116 Yogurt and cheese Accelerates ripening by
B. stearothermophilus production of more easily
Yeasts fermented sugars
Kluyveromyces fragilis 6.5 7.5 201 Sugars syrups from Increased sweetness of
K. lactis 6.5 7.0 117 deproteinized whey glucose and galactose
Candida psuedotropicalis 6.2  b allows multiple uses in ice
Fungi cream, baked goods,
Aspergillus niger 2.5 4.0 109 112 confectionery, and soft
A. oryzae 4.5 5.0 90 drinks
Fermentation to ethanol Fermentation yeasts grow
a
Dependent on strain.
more easily on the glucose
b
Not known.
produced
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
In contrast to the above, very little is known enzyme,enzyme-lactose
about the protein structure of -galactosidase
enzyme-lactose!enzyme-galactose
from eukaryotes. However, amino acid sequences
have been established for -galactosidase from sev- +glucose enzyme-galactose!
eral other bacteria and from the yeast
enzyme-galactoseþenzyme-galactoseþ
Kluyveromyces lactis (6). Comparison of the yeast
-galactosidase with the subunits of -galactosidase
þH2O!enzymeþgalactose
from prokaryotes show similarity in molecular
ð1Þ
weight (117 kDa) and extended sequence homology,
Reaction of the enzyme-galactoseþ complex with
suggesting close structural and evolutionary rela-
another sugar (such as lactose, galactose, or glucose)
tionships (6).
instead of water allows for the synthesis of oligosac-
Extracts of E. coli subjected to electrophoresis
charides by the transferase reaction, which can become
and stained histochemically for -galactosidase
significant at high substrate concentrations and/or
show multiple bands (7), and a similar phenom-
when the degree of substrate conversion is high (13).
enon has been seen with both crude and purified
There are several substrate/product analogs which
enzyme from Kluyveromyces fragilis (8). Purification
inhibit -galactosidase. Galactose is a competitive inhi-
and characterization of the isoforms from E. coli
bitor, but glucose is often ineffective except at very
have shown them to be polymers and have not
high concentrations, where it is noncompetitive (14).
revealed compositional differences (9). In the case
Thiogalactosides such as p-aminophenyl -D-thio-
of fungal enzymes, however, isoforms have been
galactoside are weak competitive inhibitors (15, 16).
observed which do differ in the extent of glycosy-
The most powerful inhibitors are the 1-4 and 1-5 galac-
lation (10).
tonolactones, especially the latter, which, it has been
suggested, may resemble the transition state form of
the substrate (17).
VI. PROPERTIES AS AN ENZYME
The enzymatic properties of -galactosidase vary with
the source. These properties are summarized in Table 3 VII. DETERMINATION OF ACTIVITY
for three different -galactosidases (one each from bac-
teria, yeasts, and molds). -Galactosidase activity can be followed quite readily
Comparison of kcat=Km shows that the synthetic by following the disappearance of substrate or the
substrate ONPG (o-nitrophenyl -D-galactopyrano- appearance of product, but it is usually easier to follow
side) is a better substrate than lactose in all cases. product formation.
The fungal enzyme from Aspergillus niger is the most Determination of lactose hydrolysis is complicated
thermostable and is unaffected by cations or sulfhydryl by transferase activity, although the latter is minimal
modifiers such as p-chloromercuribenzoate. However, (and can often be ignored) when the lactose concentra-
it is the most susceptible to product inhibition by tion is 5% or less, or only initial rates are being mea-
galactose. sured. Under these conditions, it is easy to assay
The mechanism of action is not completely activity on lactose by measuring the production of
understood, but the E. coli enzyme appears to glucose or galactose, preferably enzymatically. The
work in a fashion analogous to that of lysozyme. most commonly used method is determination of glu-
Thus, one group acts as a general acid, donating a cose with glucose oxidase coupled to peroxidase (see
proton to the glycosidic oxygen, and another, nega- Sec. VII.A below).
tively charged group stabilizes a positively charged Where a large percentage of the original lactose is
carbonium galactosyl-enzyme intermediate for reac- hydrolyzed, determination of total monosaccharides is
tion with water (11). Site-directed substitutions in a good practical indicator of the extent of hydrolysis,
the E. coli enzyme indicate that Tyr503 functions but it may underestimate the number of glycosyl bonds
as the general acid and that Glu461 functions as broken in lactose, owing to the formation of oligosac-
the stabilizer for the intermediate which alternates charides by the transferase reaction.
between a carbocation and a covalently bound Hydrolysis of lactose can also be followed by polari-
form (12). The hydrolytic reaction [Eq. (1)] can metry due to a small change in specific rotation (from
then be described as: þ52:5 to þ67:0 ). However, this method is not
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
Table 3 Enzymatic Properties of -Galactosidase
Escherichia coli Kluyveromyces fragilis Aspergillus niger
(14) (8, 15) (10, 27)
Molecular weight (kDa) 464 201 109 112
pI 4.61 5.1 4.64
pH optimum 7.2 7.4 6.2 6.4 2.5 4.0
pH stability 6 8 6.5 7.5 2 8
Temperature optimum ( C)
40 37 55 60
Activation energy (kJ/mole) 12.6 38.1 35.1
kcat ONPGa sec 1 1:38 106 3:41 103 2:19 105
kcat lactose sec 1 5:10 103 1:551 103 1:91 105
Km ONPGa (mM) 0.161 2.72 2.22
Km lactose (mM) 1.9 13.9 85 125
kcat=Km PNPGa (sec 1 M 1)8:57 109 1:25 106 98:6 106
kcat=Km lactose (sec 1 M 1)2:68 106 1:08 105 1:91 106
Ki galactose (mM) 21 27.7 4
Ki PAPTGb (mM) 5 6.05  c
Activators Naþ, Mg2þ Kþ, Mg2þ, Mn2þ None
Inhibitors
Galactono-1-4-lactone Yes Yes  c
p-Chloromercuribenzoate Yes Yes No
a
ONPG, o-nitrophenyl- -D-galactoside.
b
PAPTG, p-aminophenylthio- -D-galactoside.
c
Not known.
used much compared to chemical estimation of the analyzed for glucose, preferably by the use of glucose
products. oxidase and peroxidase in conjunction with a dye such
Synthetic substrates such as o-nitrophenyl -D- as o-dianisidine [Eqs. (2) and (3)].
galactopyranoside (ONPG) and p-nitrophenyl -D-
glucose
galactopyranoside (PNPG) are usually hydrolyzed
D-glucose+O2 ! H2O2þD-gluconolactone
oxidase
faster than lactose and provide an easy, convenient,
colorimetric assay since the nitrophenol released
ð2Þ
absorbs in the range 400 420 nm when in alkaline
solution.
peroxidase
For detection of very low levels of activity, fluori- H2O2þo-dianisidine ! oxidized o-dianisdine
metric methods of assay can be used with substrates
such as 4-methylumbelliferone -D-galactoside (8). ð max 436 nmÞð3Þ
The conditions for assay vary with the source of the
enzyme and the substrate; representative examples are
Alternatively, the galactose produced can be esti-
given below.
mated indirectly by use of galactose dehydrogenase
[Eq. (4)].
A. Assay of b-Galactosidase Activity on Lactose
galactose
with Neutral-pH Enzyme
D-galactoseþNADþ !
dehydrogenase
ð4Þ
Enzyme 100 L( 30 U/mL) is added to 4 mL of 5%
D-galactonolactoneþNADHþHþ
lactose in 0.1 M potassium phosphate buffer, pH 6.6,
containing 3.2 mM MgCl2, at 30 C, or to 4 mL milk at
the same temperature. The reaction is stopped after 10 The increase in absorbance at 340 nm is a measure
min by adding 0.1 mL of 4 N HCl and then neutralized of the NADH produced and of the lactose hydro-
by adding 0.1 mL of 6.4 N NaOH. The solution is then lyzed.
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
B. Assay of b-Galactosidase Activity on o- cipal stabilizer, but its effect is markedly lactose
Nitrophenylgalactoside (ONPG) Using dependent.
Neutral-pH Enzyme The -galactosidase from Streptoccocus thermophi-
lus can be stabilized by a number of proteins, such as
Enzyme (100 L ( 1 U/mL) is added to 4 mL of 2 bovine serum albumin and casein, via hydrophobic
mM ONPG in 0.1 M potassium phosphate buffer, pH interactions (24). The -galactosidase from K. lactis
6.6, containing 0.1 mM MnCl2 and 0.5 mM dithioer- is stabilized by several amino acids, notably histidine,
ythritol. The reaction is stopped after 5 min by adding which decreases the rate of unfolding of the enzyme
1mLof 0.5M Na2CO3 containing 15 mM EDTA, and during heat denaturation (25).
the absorbance is determined at 420 nm. Activity is
calculated using a molar extinction coefficient of 4500
M 1 cm 1 for o-nitrophenol at pH 10 (15). For acid-
VIII. PURIFICATION
pH enzymes, the substrate buffer is changed to 100
mM sodium acetate buffer, pH 4.0, and the reaction
-Galactosidase can be purified by the conventional
is stopped as described above.
methods of protein separation such as gel filtration
and ion exchange chromatography and also, very
C. Other Aspects
effectively, by affinity chromatography. Examples are
shown below.
-Galactosidase activity in tissues can be detected by
histochemical staining with 6-bromo-2-naphthyl- -
galactosidase. The product 6-bromo-2-naphthol, reacts A. Purification of b-Galactosidase from
with tetrazotized o-dianisidine (Diazo Fast Blue B) in Kluyveromyces lactis (26)
alkaline solution to give a blue-purple color at the site
of enzyme activity (19). The same method can be used The yeast cells were disrupted in a steel press, and the
to detect activity of -galactosidase in electrophoresis ruptured cells were extracted with 50 mM phosphate
gels (7). buffer, pH 7.0, containing 10 mM MgCl2 (Buffer 1).
Neutral-pH enzymes require Mg2þ or Mn2þ for After centrifugation to remove cell debris, the super-
maximum activity, but acid-pH enzymes (from fungi) natant was brought to 55% saturation with
do not require metal ions. Incorporation of a sulfhy- (NH4Þ2SO4 to precipitate the enzyme. The precipitate
dryl reagent (2-mercaptoethanol or dithiothreitol) was dialyzed against Buffer 1 containing 1 mM 2-mer-
often increases activity with neutral-pH enzymes. captoethanol to stabilize the enzyme against activity
Activity and stability of -galactosidase in milk loss. The enzyme was then purified by gel filtration
and whey can be very different from that in lactose chromatography on Sephadex G-100 using Buffer 1
solutions. Activity is often lower in milk or whey followed by ion exchange chromatography on
(20, 21) owing to the high level of calcium, which DEAE-Sephadex A50. The enzyme was applied in
exerts a strong inhibitory effect (21, 22). Stability Buffer 1 and was eluted with a gradient of 0 0.5 M
against thermal denaturation is markedly NaCl in Buffer 1. A summary of the purification is
increased up to 100-fold in milk (23). This appears shown in Table 4. Overall, this enzyme was purified
to be due to several milk constituents: salts, lactose, 79-fold with a yield of 19% and was judged homoge-
and proteins, acting in concert. Casein is the prin- neous by polyacrylamide gel electrophoresis.
Table 4 Purification of -Galactosidase from Kluyveromyces lactis
Volume Protein Activity Specific activity
Fraction (mL) (mg/mL) (U 10 3) (U/mg)
Centrifuged cell extract 46 77 14.9 4.22
Ammonium sulfate fractionation 30 48 12.7 8.25
Sephadex G-100 123 2.7 14.5 43.7
DEAE Sephadex-A50 26 0.55 2.04 143
Source: Ref. 26
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
10. F Widmer, JL Leuba. -Galactosidase from
B. Purification of -Galactosidase from
Aspergillus niger. Separation and characterization of
Escherichia coli by Affinity Chromatography
three multiple forms. Eur J Biochem 100:559 567,
(16)
1979.
11. ML Sinnott. Ions, ion pairs and catalysis by the lacZ
Cells of E. coli were suspended in 50 mM Tris-HCl, pH
-galactosidase of Escherichia coli. FEBS Lett 94:1 9.
7.5, containing 100 mM NaCl and 10 mM MgCl2 12. CG Cupples, JH Miller, RE Huber. Determination of
(Buffer 2) and disrupted by sonication. After centrifi-
the roles of Glu-461 in -galactosidase (Escherichia
gation to remove cell debris, the supernatant was
coli) using site-specific mutagenesis. J Biol Chem
brought to 50% saturation withðNH4Þ2SO4 and cen-
265:5512 5518, 1990.
trifuged, and the pellet was dissolved in Buffer 2 and
13. RR Mahoney. Galactosyl-oligosaccharide formation
dialyzed against the same buffer. The enzyme solution during lactose hydrolysis a review. Food Chem
63:147 154, 1998.
was then applied to a column of an affinity matrix
14. K Wallenfels, OP Malhotra. -Galactosidase. In: PD
consisting of the inhibitor p-aminophenyl- -D-galacto-
Boyer, H Lardy, K Myrback, eds. The Enzymes, Vol 4
pyranoside (Ki 5 mM) attached to Sepharose 4B via
(2nd ed). New York: Academic Press, 1960, p 409.
a long spacer arm (3-aminosuccinyl-3-aminodipropyla-
15. RR Mahoney, JR Whitaker. Stability and enzymatic
mine). The column was washed with Buffer 2, and the
properties of -galactosidase from Kluveromyces fra-
enzyme was eluted by 0.1 M borate buffer, pH 10; it
gilis. J Food Biochem 1:327 350, 1977.
was homogeneous as judged by gel electrophoresis.
16. E Steers Jr, P Cuatrecasas, HB Pollard. The purifica-
The yield was 95% compared with a yield of 50%
tion of -galactosidase from Escherichia coli by affi-
using conventional procedures (5).
nity chromatography. J Biol Chem 246:196 200, 1971.
17. J Conchi, AJ Hay, I Strachan, GA Levvy. Inhibition
of glycosidases by aldonolactones of corresponding
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Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.