J. Microbiol. Biotechnol. (2011), 21(3), 236–242
doi: 10.4014/jmb.1009.09010
First published online 15 January 2011
Molecular Characterization of Cold-Inducible β-Galactosidase from Arthrobacter
sp. ON14 Isolated from Antarctica
Xu, Ke1,2†, Xixiang Tang1,2†, Yingbao Gai2, Muhammad Aamer Mehmood2‡, Xiang Xiao2,3, and
Fengping Wang2,3*
1
School of Life Science, Xiamen University, Xiamen, 361005, P. R. China
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, 361005,
P. R. China
3
Key Laboratory of MOE for Microbial Metabolism and School of Life Science and Biotechnology, State Key Laboratory of Ocean
Engineering, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China
2
Received: September 8, 2010 / Revised: November 21, 2010 / Accepted: November 22, 2010
A psychrotrophic bacterium, Arthrobacter sp. ON14, isolated
from Antarctica, was shown to exhibit a high β-galactosidase
activity at a low temperature. A genomic library of ON14
was constructed and screened for β-galactosidase genes on
functional plates containing 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside (X-gal) as the substrate. Two different
β-galactosidase genes, named as galA, galB, were found in
ON14. Computational analyses of the genes revealed that
the encoded protein GalA belongs to family 2 of glycosyl
hydrolysases and is a cold-active protein, whereas GalB
belongs to family 42 of glycosyl hydrolysases and is a
mesophilic protein. Reverse transcription analyses revealed
that the expression of galA is highly induced at a low
temperature (4oC) and repressed at a high temperature
(28oC) when lactose is used as the sole carbon source.
Conversely, the expression of galB is inhibited at a low
temperature and induced at a high temperature. The
purified GalA showed its peak activity at 15oC and pH 8.
The mineral ions Na+, K+, Mg2+, and Mn2+ were identified
as enzyme activators, whereas Ca2+ had no influence on
the enzyme activity. An enzyme stability assay revealed
that the activity of GalA is significantly decreased when it
is incubated at 45oC for 2 h, and all its activity is lost when
it is incubated at 50oC.
Keywords: β-Galactosidase, cold inducible, Arthrobacter
*Corresponding author
Phone: +86-21-34207208; Fax: +86-21-34207205;
E-mail: [email protected]
†
These authors contribute equally to this paper.
‡
Present work address: Department of Bioinformatics and Biotechnology,
GC University, Faisalabad, Pakistan
Low temperatures are the most pervading environmental
factor of the Earth’s ecosystem. Psychrophilic and psychrotrophic
microorganisms are able to thrive in the coldest environments
on Earth and are the major source of cold-active enzymes
that have attracted industrial interest [5]. β-Galactosidase
(E.C. 3.2.1.23) is an enzyme that can hydrolyze lactose
into glucose and galactose, making it important to the
dairy industry, since it can be used to eliminate lactose
from milk for people who are lactose intolerant, convert
lactose to glucose to improve the sweetness of dairy products
[14], and remove lactose as a dairy industry pollutant [19].
In particular, cold-active β-galactosidases have recently
been attracting attention, as there is an increasing industrial
trend to treat dairy products under mild conditions to avoid
spoilage and changes in the taste and nutritional value, and
cold-active β-galactosidases can be inactivated at a low
temperature without heat treatment [12].
Based on hydrophobicity plots, amino acid sequence
similarities, reaction mechanisms, and the alignment of
possible common structural domains, β-galactosidases are
subdivided into four different families:1, 2, 35, and 42 [7].
Cold-active β-galactosidases are mostly produced by
psychrophiles, among which Arthrobacter is the most
frequently isolated genus [11, 15]. Cold-active β-galactosidases
have been purified and partially characterized since the
1990s, and most belong to glycosyl hydrolase family 2
[10, 11, 14, 15]. Until now, the coldest active β-galactosidase
is BglA from Arthrobacter psychrolactophilus strain F2,
with an optimum activity at 10oC [14]. Cold-active βgalactosidases are very important in the dairy industry, as they
enable dairy products to be treated under mild conditions,
in contrast to mesophilic β-galactosidases.
237
Xu et al.
The current authors previously isolated several psychrotrophic/
psychrophilic bacteria from deep-sea and Antarctic environmental
samples, and tested their enzyme-producing abilities
(unpublished data). Among these strains, a psychrotrophic
Arthrobacter strain ON14 was found to exhibit high βgalactosidase activity at a low temperature. Accordingly,
the present study reports on the cloning of two βgalactosidase genes from ON14, and the further purification
and characterization of the cold-active enzyme. Moreover,
it is shown that ON14 can adjust its isozyme expression in
response to changing temperatures.
MATERIALS AND METHODS
Bacterial Strains and Culture Conditions
Arthrobacter sp. ON14 was isolated from a soil sample taken near
the Great Wall Station in Antarctica (69o22'24''S, 76o22'40'') during the
19th Chinese Antarctic Research Expedition in 2002. The environmental
samples were stored at -20oC during the shipping process and in the
laboratory until used. A Luria-Bertani medium [1% tryptone (w/v),
0.5% yeast extract (w/v), and 1% NaCl (w/v), pH 7.4] and a M9
medium prepared using the method of Miller [13] with 0.6% (w/v)
lactose were used for the cultivation.
Cloning of β-Galactosidase Genes
The genomic DNA of Arthrobacter sp. ON14 was extracted using a
modified method of Brahamsha and Green [2], with the lysozyme
concentration increased to 5 mg/ml and incubated at 37oC for 1 h.
The isolated DNA was partially digested by Sau3AI and separated
on a 0.7% (w/v) agarose gel. The DNA fragments of around 10 kb
were excised and purified from the gel after electrophoresis and
ligated into a BamHI pre-digested pSP73 (Promega) vector [9]. The
clones were then plated on LB plates with ampicillin (100 µg/ml)
and X-gal (20 mg/ml). The positive transformants demonstrating
hydrolytic activity on the X-Gal (blue colonies) were isolated. The
plasmids were extracted from these transformants and partially
digested by Sau3AI and separated on a 1% (w/v) agarose gel. The
DNA fragments of around 4 kb were then subcloned into BamHItreated pSP73 and plated on LB plates with ampicillin (100 µg/ml)
and X-gal (20 mg/ml). The positive transformants demonstrating
hydrolytic activity on the X-Gal (blue colonies) were isolated. The
plasmids in the positive subclones were then sequenced using SP6
and T7RNA polymerase promoter-primers based on a walking strategy
(Sangon Inc. Shanghai, China).
Purification of Galactosidase-A
An E. coli strain harboring the galA gene was grown in an LB broth
supplemented with ampicillin (100 µg/ml) at 25oC until the OD600
reached 0.8-1.0. The cells were then harvested by centrifugation,
suspended in a loading buffer (25 mM sodium phosphate buffer, pH
8.0), and disrupted by sonication (SONCIS Vibra-Cell, 10 min, 40 W,
on ice). After lysis, the cell debris was removed using centrifugation
at 8,000 ×g for 10 min. The proteins were then precipitated by
adding 40% (NH4)2SO4. The precipitated proteins were collected by
centrifugation at 10,000 ×g for 20 min and dialyzed against the same
buffer to remove the salts. After dialysis, the supernatant was further
purified using an ÄKTA FPLC system (Amersham). The DEAE-
Sepharose column was prepared according to the instructions of the
manufacturer (Pharmacia). The dialyzed solution was applied to the
DEAE-Sepharose column (2.5×35 cm) equilibrated with the same
buffer, and a flow rate of 1 ml/min was maintained. The unbound
protein was washed with two bed-volumes of the starting buffer,
and the enzyme eluted using a linear gradient of NaCl (210 to
390 mM) in a 20 mM Tris-HCl buffer (pH 8.0). The protein profile
was monitored by measuring the absorbance at λ280 nm. The
elutions exhibiting enzyme activity were pooled and concentrated by
ultrafiltration using an Amicon ultracel-10K membrane (Amicon) to
give a final volume of 0.5 ml. The concentrated proteins were
further loaded onto a buffer-equilibrated Mono Q HR5/5 column
(1 ml; Amersham) and washed using a linear gradient of 0-0.4 M
NaCl in a 20 mM Tris-HCl buffer (pH 8.0) at a constant flow rate
of 0.5 ml/min. The eluted proteins were then collected and the
enzyme activity was measured under standard assay conditions. The
purity of the enzyme was confirmed by SDS-PAGE, and the
concentrations determined using the Bradford method [1].
Enzyme Assays
The β-galactosidase activity was determined by measuring the rate
of the hydrolysis of 10 mM O-nitrophenyl-β-D-galactopyranoside
(ONPG) as the substrate, as described previously [18]. The specific
activity was defined as the micromoles (µmoles) of ONP released
per minute per milligram of the protein. The protein concentrations
were determined using the Bradford method [1]. The standard curves
were generated using bovine serum albumin. The apparent optimal
reaction temperature of the purified enzyme was determined based on
incubation in a 25 mM sodium phosphate buffer (pH 8.0) containing
2.2 mM ONPG for 10 min at temperatures ranging from 0 to 60oC.
Meanwhile, the optimal pH of the enzyme was determined at 15oC
in buffers with pHs ranging from 4.0 to 10. The buffers used were
0.1 M sodium acetate-acetic acid for pH 4.0 to 6.0, 0.1 M phosphate
for pH 6.0 to 8.0, and 0.1 M potassium chloride-boric acid for pH
8.0 to 10. The thermostability of the enzyme was determined using
the modified method of Coker et al. [3]. The enzyme was incubated
at temperatures ranging from 30 to 50oC for up to 120 min, and the
residual enzyme activity then immediately assayed under standard
assay conditions. The impact of metal ions on the enzyme activity
was studied in a 20 mM phosphate buffer (pH 8.0) supplemented
with 5 mM of NaCl, KCl, MgCl2, CaCl2, MnCl2, ZnCL2, and CuCl2,
respectively. The enzyme was incubated with 5 mM of each metal
ion for 60 min at 15oC prior to performing the enzyme assay, and
the residual activity was measured under standard conditions. All
the experiments were performed in triplicate along with a control.
Expression Profile of Galactosidases
Arthrobacter sp. ON14 was incubated in a Luria-Bertani medium at
20oC until the OD600 reached 0.5-0.7. The cells were then collected
by centrifugation at 5,000 ×g for 10 min (Beckman Avanti J-E),
washed twice, and resuspended in a mineral synthetic medium [16]
containing 0.6% (w/v) lactose as the sole carbon source. Thereafter,
the cell suspension was incubated at 6, 20, or 28oC for 4 h. The
total RNA was extracted as described previously[20]. The specific
primer pairs [upstream primer 5'-CGGATCGGCGTGCGGCTGGACTT3'; downstream primer 5'-GCGGCCGGGCGTAGGGGACATTC-3')
and (upstream primer 5'-GCGCGGCATCACGACGGACTTC-3';
downstream primer 5'-GGGTGGCGGCAGCAGCAATGTT-3')] were
designed using Primer Express software (Applied Biosystems, San
A β-GALACTOSIDASE FROM ARTHROBACTER SP. ON14
238
Francisco, CA, USA) and used for GalA and GalB, respectively.
The real-time PCR was performed on a 7500 Real-time System
(Applied Biosystems) in a 20-µl reaction mixture that consisted of
1 µl of DNA as the template, 0.15 µM of each primer, and 10 µl of
AmpliTaq Supermix (Applied Biosystems, USA) with ROX and
SYBR Green I. The amplification conditions were 10 min at 55oC;
2 min at 95oC; then 40 cycles consisting of 15 s at 95oC and 1 min
at 60oC; followed by the plate read. The cycle threshold was set
automatically using the 7500 system software, version 1.3 (Applied
Biosystems).
Skim Milk Assay
The hydrolysis of lactose in milk was monitored by incubating
around 200 µg of purified GalA in 1 ml of commercial skim milk
[Inner Mongolia Meng Niu Dairy (Group) Co. Ltd, China] at 4oC for
8 h. The enzyme was inactivated by incubating at 60oC for 10 min
and the proteins were precipitated by adding 5 ml of 5% trichloroacetic
acid (TCA), plus 1.0 N NaOH was used to maintain the pH of the
solution at between 6.0 and 8.0. After centrifuging at 15,000 ×g and
4oC for 30 min, the supernatant was used for HPLC analysis. An
AKATA-purify 10 HPLC system equipped with a Carbo pac PA10
(4×250 mm) column (Pharmacia) was used, with 10 mM NaOH as
the mobile phase at a flow rate of 1 ml/min.
RESULTS
Isolation of Strains Producing Cold-Active β-Galactosidase
A variety of psychrotrophic and psychrophilic bacteria
were previously isolated from Antarctic soil samples ([22]
and unpublished data). When checking these strains for
their enzyme-producing abilities at a low temperature (4oC),
one strain named ON14 exhibited high galactosidase
activity, as when the strain colonies were placed on a XGal plate, they turned blue at a low temperature (photo not
shown). When the 16S rRNA gene sequence of ON14 was
determined, it showed a high sequence identity with A.
psychrochitiniphilus and A. psychrolactophilus. The phylogenetic
relationship between ON14 and other related Arthrobacter
reference strains is shown in Fig. 1.
Cloning of β-Galactosidase Genes from ON14
A genomic library was constructed for ON14 with an
average insert length of around 10 kb. The library contained
around 7,000 clones, corresponding to a genomic length of
around 70,000 kb and more than 14 times the genome of
ON14 (assuming that the genome size of ON14 is 5Mb). A
total of 8 β-galactosidase-producing clones (blue clones)
were screened out from the library. The plasmids were
then isolated from these clones and selected for further
subcloning and a sequencing analysis, as described above.
Gene Sequence Analysis and Preliminary Enzyme
Characterization
Two types of β-galactosidase genes were identified from
the eight clone sequences, and named galA (3,090 bp) and
Fig. 1. Phylogenetic tree showing the relationship of Arthrobacter
sp. ON14 with related Arthrobacter species.
galB (2,052 bp) (GenBank Accession No. HM178943 and
HM178942 for galA and galB, respectively). The coding
region of galA started with GTG, instead of the most
common start codon ATG. The BLAST results for the
amino acid sequence within the NCBI database showed
that the GalA from ON14 had the highest similarity with
BglA produced by Arthrobacter psychrolactophilus strain
F2 (97.28% identity) [14]. Moreover, the ON14 GalA
belonged to glycosyl hydrolysis family 2 (GH2 family),
which has several conserved regions, including glutamic
acid residues, a sugar-binding domain, an acid/base catalyst
triosephosphate isomerase barrel domain, and the GH2
signature of the GH2 family (Fig. 2).
Meanwhile, the BLAST results for the ON14 GalB
amino acid sequence within the NCBI database showed
that it had the highest identity (42.4%) with isozyme 12 of
the psychrotrophic Arthrobacter strain B7 [6]. The GalB
included two active-site residues (167 Trp and 172 Glu) in
the acid-base region (aa157-aa197) and the specific
conserved region (aa516-aa545) for members of the LacG
family. Thus, the GalB was assigned as a member of the
LacG family, which belongs to the glycosyl hydrolysis
family 42 (GH42 family) [6, 8].
Effect of Temperature on Expression of β-Galactosidase
Genes
The expressions of galA and galB in ON14 at different
temperatures were monitored using a Q-PCR, as described
in the Materials and Methods. When ON14 was transferred
from its optimal growth temperature (20oC) to a low
temperature (6oC), the expression of galA was found to be
increased about 6-fold, whereas the expression of galB was
nearly completely inhibited (Fig. 3). Conversely, when
ON14 was transferred to a higher temperature (28oC), the
expression of galA was almost totally inhibited, whereas
galB was found to be overexpressed nearly 2-fold when
compared with that at the optimal temperature (Fig. 3).
239
Xu et al.
Fig. 2. Multiple alignment of family 2 β-galactosidases from several Arthrobacter species.
They consist of five domains: Domain1, GH family 2 sugar-binding domain; Domain 2, GH family 2 immunoglobulin-like beta-sandwich domains; Domain
3, GH family 2 TIM barrel domain; Domain 4, GH family 2 immunoglobulin-like beta-sandwich domain; Domain 5, β-galactosidase small chain. ON14GalA, GalA from Arthrobacter sp. ON14 in this study; F2-BglA, BglA from Arthrobacter sp. F2; C2-2, isoenzyme from Arthrobacter sp. C2-2; SB,
isoenzyme from Arthrobacter sp. SB; B7-15, isoenzyme from Arthrobacter sp. B7-15. The black arrow indicats possible amino acids involved in thermal
stability (Val-107, Ser-134, Thr-135, Thr-136, Ala-741 in ON14-GalA). ON14-GalA (GenBank Accession No. HM178943), F2-BglA (AB243756), C22(CAD29775), B7-15(U12334), SB(AAQ19029).
Enzymatic Properties
Crude enzyme extracts were first extracted from the clones
containing the two β-galactosidase genes galA and galB,
respectively. The optimal temperature for the GalA extract
was found to be around 15oC (Fig. 4A). However, it
retained 30% of its activity at 0%, started losing its activity
above 15%, and yet still retained 50% of its activity at
40%, suggesting that GalA is a cold-active β-galactosidase
and may have good thermal stability. Meanwhile, the GalB
extract showed its peak activity at 37oC, and showed a very
low activity at temperatures close to 10oC and 60oC (Fig. 4A),
suggesting that GalB is a mesophilic β-galactosidase.
The cold-inducible GalA was purified for further studies,
owing to its possible application in the dairy industry. The
protein was purified by FPLC using DEAE-Sepharose and
Q-Sepharose, as summarized in Table 1. The SDS-PAGE
analysis revealed that the molecular mass of the protein
was close to 116 kDa (Fig. 5), which corresponded well
with the calculated value (111,453 Da). The purified GalA
also showed a similar reaction temperature range to that of
the crude enzyme, with its peak activity at 15oC (Fig. 4A).
In addition, the thermostability assay revealed that the
enzyme retained 30% of its activity at 0oC, started losing
its activity above 15%, and yet still retained 50% of its
A β-GALACTOSIDASE FROM ARTHROBACTER SP. ON14
240
Fig. 3. Differential expression of β-galactosidase genes under
different temperature inductions.
The ON14 cells were incubated at 20oC, and then shifted to 6oC or 28oC for
4 h, respectively (as described in Materials and Methods). Then, the
expressions of the galA and galB genes were monitored by Q-PCR. The
expressions of the genes at 20oC were set as 1.
activity at 40oC (Fig. 4B), confirming that GalA is a coldactive β-galactosidase. The ability of GalA to hydrolyze
lactose in commercial skim milk at a low temperature was
monitored, as described in the Materials and Methods.
After incubating GalA in the skim milk at 4oC for 8 h, the
lactose was completely digested into galactose and glucose
(data not shown).
The optimum pH for GalA was 8.0 and it exhibited
above 75% activity at pHs ranging from 6.0 to 9.0 and
even retained 70% of its activity at pH 10. When using
ONPG as the substrate, the specific activity of the purified
enzyme was shown to be 25.4 U/mg. The effects of different
metal ions on the enzyme activity were also studied, and
Na+, K+, Mg2+, and Mn2+ were found to have a positive
effect on the enzyme activity of GalA. However, Zn2+
significantly reduced the enzyme activity, and Cu2+ completely
inactivated the enzyme activity within 10 min (Table 2).
DISCUSSION
Cold-active β-galactosidases are mostly produced by
psychrotrophiles and several have been isolated since the
1990s. Loveland et al. [11] isolated Arthrobacter sp. D2
Fig. 4. Galactosidase activity assay.
The enzyme activity was determined as described in the Materials and
Methods. A. Effect of temperature on β-galactosidase activity. Black
square, purified GalA; black triangle, crude enzyme of GalA; black circle,
crude enzyme of GalB. B. The thermostability of GalA. Black circle, 30oC;
white circle, 35oC; black triangle, 40oC; white triangle, 45oC; black square,
50oC.
and D5 from Pennsylvania farmlands, which produce coldactive β-galactosidases with an optimum enzyme activity
at 25oC and 30oC, respectively. Coker et al. [3] obtained
BagS from Arthrobacter strain SB with an optimum
activity at 20oC [3]. Nakagawa et al. [14] isolated BglA
from Arthrobacter psychrolactophilus strain F2 with an
optimum activity at 10oC. In the present study, a coldactive GalA with an optimum activity at 15oC was isolated
from Arthrobacter sp. ON14. The GalA has the highest
sequence identity with BglA, both representing cold-
Table 1. Purification of the GalA.
Crude extract
40% (NH4)2SO4
DEAE-Sepharose
Q-Sepharose
Total protein
(mg)
Total activity
(U)
240.35
43.92
5.91
1.61
100.95
93.55
50.24
40.89
Specific activity
(U/mg)
0.42
2.13
8.5
25.4
Purification
(fold)
1
4.23
21.3
61.95
241
Xu et al.
Table 2. Effects of metal ions on GalA activity.
Metal ion
None
Na+
K+
Mg2+
Ca2+
Mn2
Zn2+
Cu2+
Residual activity
P value
100%
121%±5.5%
125%±7.5%
142%±4.6%
92%±4.4%
115%±4.5%
34%±2%
0%
0.02
0.029
0.004
0.086
0.027
0.00
0.00
SPSS16 analysis results and P<0.05 denote significant difference.
Fig. 5. Purified GalA determined on 10% SDS-PAGE gel.
Lane1, cell lysate; lane2, purified GalA.
loving β-galactosidases; however, they show significant
differences in their thermal stability. The enzyme activity
of GalA dramatically decreased above 45oC and almost
disappeared at 50oC, yet remained very stable below or at
40oC. In contrast, BglA and many other cold-active βgalactosidases lose most of their enzyme activity at around
35oC [3, 4, 11, 14], making GalA relatively thermostable.
However, there seems to be an apparent contradiction: the
enzyme GalA starts to be inactivated at 45oC, yet the
thermostability assay revealed that the enzyme was partially
stable at 40oC (Fig. 4A and 4B). A possible explanation is
reversible unfolding and dissociation of the active enzyme
within a certain temperature range (i.e., below 50oC) [17].
At temperatures above 15oC, GalA is gradually dissociated
and loses partial activity, yet its monomers can be quickly
reassociated into the active form when it is taken back
to a low temperature. Meanwhile, the dissociated GalA
proteins at a high temperature can quickly associate into
the active enzyme unit [3] when cooling down to a low
temperature. This may explain the contradiction between
the thermodependent activity and stability of GalA.
Comparisons of several cold-active enzymes, including
GalA, BglA, and isozymes from Arthrobacter strain C2-2,
SB, and B7-15, found that the thermal stabilities of GalA,
C2-2, and B7-15 were very similar, whereas that of BglA
and SB was similar [3, 10, 14, 21]. Five amino acid
residues, Val-107, Ser1-34, Thr1-35, Thr-136, and Ala741,
were identified as potentially important residues for the
enzyme thermal stability, as they corresponded with
residues in GalA, C2-2-1, and B7-15, yet differed in BglA
and BgaS (Fig. 2). However, the roles of these residues in
the enzyme thermal stability need further study.
The co-existence of more than two β-galactosidase
isozymes in one single bacterial strain has been previously
reported [3, 10, 11, 15]. In the present study, two β-
galactosidase genes, galA (3,090 bp) and galB (2,052 bp),
were isolated from Arthrobacter sp. ON14 and found to
encode isozymes belonging to the GH2 and GH42 family,
respectively. Whereas GalA was found to be cold-active,
GalB was active under mesophilic conditions. Different
types of β-galactosidase are distributed in different areas in
cells, and carry out different physiological functions [18,
23]. Generally, intracellular β-galactosidases are related to
carbon metabolism and provide material for the construction
of the cell wall. GalA and GalB were both found to be
intracellular, as very little enzyme activity was detected
outside the bacterial cells (unpublished data). Thus, questions
arise as to why the cells need two enzymes with similar
functions, how the enzymes are regulated, and whether
they are related to the environmental adaptation of the
cells? The expressions of both galA and galB were
monitored according to different temperatures, using QPCR analyses with lactose as the sole carbon source. A
remarkable induction of galA and inhibition of galB were
observed at a low temperature (4oC) and vice versa at a
high temperature (28oC), suggesting that ON14 is more
likely to use GalA at a low temperature as it is coldactive, and then shifts to the overproduction of GalB to
cope with an increasing temperature. This would seem to
be the most economic adaptation to improve the utilization
efficiency of lactose by the strain. Thus, the presence of
two β-galactosidase isozymes in Arthrobacter sp. ON14
provides more flexibility and adaptability in response to
ever-changing environmental temperatures.
The metal ions experiments revealed that Na+, K+, Mg2+,
and Mn2+ are activators, with Mg2+ being a possible cofactor for GalA; Zn2+ and Cu2+ are inhibitors; and Ca2+ has
no effect on the enzyme activity. The effects of metal ions
on β-galactosidase are important to consider, as metal ions
should not inhibit the enzyme activity during dairy processes.
The ability of GalA to hydrolyze lactose in commercial
skim milk at a low temperature was also demonstrated.
Thus, GalA may have potential applications in the dairy
industry, as it allows dairy products to be treated under
mild conditions, in contrast to mesophilic β-galactosidases.
A β-GALACTOSIDASE FROM ARTHROBACTER SP. ON14
Acknowledgments
This study was supported by the National Infrastructure of
Natural Resources for Science and Technology Program of
China (No. 2005DKA21209), National High-Tech Program
(2007AA091904), and China Ocean Mineral Resources
R&D Association (COMRA DYXM-115-02-2-03).
11.
12.
13.
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