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Journal of Animal Production Advances
Cold Active β-Galactosidase of Hafnia Alvei KNOUC302
Isolated from Raw Milk: Identification of the Bacteria,
Cloning Partial Gene of β-Galactosidase in Escherichia Coli
Nam E. S. and Ahn J. K.
J Anim Prod Adv 2012, 2(2): 101-108
Online version is available on: www.grjournals.com

ISSN: 2251-7677
NAM AND AHN
Original article
Cold Active β-Galactosidase of Hafnia Alvei
KNOUC302 Isolated from Raw Milk: Identification
of the Bacteria, Cloning Partial Gene of β-
Galactosidase in Escherichia Coli
1 Nam E. S. and *2 Ahn J. K.
1 Sogang-Binggrae Food Advanced Analysis Center, Sogang University, Seoul 121-742, Republic of Korea
2 Department of Agricultural Sciences, Korea National Open University, Seoul 110-791, Republic of Korea
Abstract
Psychrophilic bacteria producing lactose hydrolyzing enzyme were isolated from the samples of raw milk
collected from dairy farms of Kyung-gi province in Korea. Among isolated strains, KNOUC302 showing the
best activity of lactose hydrolysis was identified, and its β-galactosidase gene was cloned. This strain was
aerobic, asporogenic bacilli, immobile, Gram negative, catalase positive and oxidase positive. It grew optimally
at 20 ºC and pH=7.0-7.2. The composition of major fatty acids in the cell membrane of KNOUC 302, were C 12:0
(10.2 %), C 14:0 3OH (25.41 %), C 16:0 (9.94 %) and C 16:1 ω 7 c (29.75 %). Based on morphological and biochemical
properties, the strain could be identified as Hafnia sp. and finally as Hafnia alvei by phylogenetic analysis based
on 16S rDNA sequence. A part of β-galactosidase gene, 779 bp, coding 296 a.a from N-terminal of the β-
galactosidase was isolated from Hafnia alvei KNOUC302 by partial digestion of chromosomal DNA with
Sau3AI and cloned in E. coli Top 10F’. The incomplete gene of 779 bp was supplemented, from vector during
cloning, with 109 bp nucleotides providing terminal codon to a complete ORF (KNOUC302 β-gal). The gene
(KNOUC302 β-gal) consists of 918 bp encoding the protein of 306 amino acids and 34,806 Da of deduced Mw.
Key words: Psychrophilic, β-galactosidase gene, Hafnia alvei, cloning
* Corresponding author: ajk@knou.ac.kr
Received on: 06 Jan 2012
Revised on: 26 Jan 2012
Accepted on: 29 Jan 2012
Online Published on: 01 Feb 2012
101 J. Anim. Prod. Adv., 2012, 2(2):

COLD ACTIVE β–GALACTOSIDASE OF HAFNIA ALVEI KNOUC302 ISOLATED FROM …
Introduction
Psychrophiles exhibiting optimal growth at low
temperature (1) produce cold-adapted enzymes,
which exhibit high catalytic activities at low
temperature to adapt to cold habitats, and thus,
psychrophiles have attracted attention as sources of
enzymes with potential for low-temperature
catalysis (2). Many psychrophiles were reported to
produce variety of cold-active enzymes useful in
food processing, biomass conversion, molecular
biology, environmental biosensors, bioremediation,
cleaning of contact lense, and several other
processes (3-7).
In particular, cold active enzymes are attractive
in food industry for the processing of fruit juices
and milk, because there is an increasing industrial
trend to treat food stuffs under mild conditions to
avoid spoilage, changes in taste and nutritional
values at ambient temperature, and because coldactive
enzymes can be inactivated at moderate
temperatures without harsh heating treatment (8, 9).
Among cold-active enzymes, β-galactosidase
(EC.3.2.1.23), which hydrolyzes lactose to glucose
and galactose, is one of the important foodindustrial
enzymes. It can be used to degrade
lactose for several purposes; e.g., (1) removal of
lactose from refrigerated milk for people who are
lactose intolerant, (2) conversion of lactose to
glucose and galactose, which are more fermentable
sugars than lactose, and (3) removal of lactose from
pollutants of dairy industry (10).
There have been many reports on cold active β-
galactosidase from psychrotrophic bacteria of
Arthrobacter psychrolactophilus (11),
Carnobacterium pisciocola BA (12) and
Pseudoalteromonas haloplakis (2), Planococcus
(13), and a psychotropic yeast of Guehomyces
pullulans (7). The genes coding cold active β-
galactosidase have been detected in Arthrobacter
sp. (14), Carnobacterium sp. (12) and Planococcus
sp. (13). However there has not been a useful one
yet for application in milk processing and further
research is required to find a proper one for
practical use.
We isolated a psychrophilic bacteria, Hafnia
alvei, producing β-galactosidase, cloned gene of the
enzyme in E. coli, and the results are being
reported.
Materials and Methods
Screening and cultivation condition
Raw milk samples produced at dairy farms of
Northern Kyunggi province in Korea were used for
isolation of psychrophilic bacteria hydrolysing
ONPG and lactose. Milk sample was enriched in the
Brain heart infusion broth (BHI; Difco
Laboratories, Detroit, Mich) containing 1 % (w/v)
lactose, and incubated at 4 ºC aerobically by
shaking (200 rpm) for 30 days, and spread on BHI
agar containing 1 % (w/v) lactose, and 0.01 % (w/v)
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
(X-gal; Duchefa Biochemei, Holland). After
incubation at 15 ºC for 3 days, blue colonies were
selected, and then cultivated in Brain heart infusion
broth and agar for determining β-galactosidase
activity and identification.
Assay of β-galactosidase activity
β-galactosidase activity was determined by
measuring the hydrolysis of o-nitrophenyl -Dgalactopyranoside
(ONPG; Sigma) as the substrate
by the procedure presented by Miller (15). An
aliquot of cell free extracts (0.5 mL) was added to
2.5 mL of ONPG solution (0.04 M, in Na-phosphate
buffer of 0.01 M and pH 6.8) and incubated at 4 ºC
for 2 hrs. The reaction was stopped by addition of 3
mL of 0.5 M Na 2 CO 3 and the absorbance at 420 nm
was measured. One unit of enzyme activity is
defined as the activity hydrolyzing 1μmol of ONPG
per min by cell free extract from 1 mL of culture
cell whose A 600 was adjusted to 8.0. Hydrolysis of
lactose was measured by glucose detection kit
(Glucose B-test Wako; Wako Pure Chemicals
Industries, Ltd, Osaka, Japan). 0.04 mL of cell free
extracts was added to 1.6 mL of skim milk, and
incubated for 5 days at 4 ºC. One unit of activity for
hydrolysis of lactose was defined as the amount of
enzyme producing 1 μmol of glucose per day by 1
mL of cell culture whose A 600 was 8.0.
Morphological and biochemical
characterization of microorganism
Among isolated strains, the strain showing the
highest β-galactosidase activity was identified by
102 J. Anim. Prod. Adv., 2012, 2(2):101-108

Gram staining, morphological, biochemical, and
physiological tests. Cell was grown on BHI agar to
determine optimum growth condition for
temperature (5-40 ºC) and in BHI broth for pH(4.0-
8.0). pH of BHI broth was adjusted with HCl or
NaOH. Acid production from carbohydrate, and
utilization of sole carbon sources were determined
using API tests (BioMerieux), including API 20E
(identification system for Enterobacteriaceae and
other Gram-negative rods) and API 20NE
(identification system for gram-negative nonenterobacterial
rods) galleries.
Analysis of cellular fatty acid composition
The cell biomass for cellular fatty acid
composition analysis was collected from BHI agar
plates after incubation for 5 days. Cells were
harvested, and the cellular fatty acid was saponified,
methylated and extracted following the instructions
in the manual for Sherlock Microbial Identification
System (MIDI, USA). The fatty acids were
analyzed by gas chromatography (Hewlett Packard
6890, USA) and identified using the Microbial
Identification software package (16).
Determination of 16S rDNA sequence and
phylogenetic analysis
Genomic DNA was isolated, and 16S rDNA
was amplified by PCR and sequenced following the
method described by Rainey et al. (17). Universal
primers of fD1 (5’-GAGTTTGATCCTGGCTCAG-
3’) and rD1 (5’-AGAAAGGAGGTGATCCAGCC-
3’) were used for PCR. PCR products were purified
by ethanol precipitation and electrophoresis with a
model 377 Genetic Analyzer (Perkin-Elmer Co.).
The 16S rDNA sequence was aligned against the
previously determined sequences in Ribosomal
Data of GenBank. The phylogenetic tree for the
dataset was inferred using the neighbor-joining
method (18).
Assaying effect of temperature and pH on the
β-galactosidase activity
Effect of temperature on β-galactosidase
produced in cell free extracts of KNOUC 302 was
analyzed by measuring the enzyme activity at
various temperatures (4 to 60 ºC) in 0.01 M sodium
phosphate buffer (pH 6.8). Optimum pH for the β-
galactosidase activity was determined by measuring
the activity at various pHs(4.6 to 9.6) at 4 ºC in
103 J. Anim. Prod. Adv., 2012, 2(2):
NAM AND AHN
sodium acetated buffer (0.01 M, pH 4.25-pH 6.0)
and sodium phosphate buffer (0.01 M, pH 6.0-pH
7.68). Enzyme stability was determined by
measuring the residual activity during incubation of
cell free extracts of KNOUC302 in sodium
phosphate buffer (0.01 M, pH6.8) at 4 ºC and 37 ºC
for 7 days.
Cloning and sequence determination of β-
galactosidase gene
The chromosomal DNA of strain KNOUC302
cells was isolated using a Genomic DNA Prep Kit
(A&A Biotechnology, Poland) according to the
protocol for gram-negative bacteria. The DNA was
partially digested using Sau3A1 endonuclease, and
4-10 kb fragments were collected and purified after
electrophoresis in 0.8 % agarose gel using the DNA
Gel Out Kit (A&A Biotechnology, Poland). To
prepare genomic library, these DNA fragments
were ligated into BamH1 site in pRSET (Promega,
USA), transformed to E. coli TOP10F’ and
incubated at 15 ºC for 3 days on LB agar containing
100 ug ampicillin/mL, 100 ug/mL X-Gal without
addition of IPTG. Colony was randomly taken for
analysis. Sequence of the cloned DNA fragment
was determined using Big Dye Automatic
sequencer ABI 377 (Perkin Elmer, USA) and
PE9600 Thermocycler (Perkin Elmer, USA).
Results and Discussion
Isolation and identification of bacterium
producing cold-active β-galactosidase
From raw milk samples collected from dairy
farms of Northern Kyunggi province in Korea. 11
strains having the activity of X-gal hydrolysis at 4
ºC were isolated and KNOUC302 showed a good
activity of ONPG hydrolysis and the highest
activity of lactose hydrolysis (Table 1). Strain
KNOUC302 was chosen, and it’s 16S rDNA,
morphological and biochemical properties were
examined for identification. The 16S rDNA of
strain KNOUC302 was composed of 1,498bp
(GenBank accession No. JN014467). BLAST
searching revealed the highest similarity of 16S
rDNA of strain KNOUC302 with those of
Obesumbacterium proteus DSM 2777 T (98.9%
identity) and Hafnia alvei ATCC 13337 T (99.8%
identity) in phylogenetic tree (Fig. 1).
Obesumbacterium sp. and Hafnia alvei are closely

COLD ACTIVE β–GALACTOSIDASE OF HAFNIA ALVEI KNOUC302 ISOLATED FROM …
related to each other (19). However
Obesumbacterium has different shape of long
pleomorphic rod with that of Hafnia forming
uniformly rod shape (20), and most strains of
Obesumbacterium ferment only D-mannose and
trehalose, rarely other carbohydrates including
lactose (21, 22).
Table 1: Hydrolysis of ONPG and lactose of isolated
strains from raw milk
No. Strain No.
Hydrolysis of
ONPG 1) Lactose 2)
1 KNOUC301 0.024 N.D
2 KNOUC302 0.530 28.073
3 KNOUC303 0.770 N.D
4 KNOUC304 0.557 14.194
5 KNOUC305 1.087 3.943
6 KNOUC306 1.407 N.D
7 KNOUC307 1.118 18.932
8 KNOUC308 1.098 N.D
9 KNOUC309 0.533 N.D
10 KNOUC310 0.309 N.D
11 KNOUC311 0.425 N.D
1)
One unit of enzyme activity is defined as the activity
hydrolyzing 1μmol of ONPG per min by cell free extract from
1ml of culture whose A 600 is 8.
2) One unit of enzyme activity is the one hydrolyzing 1μmol of
lactose per day at 4℃ by cell free extracts from 1ml of culture
whose A 600 is 8.
acids of Hafnia alvei α773 were reported to be C 16:0 ,
C 16:1 , C 17:0 and 18:1 (24). Though cellular fatty acids
composition of KNOUC 302 does not completely
coincide with those of Hafnia alvei α773, the
morphological and biochemical properties of
KNOUC302 are matching well to the result of
phylogenetic analysis presenting that strain
KNOUC is genetically close to Hafnia alvei ATCC
13337. Therefore, strain KNOUC302 would be the
species Hafnia alvei. We finally identified
KNOUC302 as Hafnia alvei, and named the strain
as Hafnia alvei KNOUC302.
Effect of temperature and pH on β-
galactosidase in cell free extract of KNOUC302
The selected strain, KNOUC302 was tested on
optimum temperature and pH, and stability for its β-
galactosidase in crude cell extracts. The optimal
temperature and pH of strain KNOUC302 β-
galactosidase in cell free extracts was 15 ºC and
pH7.5 (Fig. 2). The β-galactosidase retended 80%
activity of optimum temperature at 4 ºC and 10 ºC
and 60 % activity of optimum pH at pH of milk (pH
6.8). Stability of the β-galactosidase was examined
by incubating at 4 ºC and 37 ºC for 7 days. The β-
galactosidase was stable at 4 ºC for 7 days, but its
activity decreased to less than 60 % of initial
activity in 7 days at 37 ºC(Fig. 2). The properties of
KNOUC302 β-galactosidase are satisfying the
requirements for hydrolysis of lactase in milk at low
temperature. Psychrotrophic bacteria, Planococcus
sp. L4 (25) and Arthrobacter sp. (26, 27) produced
β-galactosidase whose optimum temperature and
optimum pH were 20 ºC and pH 6.8, and 10-15 ºC
and pH 8 respectively. However other
psychrotrophic bacteria, Arthrobacter
psychrolactophilus (11), Carnobacterium pisciocola
BA (12), Pseudoalteromonas haloplakis (2) and
Planococcus sp. (13) produced β-galactosidase
reacting optimally at high temperature of 40 ºC, 30
ºC, 45 ºC and 42 ºC respectively. The β-
galactosidase of Arthrobacter psychrolactophilus
(11), Pseudoalteromonas haloplakis (2) and
Planococcus sp. (13) reacted optimally at pH 7.2,
pH 6.4, pH 8 and pH 6.4 respectively. Optimum
temperature and pH of β-Galactosidase of
Enterobacter cloacae, belonging to the same family
with strain KNOUC 302 were 50 ºC and pH 9.0
(27).
As in Table 2 presenting morphological
biochemical properties, the strain KNOUC302 was
Gram negative, rod, nonsporing, facultative
anaerobic, oxidase negative, and reduced nitrate to
nitrite. These properties classify KNOUC302 as
family Enterobacteriaceae (22), and uniformly red
shape of KNOUN302 and utilization of many
carbohydrates by KNOUC302 let KNOUC302 be
out of genus Obesumbacterium. KNOUC302 failed
in production of indole and H 2 S, and was negative
in fermentation of raffinose, sorbitol, and inositol
that are the typical characteristics of Genus Hafnia
(23). And KNOUC302 showed negativity in indole
production and urease, and positivity in hydrolysis
of PNPG and ONPG that are fitting to species
Hafnia alvei (21, 23). The main fatty acids of
KNOUC 302 cell were C 12:0, C 14:0 3-OH, C 16:0 and
C 16:1 ω 7 c comprising 10.2 %, 25.41 %, 9.94 % and
29.75 % respectively (Table 3). The main fatty
104 J. Anim. Prod. Adv., 2012, 2(2):101-108

NAM AND AHN
Table 2: Morphological and biochemical properties of the strain KNOUC 302
Characteristics Reaction Characteristics Reaction
Gram staining
Shape
Motility
Spore formation
Oxidation-fermentation
Optimum temp.
Optimum pH
Growth at 5℃
37℃
Catalase
Oxidase
Hydrolysis of PNPG
ONPG
Nitrate reduction
Indol production
H2S formation
Citrate utilization
Urease
β-Hemolysis
MacConkey
Utilization of D-glucose
Mannitol
-
Rod
-
-
O/F
20℃
6.8~7.2
+
-
+
-
+
+
+
-
-
-
-
-
+
+
+
Maltose
Rhamnose
Sucrose
Mannose
Galactose
Gelatin
Lactose
Fructose
Xylitol
Ducitol
Adonitol
Raffinose
Glycerol
Erythrol
Acetylglucosamine
Starch
Amigdalin
Tagatose
Sorbitol
Xylose
Arabinose
+
+
-
+
-
-
+
-
-
+
-
-
-
-
+
-
-
+
-
+
+
Presumptive identification
Hafnia sp.
+, positive reaction; -, negative reaction
66.0
80.7
0.01
98.5
99.9
100
Obesumbacterium proteus DSM 2777 T (AJ233422)
Hafnia alvei ATCC 13337 T (M59155)
KNOUC302
Yersinia pestis D-28 (X75274)
Rahnella aquatica DSM 4594 T (AJ233426)
Serratia marcescens DSM 30121 T (AJ233431)
Serratia rubidaea DSM 4480 T (AJ233436)
Erwinia amylovora DSM 30165 T (AJ233410)
Klebsiella pneumoniae DSM 30104 T (AJ233420)
Citrobacter freundii DSM 30039 T (AJ233408)
Pectobacterium carotovorum DSM 30168 T (AJ233411)
Pectobacterium cacticidum LMG 17936 T (AJ223409)
Brenneria salicis DSM 30166 T (AJ233419)
Vibrio cholerae CECT 514 T (X76337)
Fig. 1: Phylogenetic tree based on 16S rDNA sequences showing the positions of strain KNOUC302, the representative
of some other related taxa. Bootstrap values (1000replicatioons) are shown as percentage at each node only if they are 50
% greater. Scale bar represents 0.01 substitutions per nucleotide position.
105 J. Anim. Prod. Adv., 2012, 2(2):

Residual activity(%)
Relative activity(%)
Relative activity (%)
COLD ACTIVE β–GALACTOSIDASE OF HAFNIA ALVEI KNOUC302 ISOLATED FROM …
Cloning β-galactosidase gene of strain
KNOUC302 in E. coli
In the prepared genomic library, there was a
colony having the vector harboring an ORF coding
a protein of β-galactosidase. The ORF is composed
of 888 bp encoding 295 amino acids whose deduced
Mw is 33,709 Da (Fig. 3). But the ORF is not a
complete β-galactosidase gene of Hafnia alvei
KNOUC302. The front sequence of 779 bp,
beginning from initial codon, was from
chromosome of Hafnia alvei KNOUC302, and the
rear sequence of 109 bp to terminal codon was from
vector (pRSET). The nucleotide was named as
KNOUC302 β-gal. A BLAST search in the
databases of NCBI revealed that the 779 bp front
sequence of KNOUC302 β-gal coded the amino
terminal of β-galactosidase belonging to glycoside
hydrolase family 2. The front 779 bp sequence of
KNOUC302 β-gal, from chromosome of Hafnia
alvei KNOUC302, showed the following identities
to β-galactosidase genes from microorganisms of
family Enterobacteriaceae: Escherichia coli K12
(297/401, 74 %, GenBank Accession No.
AP009378), Rahnella sp. Y9602 (364/485, 75 %,
GenBank Accession No. CP002505), Serratia
proteamaculans 568(311/409, 76 %, GenBank
Accession No. CP000826) and Enterobacter
aerogenes KCTC2190 (289/388, 74 %, GenBank
Accession No. CP002824).
galactosidase of Arthrobacter sp. 32c (29),
Pseudoalteromonas haloplanktis (2) and
Planococcus sp. L4 (25) were 2085 bp, 3117 bp and
2031 bp respectively.
100
80
60
40
20
0
0 10 20 30 40
100
80
60
40
20
120
100
Temp.
0
4 5 6 7 8
pH
Table 3: Composition of major fatty acids of strain
KNOUC302
Fatty acid Contents (%)
C 12:0 10.20
C 14:0 6.31
C 14:0 3OH 25.41
C 16:1 ω 7 c 29.75
C 16:0 9.94
C 18:1 ω 7 c 5.05
The size of β-galactosidase genes from Escherichia
coli K12 (GenBank Accession No. AP009378),
Rahnella sp. Y9602 (GenBank Accession No.
CP002505), Serratia proteamaculans 568(GenBank
Accession No. CP000826) and Enterobacter
aerogenes KCTC2190 (GenBank Accession No.
CP002824) were 3075 bp, 3099 bp, 3090 bp and
3108 bp respectively. Genes encoding cold active β-
0
0 1 2 4 7
Fig. 2: Effects of temperature (A) and pH (B) on the
activity, and stability (C) of β-galactosidase in cell free
extracts of Hafnia alvei KNOUC302.
* Effect of temperature was tested in Na-phosphate buffer
(0.01 M, pH= 6.8).
* Effect of pH was examined in Na-acetate (0.01 M) for
pH=4.25 to 6.0, and in Na-phosphate buffer (0.01 M) for pH
6.0 to 7.68 at 4 ℃.
* Values are means of triplicates ± S.D.
106 J. Anim. Prod. Adv., 2012, 2(2):101-108
80
60
40
20
Days

NAM AND AHN
Conclusion
In this study, Hafnia alvei KNOUC302, a strain
that produces lactose hydrolyzing enzyme at low
temperature, was isolated from the samples of raw
milk collected from dairy farms of Kyung-gi
province in Korea. The results obtained in this study
show that Hafnia alvei KNOUC302 grew optimally
at 20 ºC and pH=7.0-7.2. The gene (KNOUC302 β-
gal) consists of 918 bp encoding the protein of 306
amino acids and 34,806 Da of deduced Mw. In the
search for cold-adapted β-galactosidase, we
conclude that the β-D-galactosidase from Hafnia
alvei KNOUC302 is a cold-active enzyme, and that
the enzyme could have advantageous applications in
the food industry, the treatment of chilled dairy
products while avoiding flavor tainting and the risk
of microbial contamination.
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Fig. 3: DNA sequence of Hafnia alvei KNOUC302 β-
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amino acid sequence.
*The fragment from pRSET vector came from pRSET C,
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