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