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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
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