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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
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