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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.
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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
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