Biology Reference
In-Depth Information
Characterization of the
-D-galactosidase Gene
The psychrotrophic
Arthrobacter
sp. 32c chromosomal library was prepared in
E. coli
TOP10F'. The plasmid pBADmycHisA was used to construct the library, and ampicil-
lin-resistant transformants were selected and screened for the ability to hydrolyze X-
Gal. Several transformants out of approximately 5,000 were selected as blue colonies
on plates containing X-Gal. Restriction analysis of plasmid inserts from these transfor-
mants indicated that they had been derived from the same fragment of chromosomal
DNA. Sequence data from the shortest construct, named pBADmycHisALibB32c,
contained 5,099 bp insert with an open reading frame (2,085 bp) encoding protein,
which shares high homology to a β-D-galactosidase (NCBI Access No. FJ609657).
The sequence of
Arthrobacter
sp. 32c β-D-galactosidase was analyzed and found to
encode a 694 amino acid protein with a predicted mass of 76.142 kDa and a theo-
retical pI of 5.59. The analysis of DNA sequence upstream the
Arthrobacter
sp. 32c
β-D-galactosidase gene with the promoter prediction tool (BPROM software, http://
www.softberry.com webcite) revealed a potential promoter sequence with cttaca and
tacaat as -35 and -10 sequences, respectively. A putative ribosomal binding site was
apparent eight bases before the initiating methionine codon. The insert fragment and
β-D-galactosidase gene had a high G+C content, 67 mol%, and 66 mol%, respectively,
which is typical of
Arthrobacter
species.
A comparison of the
Arthrobacter
sp. 32c β-D-galactosidase gene sequence with
those from the NCBI database showed that it was most closely related to the
Ar-
throbacter
sp.
FB24
gene (77.13% sequence identity) and to the
A. aurescens
TC1
gene (71.8% sequence identity) (Figure 1B). The deduced amino acid sequence from
Arthrobacter
sp. 32c β-D-galactosidase gene was also used to compare with other
amino acid sequences deposited in the NCBI database. The
Arthrobacter
sp. 32c β-D-
galactosidase was found to be a member of the glycoside hydrolase family 42 and
contained an A4 beta-galactosidase fold. The enzyme shares 84% of identity and 91%
of similarity to the sequence of the
Arthrobacter
sp. FB24, 74% identity and 84% sim-
ilarity to the sequence of the
Arthrobacter aurescens
TC1 and only 51% identity and
65% similarity to the sequence of the
Janibacter
sp. HTCC2649 β-D-galactosidase.
β
Overexpression and Purification of Recombinant
Arthrobacter
sp. 32c
β
-D-
galactosidase
In order to produce and investigate the biochemical properties of
Arthrobacter
sp. 32c
β-D-galactosidase, we constructed bacterial and yeast expression systems. The recom-
binant arabinose-inducible pBAD-Myc-HisA-β-gal32c plasmid was used for the ex-
pression of the
Arthrobacter
sp. 32c β-D-galactosidase gene in
E. coli
LMG194/plysN
[29]. The highest enzyme biosynthesis yields were achieved by adding arabinose to
the final concentration of 0.02% w/w, at A
600
0.5 and by further cultivation for 5 hr. Af-
ter purification a single protein migrating near 70 kDa was observed following sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie blue
(Figure 2A, lane 3). It was in good agreement with the molecular mass deduced from
the nucleotide sequence (75.9 kDa). The applied overexpression system was quite
efficient, giving 27 mg (Table 1) of purified β-D-galactosidase from 1 l of induced
culture. The relative molecular mass of native enzyme estimated by gel filtration on a
