Biology Reference
In-Depth Information
tion at 5,000 × g for 20 min and the cell pellet was resuspended in 30 ml of buffer A
(20 mM K
2
HPO
4
-KH
2
PO
4
, pH 7.5) and frozen at 20°C for 15 min. After thawing at
room temperature, the samples were centrifuged at 10,000 × g. The supernatant con-
taining the desired protein was applied onto affnity matrix of agarose coupled with
p
-aminobenzyl-1-thio-β-D-galactopyranoside (PABTG-agarose, Sigma) (10 ml col-
umn) equilibrated with four volumes of buffer A. The column was washed with 300
ml of the buffer A, and the recombinant β-D-galactosidase was eluted three times with
10 ml of 0.05 M sodium borate (pH 10.0) buffer at a flow rate of 0.5 ml/min. Active
fractions containing the β-D-galactosidase were collected and dialyzed three times
against 3 l of buffer D (100 mM NH
4
HCO
3
).
In case of the purifi cation of the extracellular produced β-D-galactosidase in
P.
pastoris
cultures, the yeast cells were separated from the post-culture medium through
centrifugation. Next, the ammonium sulfate was added to the post-culture medium to
60% w/w, at 4°C. The precipitated proteins were centrifugated at 20,000 × g, dissolved
in buffer A and dialyzed overnight against the same buffer. For β-D-galactosidase
purifi cation the dissolved sample was applied further directly onto affnity matrix of
agarose coupled with
p
-aminobenzyl-1-thio-β-D-galactopyranoside and purifi ed as
described above for bacterial system. The concentration of purifi ed protein was de-
termined by the Bradford method using bovine serum albumin (BSA) as a standard.
β
-D-galactosidase Activity Assays
The activity of purified
Arthrobacter
sp. 32c β-D-galactosidase was determined by
the use of chromogenic substrates as described elsewhere [4, 14]. The o-nitrophe-
nol released from 10 mM of
o
-nitrophenyl-β-
D
-galactopyranoside (ONPG) by β-D-
galactosidase at 0-70°C and pH range 4.5-9.5 (0.02 M citrate buffer for pH 4.5 and
5.5; 0.02 M K
2
HPO
4
-KH
2
PO
4
for pH 6.5 and 7.0 and 0.02 M Tris-HCl for pH 8.5
and 9.5) was measured at 405 nm. The reaction was stopped after 10 min with 1 M
Na
2
CO
3
. One unit is defined as one micromolar of
o
-nitrophenol released per minute.
Substrate specifi city was estimated using 1 mM solution of chromogenic sub-
strates: The ONPG,
p
-nitrophenyl-β-D-galactopyranoside (PNPG),
o
-nitrophenyl-β-
D-glucopyranoside (ONPGlu), and
p
-nitrophenyl-β-D-glucopyranoside (PNPGlu).
Activity determination was carried out under standard conditions in 0.02 M K
2
HPO
4
-
KH
2
PO
4
(pH 6.5) buffer at 10, 20, 30, 40, or 50°C. The activity of the β-D-galactosidase
towards lactose was monitored by HPLC analysis (column Bio-rad, Aminex HPX-
87H) where 1% solutions of lactose, glucose, fructose, and galactose were used as
standards.
In the combined enzyme assay glucose isomerase from
Streptomyces murinus
(Sigma G4166) was used in the amount of 0.01 g/ml of 5% w/v solution of lactose
(0.02 M K
2
HPO
4
-KH
2
PO
4
, pH 6.5). The
Arthrobacter
sp. 32c β-D-galactosidase was
used at concentration of 200 U/ml of the mixture. The reaction mixture was set at 37°C
for 72 hr and products were analyzed by HPLC every 12 hr.
Effects of 5 mM dithiothreitol, 5 mM of 2-mercaptoethanol, 5 mM of L-cysteine,
5 mM of reduced glutathione, and metal ions (Na
+
, K
+
, Mn
2+
, Mg
2+
, Ca
2+
, Fe
2+
, Zn
2+
,
Cu
2+
, Co
2+
and Ni
2+
; each at concentration of 5 mM) on
Arthrobacter
sp. 32c β-D-
galactosidase activity were determined under standard conditions.
