Biology Reference
In-Depth Information
In the
P. pastoris
expression system the methanol induced and constitutive biosyn-
thesis variants for larger scale production of the enzyme were tested. By cloning the
gene in the form of translational fusion with the
S. cerevisiae
α-factor leader sequence
under the control of either the methanol induced promoter AOX1 or under the con-
stitutive promoter GAP, pPICZαA-32cβ-gal, and pGAPZαA-32cβ-gal recombinant
expression plasmids were constructed.
P. pastoris
GS115 strain was transformed with
linearized pPICZαA-32cβ-gal or pGAPZαA-32cβ-gal plasmids. The obtained
P. pas-
toris
GS115 recombinant strains harboring pGAPZαA-32cβ-gal or pPICZαA-32cβ-
gal recombinant plasmids were used for extracellular production of the
Arthrobacter
sp. 32c β-D-galactosidase (Figure 2B, lane 2 and Figure 2C, lane 2). The applied
overexpression systems were effi cient, giving approximately 137 and 97 mg (Table
1) of purifi ed β-D-galactosidase (Figure 2B and C, lanes 4) from 1 l of induced cul-
ture for the AOX1 and constitutive system, respectively. Noteworthy is the fact that
all attempts in extracellular expression of β-D-galactosidase from
Pseudoalteromonas
sp.22b [10, 11] previously described by us did not succeed (data not shown). The
corresponded β-D-galactosidase is a tetramer composed of 115 kDa subunits. All the
amount of produced protein with fused secretion signal was accumulated in the cells.
We also tried to produce the
Pseudoalteromonas
sp. 22b β-D-galactosidase in the form
of fusion protein with other secretion sequences: PHO5 and STA2. All attempts gave
negative results. It seems that molecular mass of desired recombinant protein is lim-
ited for extracellular production by
P. pastoris
host.
Characterization of
Arthrobacter
sp. 32c
-D-galactosidase
The temperature profiles of the hydrolytic activity of the recombinant
Arthrobacter
sp. 32c β-D-galactosidase showed that the highest specific activity with ONPG was
at 50°C (155 U/mg). Lowering or raising temperature from 50°C resulted in the re-
duction of β-D-galactosidaseactivity. Recombinant β-D-galactosidase exhibited 15%
of the maximum activity even at 0°C and approximately 60% at 25°C (Figure 3). In
order to determine the optimum pH for recombinant β-D-galactosidase, we measured
the enzyme activity at various pH values (pH 4.5-9.5) at 0-70°C, using ONPG as a
substrate. β-D-galactosidase exhibited maximum activity in pH 6.5 and over 90% of
its maximum activity in the pH range of 6.5-8.5 (Figure 3).
To examine the possible metal ion requirements, the enzyme preparation was treat-
ed with EDTA to remove metal ions. No activity was lost during treatment with 100
mM EDTA after 2 h. The activity was not considerably affected by metal ions (5 mM):
Na
+
, K
+
, Mg
2+
, Co
2+
, Ca
2+
. The enzyme activity was completely inhibited by Cu
2+
or
Zn
2+
(5 mM) and was strongly inhibited by Mn
2+
(11%), Fe
2+
(25%), and Ni
2+
(38%) in
comparison to the activity of the enzyme in the absence of cations (100%) (Table 2).
The activity of the β-D-galactosidase was not considerably affected by ditiothreitol,
β-mercaptoethanol, and L-cysteine, whereas reduced glutathione almost completely
inactivated the enzyme (Table 3). The examination of the ethanol infl uence on the
Arthrobacter
sp. 32c β-D-galactosidaseactivity with ONPG as the substrate shows
that addition of ethanol up to 20% still slightly stimulates the enzyme activity (Table
4). The relative enzyme activity was increasing up to 120% in the presence of 8% v/v
ethanol at pH 5.5.
β
