Biology Reference
In-Depth Information
Table 4.
Effect of ethanol concentration on recombinant
Arthrobacter
sp. 32c
β
-D-galactosidase
activity.
Ethanol [% v/v]
Relative activity [%] pH 5.5
Relative activity [%] pH 6.5
0
100
100
1
109 ± 2.0
102 ± 2.4
2
111 ± 2.2
107 ± 3.0
4
114 ± 2.7
109 ± 2.6
6
116 ± 2.5
110 ± 2.4
8
120 ± 2.1
111 ± 2.4
10
119 ± 2.3
109 ± 2.5
12
117± 1.9
107 ± 2.6
14
109 ± 2.2
105 ± 2.4
16
108 ± 2.1
103 ± 2.5
18
105 ± 2.7
102 ± 2.7
20
103 ± 2.9
101 ± 3.1
A study of the substrate specifi city of the
Arthrobacter
sp. 32c β-D-galactosidase
was performed with the use of various chromogenic nitrophenyl analogues. The re-
combinant
Arthrobacter
sp. 32c β-D-galactosidase displayed four times higher level
of activity with PNPG than with ONPG as substrate. The activities with PNPGlu
and ONPGlu were signifi cantly lower with only 1.4% and 0.5% of the activity with
ONPG, respectively.
In order to further characterize the biochemical properties of the enzyme the high-
est specifi c activity k
cat
, the K
M
values and the catalysis effi ciency k
cat
/K
M
in reaction
with ONPG and lactose were calculated. The highest observed specifi c activity with
ONPG was 212.4 s
-1
at 50°C. The half saturation coeffi cient (K
M
) was highest at 10°C
(5.75 mM), decreased to 2.62 mM at 50°C and rose again to 5.11 mM at 55°C. The
highest catalysis effi ciency was achieved at 50°C (81.7 s
-1
mM
-1
). The same kinetic
parameters were also determined with lactose (Table 5). Hereby the half saturation
coeffi cient was signifi cantly higher, the reaction velocity constant was signifi cantly
lower and the reaction effi ciency was very low. To investigate the reason for such
results another test was performed, where glucose was transformed in the reaction
mixture by glucose isomerase that converted it to fructose, while galactose remained
in the mixture. In this test the reaction effi ciency was signifi cantly higher and over
30% from the 5% w/v of lactose was hydrolyzed to glucose and galactose for 12 hr
and over 75% of the lactose was found to be hydrolyzed after 72 hr. These results were
similar to another test where the recombinant
P. pastoris
strain extracellularly produc-
ing
Arthrobacter
sp. 32c β-D-galactosidase (pGAPZαA-32cβ-gal) was cultivated on
lactose containing broth. It seems obvious that
Arthrobacter
sp. 32c β-D-galactosidase
is inhibited by glucose. Nevertheless this shows that the enzyme might successfully
catalyze the conversion of lactose to corresponding monocarbohydrates in a fermenta-
tion broth where glucose is consumed by cells of the fermenting strain.
