Biology Reference
In-Depth Information
from psychrophilic
Planococcus
sp. strains have low molecular weight of about 75
kDa of monomer and about 155 kDa of native protein. The β-D-galactosidase isolated
from
Planococcus
sp. L4 is particularly thermolabile, loosing its activity within only
10 min at 45°C [14] and therefore larger scale production of this enzyme by recom-
binant yeast strains cultivated at 30°C might be economically not feasible. Only the
β-D-galactosidase from
Planococcus
sp. isolate SOS orange [4] displays interesting
activity and might be considered in biotechnological production on a larger scale.
In comparison with known β-D-galactosidases, the
Arthrobacter
sp. 32c β-D-
galactosidase is a protein with a relatively low molecular weight. Molecular sieving
revealed that the active enzyme is a trimmer with a molecular weight of approximately
195 ± 5 kDa. Relatively low molecular weight of the protein did not interfere with
extracellular production of the protein by
P. pastoris
. Therefore the constructed re-
combinant strains of
P. pastoris
may serve to produce the protein extracellularly with
high effi ciency and in a cheap way. The calculated production cost of 1 mg of purifi ed
β-D-galactosidase was estimated at 0.03 €.
The same
Pichia pastoris
expression systems had been unsuccessfully used for extra-
cellular expression of previously reported β-D-galactosidase from
Pseudoalteromonas
sp.
22b [10, 11]. This enzyme is much bigger than
Arthrobacter
sp. 32c β-D-galactosidase
and forms a tetramer of approximately 490 kDa. It is worth noting that we have tried to
secrete this enzyme with three different secretion signals (α-factor from
Saccharomy-
ces cerevisiae
, glucoamylase STA2 from
Saccharomyces diastaticus
or phosphatase
PHO
5
from
S. cerevisiae
) with no success. It seems that the molecular mass of the
desired recombinant protein is limited to extracellular production by
P. pastoris
host,
whereas the used secretion signal is without any infl uence. Based on our experience
with
Pichia pastoris
expression systems we assert that the larger protein the lower
expression yield can be achieved.
In comparison with the known β-D-galactosidase from
Planococcus
sp. isolate
SOS orange [10], β-D-galactosidase from
Arthrobacter
sp. 32c is more thermostable
and it has a similar activity profi le. Moreover, as shown in this study, it can be pro-
duced extracellularly in high amounts by yeast strain. The displayed activity profi le of
the
Arthrobacter
β-D-galactosidase, especially the activity at pH range from 5.5 to 7.5,
over 50% of relative activity at 30°C and enhancement of the activity by the presence
of ethanol suggest that this enzyme is compatible with the industrial process condi-
tions for ethanol production by yeast. The construction of corresponding
S. cerevisiae
recombinant strains and fermentation tests for the production of ethanol from cheese
whey by the application of this β-D-galactosidase are pending.
The
Arthrobacter
β-D-galactosidase was strongly inhibited by glucose and
therefore the catalysis effi ciency was very low. Removal of this product resulted
in 75% hydrolysis of a solution containing 5% of lactose after 72 hr in a combined
enzyme assay. These results clearly indicate that the enzyme can be used for the
production of sweet lactose free milk where hydrolysis of lactose to glucose and
galactose is performed by simultaneous isomerisation of glucose to fructose by glu-
cose isomerase.
