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Table 5. Kinetic parameters of Arthrobacter sp. 32c
β
-D-galactosidase.
Substrate
Temperature [ºC]
K m [mM]
k cat [s -1 ]
k cat /Km [s -l mM -1 ]
ONPG
10
5.75 ± 0.34
52.4 ± 0.72
9.12 ± 0.71
20
4.86 ± 0.37
81.0 ± 1.03
16.67 ± 1,60
30
3.46 ± 0.29
123.9±1.21
35.81 ± 3.66
40
3.15 ± 0.27
169.9 ± 1.44
53.92 ± 5.56
50
2.62 ± 0.21
212.4 ± 1.67
81.07 ± 7.76
55
5.11 ± 0.32
71.2 ± 0.98
13 .93 ± 1.14
lactose
10
77.54 ± 1.77
1.76 ± 0. 11
0.023 ± 0.002
20
67.82 ± 1.74
2.36 ± 0.14
0.035 ± 0.003
30
52.67 ± 1.71
4.81 ± 0.22
0.091 ± 0.007
40
44.31 ± 1.73
5.73 ± 0.21
0.129 ± 0.010
50
39.73 ± 1.72
6.98 ± 0.23
0.176 ± 0.014
DISCUSSION
The β-D-galactosidase from Arthrobacter sp. 32c characterized in this study has in-
teresting industrial properties. It displays optimum activity at pH 6.5 and catalyses
the hydrolysis of 1,4-β-D-galactoside linkages at pH 4.5-9.5 with high efficiency. Its
optimum activity was observed at about 50°C. Nevertheless it showed over 50% of
activity at pH 5.5-7.5 at 30°C and was not considerably inactivated by Ca 2+ ions what
in fact can be of interest in industrial ethanol production from cheese whey by means
of brewing Saccharomyces cerevisiae strains or by recombinant strains that simultane-
ously utilize glucose and galactose.
The β-D-galactosidases naturally produced by psychrophilic microorganisms are
either intracellular or expressed at low levels. In order to make progress in cheaper
production of β-D-galactosidases of industrial interest, we choose highly effi cient P.
pastoris expression systems for consideration to produce enzyme extracellularly. The
P. pastoris has been successfully used many times in extracellular protein production,
however, there are only several examples of cold-adapted proteins and none cold-
adapted β-D-galactosidase produced by this host. We have found only one published
example of P. pastoris extracellular β-D-galactosidase production for a thermostable
enzyme from Alicyclobacillus acidocaldarius [23].
There are several examples of cold active β-D-galactosidases isolated from
Pseudoalteromonas strains [5, 10, 11] and Arthrobacter strains [7-9, 12, 13] with
molecular mass above 110 kDa of monomer and forming an active enzyme of over
300 kDa. Most of them belong to the family 42 β-D-galactosidases. However, the
β-D-galactosidase belonging to family two obtained from the Antarctic Arthrobacter
isolate appears to be one of the most cold-active enzymes characterized to date [8].
All of the known cold-adapted β-D-galactosidases, except two of them isolated from
Planococcus sp. strains [4, 14] and from Arthrobacter sp. 32c (this study), form very
large oligomers and therefore are of minor interest in industrial application probably
because of many problems in effective overexpression. The β-D-galactosidases isolated
 
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