Hereby the half saturation coefficient was significantly higher, the reaction veloCity constant was significantly lower and the reaction efficiency 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 efficiency was significantly higher and over 30% from the 5% w/v of lactose was hydrolysed to glucose and galactose for 12 hours and over 75% of the lactose was found to be hydrolysed after 72 hours. These results were similar

to another test where the recombinant P. pastoris strain extracellularly producing 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 AZD3965 in vitro SC75741 molecular weight this shows that the enzyme might successfully catalyse the conversion of lactose to corresponding monocarbohydrates in a fermentation

broth where glucose is consumed by cells of the fermenting strain. Table 5 Kinetic parameters of Arthrobacter sp. 32c β-D-galactosidase. Substrate Temperature [°C] Km [mM] kcat [s-1] kcat/Km [s-1mM-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 interesting 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 Ca2+ 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 for that simultaneously utilize glucose and galactose. 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 efficient P. pastoris expression systems for consideration to produce enzyme extracellularly. P. pastoris has been successfully used many times in extracellular Selleck XAV939 protein production, however, there are only several examples of cold-adapted proteins and none cold-adapted β-D-galactosidase produced by this host.