Enzymatic
synthesis with K. lactis and B. bifidum β-gal was performed
in 50 mM sodium phosphate buffer with 2 mM MgCl2 at pH
6.5. A. oryzae and B.
circulans β-gal assays were carried out in 50
mM sodium acetate buffer at pH 4.5. The enzymatic activity used for
the four enzymes was 15 U mL–1.
Transgalactosylation
reactions were carried out in an orbital Thermomixer comfort (Eppendorf)
at 50 °C and 750 rpm using trehalose (25% w/v), as an acceptor,
and lactose (25% w/v), as a donor. Reactions were incubated during
24 h, taking aliquots at 0, 2, 4, 6, and 24 h. Assays were stopped
by heating in boiling water for 5 min. In all cases, reaction blanks
using only lactose were performed under the same conditions. Transgalactosylation
reactions were monitored through gas chromatography coupled to a flame
ionization detector (GC-FID), as described below. After GC-FID analysis,
β-gal from K. lactis and B. bifidum did not show any formation of new trehalose
derivatives. For this reason, analysis, isolation, and characterization
were carried out using A. oryzae and B. circulans β-gal.
synthesis with K. lactis and B. bifidum β-gal was performed
in 50 mM sodium phosphate buffer with 2 mM MgCl2 at pH
6.5. A. oryzae and B.
circulans β-gal assays were carried out in 50
mM sodium acetate buffer at pH 4.5. The enzymatic activity used for
the four enzymes was 15 U mL–1.
Transgalactosylation
reactions were carried out in an orbital Thermomixer comfort (Eppendorf)
at 50 °C and 750 rpm using trehalose (25% w/v), as an acceptor,
and lactose (25% w/v), as a donor. Reactions were incubated during
24 h, taking aliquots at 0, 2, 4, 6, and 24 h. Assays were stopped
by heating in boiling water for 5 min. In all cases, reaction blanks
using only lactose were performed under the same conditions. Transgalactosylation
reactions were monitored through gas chromatography coupled to a flame
ionization detector (GC-FID), as described below. After GC-FID analysis,
β-gal from K. lactis and B. bifidum did not show any formation of new trehalose
derivatives. For this reason, analysis, isolation, and characterization
were carried out using A. oryzae and B. circulans β-gal.