초록
<P><B>Background</B></P><P>D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze <I>in vitro</I> the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose.</P><P><B>Results</B></P><P>In this study, the <I>araA</I> gene from psychrotolerant Antarctic bacterium <I>Arthrobacter</I> sp. 22c was isolated, cloned and expressed in <I>Escherichia coli</I>. The active form of recombinant <I>Arthrobacter</I> sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant <I>Arthrobacter</I> sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg<SUP>2+</SUP>, Mn<SUP>2+</SUP> or Ca<SUP>2+</SUP> and slightly inhibited by Co<SUP>2+</SUP> or Ni<SUP>2+</SUP>. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant <I>Pichia pastoris</I> yeast strain secreting β-D-galactosidase <I>Arthrobacter chlorophenolicus</I> was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the <I>P. pastoris</I> strain secreting β-D-galactosidase in a whey permeate supplemented with <I>Arthrobacter</I> sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose.</P><P><B>Conclusions</B></P><P>The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a <I>P. pastoris</I> strain secreting β-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.</P>