A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c.
BACKGROUND
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 in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose.


RESULTS
In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter 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 Arthrobacter 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 Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. 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 Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus 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 P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter 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.


CONCLUSIONS
The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris 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.
Department of Microbiology, Gdańsk University of Technology, Poland. marwanar@pg.gda.pl
Marta
M
Wanarska
Józef
J
Kur
D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006601:Hexoses / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D006601:Hexoses; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; D005947:Glucose; C030192:tagatose; D005690:Galactose