Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima.
OBJECTIVE
Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima for the hydrolysis of lactose and the production of galacto-oligosaccharides.


RESULTS
A putative beta-galactosidase gene of Thermotoga maritima was expressed in Escherichia coli as a carboxyl terminal His-tagged recombinant enzyme. The gene encoded a 1100-amino acid protein with a calculated molecular weight of 129,501. The expressed enzyme was purified by heat treatment, His-tag affinity chromatography, and gel filtration. The optimum temperatures for beta-galactosidase activity were 85 and 80 degrees C with oNPG and lactose, respectively. The optimum pH value was 6.5 for both oNPG and lactose. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation and its half-life times at 80 and 90 degrees C were 16 h and 16 min, respectively. Mn2+ was the most effective divalent cation for beta-galactosidase activity on both oNPG and lactose. The Km and Vmax values of the thermostable enzyme for oNPG at 80 degrees C were 0.33 mm and 79.6 micromol oNP min(-1) mg(-1). For lactose, the Km and Vmax values were dependent on substrate concentrations; 1.6 and 63.3 at lower concentrations up to 10 mm of lactose and 27.8 mm and 139 micromol glucose min(-1) mg(-1) at higher concentrations, respectively. The enzyme displayed non-Michaelis-Menten reaction kinetics with substrate activation, which was explained by simultaneous reactions of hydrolysis and transgalactosylation.


CONCLUSIONS
The results suggest that the thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galacto-oligosaccharides.


CONCLUSIONS
The findings of this work contribute to the knowledge of hydrolysis and transgalactosylation performed by beta-galactosidase of hyperthermophilic bacteria.
Department of Bioscience and Biotechnology, Sejong University, Seoul, Korea.
C S
CS
Kim
E-S
ES
Ji
D-K
DK
Oh
D003001:Cloning, Molecular / Q000379:methods; D003067:Coenzymes / Q000502:physiology; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000096:biosynthesis; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D009844:Oligosaccharides / Q000096:biosynthesis; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D003067:Coenzymes; D009844:Oligosaccharides; D001616:beta-Galactosidase; D007785:Lactose; D005690:Galactose