[Thermoanaerobacter ethanolicus gene cluster containing the alpha- and beta-galactosidases genes melA and lacA, and properties of recombinant lacA].
The nucleotide sequence of a 4936 bp Thermoanaerobacter ethanolicus genomic DNA fragment containing the thermostable beta-galactosidase gene lacA and two incomplete open reading frames has been determined. The product of the first frame is highly homologous to alpha-galactosidases (melibiases), the product of the third frame is homologous to the alpha-D-mannosidases. The terminal area of the lacA, immediately following the stop-codon, harbors presumably a transcription termination site. Based on the location of the putative alpha-galactosidase gene melA and of the beta-galactosidase gene lacA on the T. ethanolicus chromosome, their combined transcription could be presumed. The calculated molecular mass of LacA is 86 kDa. LacA belongs to GH family 2 (GH2). Maximal activity of the purified recombinant enzyme was observed between pH values of 5.7 and 6.0 and temperatures of 75-80 degrees C. The highest activity, 480 units mg(-1), was found on lactose (Km 30 mM), the activities on pNPhGal and oNPhGal amounting to 330 and 420 units mg(-1), respectively. Immobilization on aldehyde silochrome increases the thermostability of the enzyme and keeps its high activity.
Volkov
I Iu
IIu
Lunina
N A
NA
Berezina
O V
OV
Velikodvorskaia
G A
GA
Zverlov
V V
VV
D000595:Amino Acid Sequence; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics
D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000519:alpha-Galactosidase; D001616:beta-Galactosidase