ABSTRACT
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.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Genes, Bacterial/genetics , Multigene Family/genetics , Thermoanaerobacter/enzymology , alpha-Galactosidase/chemistry , beta-Galactosidase/chemistry , Amino Acid Sequence , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Substrate Specificity , Thermoanaerobacter/genetics , alpha-Galactosidase/genetics , beta-Galactosidase/geneticsABSTRACT
The properties of substrate-binding modules of glycosyl hydrolases have been reviewed. Variation of the properties of these modules makes them promising as components of chimeric proteins, which is a rapidly developing field of biotechnology. Examples of applying substrate-binding modules of glycosyl hydrolases to immobilization of proteins and whole cells on polysaccharides and purification of proteins are described. Promising methods for (1) detecting various compounds using hybrids of substrate-binding modules with antibodies and (2) locating polysaccharides in live tissues are reviewed as well.
