Cloning and expression of a beta-glycosidase gene from Thermus thermophilus. Sequence and biochemical characterization of the encoded enzyme.
A 3.2 kilobase pair DNA fragment from Thermus thermophilus HB27 coding for a beta-galactosidase activity was cloned and sequenced. A gene and a truncated open reading frame orf1 encoding respectively a beta-glycosidase (ttbeta-gly) and probably a sugar permease were located directly adjacent to each other. The deduced aminoacid sequence of the enzyme Ttbeta-gly showed strong identity with those of beta-glycosidases belonging to the glycosyl hydrolase family 1. The enzyme was overexpressed in Escherichia coli and was purified by a two-step purification procedure. The recombinant enzyme is monomeric with a molecular mass of 49-kDa. It catalyzes the hydrolysis of beta-D-galactoside, beta-D-glucoside and beta-D-fucoside derivatives. However, the kcat/Km ratio is much higher for p-nitrophenyl-beta-D-glucoside and p-nitrophenyl-beta-D-fucoside than for p-nitrophenyl-beta-D-galactoside. The specificity towards linkage positions of the disaccharides tested decreased in the following order: beta1-3 (100%) > beta1-2 (71%) > beta1-4 (40%) > beta1-6 (10%). Ttbeta-gly is a thermostable enzyme displaying an optimum temperature of 88 degrees C and a half life of 10 min at 90 degrees C. It performs transglycosylation reactions at high temperature with a yield exceeding 63% for transfucosylation reactions. On the basis of this work, the enzyme appears to be an attractive tool in the synthesis of fucosyl adducts and fucosyl sugars.
Dion
M
M
Unité de Recherche sur la Biocatalyse, Faculté des Sciences et Techniques, Université de Nantes, France.
Fourage
L
L
Hallet
J N
JN
Colas
B
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D013696:Temperature; D016963:Thermus thermophilus / Q000235:genetics*; D001617:beta-Glucosidase / Q000235:genetics*
D001426:Bacterial Proteins; D001617:beta-Glucosidase