Cloning and expression of a β-glycosidase gene from Thermus thermophilus.: Sequence and biochemical characterization of the encoded enzyme

被引:60
作者
Dion, M [1 ]
Fourage, L [1 ]
Hallet, JN [1 ]
Colas, B [1 ]
机构
[1] Univ Nantes, Fac Sci & Tech, Unite Rech Biocatalyse, F-44322 Nantes 3, France
关键词
beta-glycosidase gene; Thermus thermophilus; transglycosylation;
D O I
10.1023/A:1006997602727
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (tt beta-gly) and probably a sugar permease were located directly adjacent to each other. The deduced aminoacid sequence of the enzyme Tt beta-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: beta 1-3 (100%). beta 1-2 (71%). beta 1-4 (40%). beta 1-6 (10%). Tt beta-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.
引用
收藏
页码:27 / 37
页数:11
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