Cloning and nucleotide sequence of the thermostable β-galactosidase gene fromPyrococcus woesei inEscherichia coli and some properties of the isolated enzyme

Abstract

Pyrococcus woesei (DSM 3773) beta-galactosidase gene amplified by polymerase chain reaction was cloned into KpnI and HindIII binding sites of pET-30LIC expression plasmid. The obtained pGal2 (6785 bp) transcription vector was then transferred to Escherichia coli B121 (DE3) cells. High identity (99.9%) of DNA sequences suggests that beta-galactosidases from P. woesei and Pyrococcus furiosus are closely related. This enzyme from E. coli transformant is a unique thermostable protein in the cells and can be successfully separated by thermal precipitation of other bacterial proteins at 85 degrees C. The crude beta-galactosidase remaining in the solution comprises about 21% of the total amount of proteins extracted from E. coli cells and has maximal activity at pH 5.4 and temperature of 93 degrees C. Isolated enzyme is active at temperatures up to 110 degrees C and the activity loss after 4 h of incubation at 85 and 93 degrees C did not exceed 11 and 15% of the initial value respectively.

Cloning, Expression, and Purification of the His6-Tagged Thermostable β-Galactosidase from Pyrococcus woesei in Escherichia coli and Some Properties of the Isolated Enzyme

“…Recombinant enzyme expressed in E. coli can easily be prepared in milligram quantities, and it appears to have the same activity as that purified from native sources or that expressed in native form (1). His 6 -tagged ␤-galactosidase was purified in a single chromatography step using a Ni 2ϩ -TED-Sepharose column (95% purity).…”

“…In our previous study the gene coding ␤-galactosidase (EC 3.2.1.23) of the hyperthermophilic archaea P. woesei was cloned and sequenced (1). In this study the 1550-bp NdeI and HindIII fragment of ␤-galactosidase gene derived from pGal2 plasmid (1) was first cloned into pET15b expression plasmid giving pET15b␤-Gal recombinant plasmid (Fig.…”

Beta-Galactosidase-Producing Thermophilic Bacterium, Thermus thermophilus KNOUC114: Identification of the Bacterium, Gene and Properties of Beta-Galactosidase

“…The enzyme functioning well at pasteurization temperature of raw milk has greater possibility to ensure the hygienically outstanding quality of lactose hydrolyzed milk useful for lactose intolerant persons prevalent in the world, especially in East Asia and Africa. An extremely thermostable β-galactosidase produced by a hyperthermophilic archaea of Pyrococcus woesei active up to 110℃ and optimally at 93℃ was reported (Dabrowski et al 1998). Enzymes from hyperthermophiles have a strong advantage of excellent thermostability, however the utility of hyperthermophilic enzymes can be limited in industrial application due to their poor activity at temperature compatible with the stability of substrates, end products and industrial process.…”

Heterologous gene expression in Thermus thermophilus: β-galactosidase, dibenzothiophene monooxygenase, PNB carboxy esterase, 2-aminobiphenyl-2,3-diol dioxygenase, and chloramphenicol acetyl transferase

“…The majority of studies of genes from Thermus and other species of extreme thermophiles and hyperthermophiles involve the use of Escherichia coli as the host for the cloning and expression of these genes [14][15][16][17]. This approach has been quite fruitful, but it also has significant limitations.…”