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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.

The enzyme with β‐galactosidase activity from E. coli BL21(DE3) transformant containing the gene encoding enzyme from Pyrococcus woesei (DSM 3773) was isolated using cell extraction in 0.01 M phosphate buffer (pH 7.2), protein thermopredpitation at 85C, precipitation at acetone/extract ratio of 1:1 (v/v) and gel filtration on Sephadex G‐200. The increase in the enzyme specific activity was determined using ONPG as substrate. The activity increased from 2.9 × 103 U/mg protein to 37 × 103 U/mg. Thermoprecipitation removed 78% of E. coli protein and retained 92% of the cell extract activity. The acetone precipitation and gel filtration applied in the next purification steps led to homogeneous enzyme with specific activity of 37,700 U/mg protein. The isolated enzyme had a half‐life of 23 h and 9 h during incubation at 85C and 100C, respectively, in 0.1 M citrate‐phosphate buffer (pH 5.4).

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