Dabrowski S, Maciuńska J, Synowiecki J
Department of Microbiology, Technical University of Gdańsk, Poland.
Mol Biotechnol. 1998 Dec;10(3):217-22. doi: 10.1007/BF02740841.
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.
通过聚合酶链反应扩增的沃氏火球菌（DSM 3773）β-半乳糖苷酶基因被克隆到pET-30LIC表达质粒的KpnI和HindIII结合位点。然后将获得的pGal2（6785 bp）转录载体转入大肠杆菌B121（DE3）细胞。DNA序列的高度一致性（99.9%）表明沃氏火球菌和激烈火球菌的β-半乳糖苷酶密切相关。来自大肠杆菌转化体的这种酶是细胞中一种独特的热稳定蛋白，并且可以通过在85℃热沉淀其他细菌蛋白而成功分离。溶液中剩余的粗β-半乳糖苷酶约占从大肠杆菌细胞中提取的总蛋白量的21%，在pH 5.4和93℃温度下具有最大活性。分离出的酶在高达110℃的温度下有活性，在85℃和93℃孵育4小时后活性损失分别不超过初始值的11%和15%。
