Cloning and expression in Escherichia coli of an extremely thermostable oligo-1,6-glucosidase gene from Bacillus thermoglucosidasius.

The gene for an extremely thermostable oligo-1,6-glucosidase (dextrin-6-alpha-D-glucanohydrolase; EC 3.2.1.10) of obligately thermophilic Bacillus thermoglucosidasius KP1006 was cloned within a 4.2-kilobase HindIII-PvuII fragment of DNA by using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. The gene was transcribed, presumably from its own promoter, in E. coli. E. coli with the hybrid plasmid accumulated oligo-1,6-glucosidase mainly in the cytoplasm. The level of enzyme production was comparable to that observed for B. thermoglucosidasius. The enzyme coincided absolutely with the B. thermoglucosidasius enzyme in its molecular weight (60,000), in its electrophoretic behavior on denaturing and nondenaturing polyacrylamide gels, in the temperature dependency of its stability and activity, and in its antigenic determinants.