Molecular studies on thiaminase I

The thiaminase I gene of Bacillus thiaminolyticus was cloned on a 1.6 kb DNA fragment (enzyme molecular weight 42 000), and was expressed in both Escherichia coli and Bacillus subtilis. When a selection drug was absent, the plasmid was maintained stably for approx. 100 generations in wild-type E. coli. Instability of the thiaminase gene was demonstrated in the thiamin pyrophosphate-requiring mutant of E. coli from which the plasmid was deleted rapidly. Wild-type E. coli accumulated the enzyme in its periplasm. A method for the detection of thiaminase I enzyme in SDS-polyacrylamide gel was developed. Thiaminase I of B. thiaminolyticus was found to exist in two sizes, 44 and 42 kDa, among different strains. Moreover, thiaminase of 42 kDa became approximately 41 kDa after a long-term culture, most likely because of the action of proteinases. Thiaminase expressed in E. coli from a thiaminase-positive recombinant plasmid was 42 kDa, and showed the same mobility on SDS-polyacrylamide gele electrophoresis as the enzyme isolated from the young culture of the parent strain of B. thiaminolyticus used for cloning. This value was, therefore, considered to represent intact thiaminase that had escaped from the attack of bacilli proteinases.