ABSTRACT
The genus Bacillus is one of the most important gram-positive bacteria that can secrete a wide variety of proteins directly into the culture medium. The RepA and RepB proteins function as positive and negative factors for the replication of repA and repB plasmids, and give low- and high-copy numbers in bacillus, respectively. Two types of mixed oligonucleotides, corresponding to these amino acid sequences were synthesized for the gene cloning. By colony hybridization, the 2.5-kb EcoRl fragment was cloned in E. coli. The ALDH-T was very stable within the pH range of 7 to 9, and the buffer with the pH 7.8 was used throughout the enzyme purification. Throughout the purification, heat treatment was quite efficient in enriching the thermostable enzyme from other cellular proteins. The ion-exchange chromatography elution pattern showed that the highest protein peak corresponded to that of ALDH activity, and ALDH-T was eluted at an ionic strength of approximately 120-150 mM KC1.
