Cloning and expression of raw-starch-digesting alpha-amylase gene from Bacillus circulans F-2 in Escherichia coli.
The raw potato-starch-digesting alpha-amylase gene of Bacillus circulans F-2 was cloned for the first time in Escherichia coli C600, using plasmid pYEJ001. The recombinant plasmid, named pYKA3, has a 5.4 kb insert from a chromosome of the donor bacterium. Subcloning of this amylase gene gave plasmid pHA300 which carried 3.15 kb of the inserted DNA. The transformed bacterium, E. coli C600 (pYKA3), produced the amylase in the periplasmic space, whereas it is secreted outside the cell in the donor bacterium. The cloned raw-starch-digesting alpha-amylase has a molecular weight of 93,000 on SDS-PAGE, and its action pattern was absolutely the same as that of the potent raw-starch-digestible amylase produced by B. circulans F-2. The periplasmic amylase produced by the transformed E. coli (pHA300) could digest raw starch granules such as potato, corn and barley raw starch granules, indicating that the raw-starch-digesting amylase is active in E. coli. Furthermore, this amylase crossreacted with the rabbit antiserum raised against the raw potato-digesting alpha-amylase of B. circulans F-2. From these results it was concluded that the cloned amylase is the same amylase protein as B. circulans F-2 amylase, which has a potent raw-starch digestibility. Thus, this paper is to our knowledge the first describing the molecular cloning of raw-starch-digesting alpha-amylase from Bacillus species and its successful expression in E. coli.
Kim
C H
CH
Department of Agricultural Chemistry, University of Tokyo, Japan.
Sata
H
H
Taniguchi
H
H
Maruyama
Y
Y
D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013213:Starch; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013213:Starch; D000516:alpha-Amylases