TY - JOUR
T1 - Cloning and expression of raw-starch-digesting α-amylase gene from Bacillus circulans F-2 in Escherichia coli
AU - Kim, Cheorl Ho
AU - Sata, Hiroshi
AU - Taniguchi, Hajime
AU - Maruyama, Yoshiharu
PY - 1990/4/6
Y1 - 1990/4/6
N2 - The raw potato-starch-digesting α-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 α-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 α-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 α-amylase from Bacillus species and its successful expression in E. coli.
AB - The raw potato-starch-digesting α-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 α-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 α-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 α-amylase from Bacillus species and its successful expression in E. coli.
KW - (Bacillus circulans DNA)
KW - (E. coli)
KW - Cloned DNA
KW - Gene expression
KW - Promoter region
KW - Restriction enzyme map
KW - α-Amylase
UR - https://www.scopus.com/pages/publications/0025321579
U2 - 10.1016/0167-4781(90)90060-F
DO - 10.1016/0167-4781(90)90060-F
M3 - Article
C2 - 2182125
AN - SCOPUS:0025321579
SN - 0167-4781
VL - 1048
SP - 223
EP - 230
JO - Biochimica et Biophysica Acta - Gene Structure and Expression
JF - Biochimica et Biophysica Acta - Gene Structure and Expression
IS - 2-3
ER -