Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis.
The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.
Inoue
Yasushi
Y
Showa Sangyo Co., Ltd., 1-16 Sakura, Tsukuba, Ibaraki 305-0003, Japan. brc@showa-sangyo.co.jp
Yasutake
Nozomu
N
Oshima
Yoshie
Y
Yamamoto
Yoshie
Y
Tomita
Tetsuji
T
Miyoshi
Shinsuke
S
Yatake
Tsuneya
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D014199:Trehalose / Q000378:metabolism
D008320:Maltose; D014199:Trehalose; D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase; C021535:maltose phosphorylase