Cloning, sequencing, and expression of a beta-amylase gene from Bacillus cereus var. mycoides and characterization of its products.
The cloned gene was composed of 1638 bp for coding plus promoter like and SD-like sequences ahead of it. The deduced amino acid sequence had high similarity with known beta-amylases. The N-terminal sequence of the cloned beta-amylase seemed to be a signal peptide. The gene was introduced into Bacillus subtilis 1A289 using pHY300PLK as a vector and the expressed protein was recovered from the culture media. The enzyme fraction produced was divided into two components upon the DEAE column chromatography. The amino acid sequence of one fraction (FrI) was the same as the mature enzyme, and the other (FrII) lacked the N-terminal amino acid residue (Ala) of the mature enzyme. The kinetic parameters of the hydrolysis catalyzed by the enzyme component FrI were measured, and the subsite affinities of the enzyme were evaluated. In conclusion, it was shown that the recombinant enzyme was the same as the mature enzyme functionally and proteochemically.
Yamaguchi
T
T
Laboratory of Biophysical Chemistry, College of Agriculture, Osaka Prefecture University, Japan.
Matsumoto
Y
Y
Shirakawa
M
M
Kibe
M
M
Hibino
T
T
Kozaki
S
S
Takasaki
Y
Y
Nitta
Y
Y
D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D005069:Evaluation Studies as Topic; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D001614:beta-Amylase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D009844:Oligosaccharides; D011994:Recombinant Proteins; C021705:maltooligosaccharides; D001614:beta-Amylase