Expression of periplasmic alpha-amylase of Xanthomonas campestris K-11151 in Escherichia coli and its action on maltose.
A gene encoding the periplasmic alpha-amylase of Xanthomonas campestris K-11151 was cloned into Escherichia coli using pUC19 as a vector. An ORF of 1578 bp was deduced to be the amylase structural gene. The primary structure of the enzyme had little identity with other alpha-amylases, except with the enzyme from Bacillus megaterium. The enzyme was expressed in E. coli from the lac promoter of pUC19 and was found to be transported to the periplasmic space. The expressed enzyme showed the same thermal stability, optimum temperature and substrate specificity as the enzyme from X. campestris. The enzyme formed maltotetraose, but not 6(1)- nor 6(2)-maltosyl-maltose, from maltose by the reverse reaction, and the tetraose was then hydrolysed to maltotriose and glucose. The addition of maltotriose enhanced the production of glucose from maltose. In addition, maltose was formed by the condensation of glucose by the enzyme. Thus, the periplasmic alpha-amylase of X. campestris was shown to produce glucose from maltose by hydrolysing maltotetraose and possibly higher maltooligosaccharides, which were the products of a condensation reaction, as a major pathway, and by direct hydrolysis of maltose as a minor pathway.
Abe
Jun-Ichi
JI
Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto-1 21-24, Kagoshima 890, Japan.
Shibata
Yuko
Y
Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto-1 21-24, Kagoshima 890, Japan.
Fujisue
Mami
M
Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto-1 21-24, Kagoshima 890, Japan.
Hizukuri
Susumu
S
Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto-1 21-24, Kagoshima 890, Japan.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D004790:Enzyme Induction; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000378:metabolism; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D016959:Xanthomonas campestris / Q000201:enzymology / Q000235:genetics*; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; D009844:Oligosaccharides; D008320:Maltose; C009819:maltotetraose; D000516:alpha-Amylases; D005947:Glucose