Molecular cloning, characterization and nucleotide sequence of the gene for secreted alpha-amylase from Xanthomonas campestris pv. campestris.
alpha-Amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) of apparent molecular mass 45 kDa was secreted by Xanthomonas campestris pv. campestris grown in medium containing starch or maltose. We isolated its structural gene from a recombinant lambda library and located it on a 2.7 kb DNA fragment. Nucleotide sequencing of the fragment revealed a potential ORF encoding a protein of 475 amino acid residues, including a potential signal sequence of 35 amino acids. The signal processing site was confirmed by N-terminal amino acid sequence analysis of the exported alpha-amylase. The deduced amino acid sequence of the mature protein is very similar to that of the alpha-amylase of Aeromonas hydrophila. It also contains all four amino acid sequences highly conserved in the alpha-amylases from a wide range of organisms. Expression of the amy gene in Escherichia coli was poor from its own promoter, but was enhanced by the upstream promoter on the vector. The alpha-amylase synthesized in E. coli was located in the periplasm.
Hu
N T
NT
Agricultural Biotechnology Laboratories, National Chung Hsing University, Taiwan, Republic of China.
Hung
M N
MN
Huang
A M
AM
Tsai
H F
HF
Yang
B Y
BY
Chow
T Y
TY
Tseng
Y H
YH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D016959:Xanthomonas campestris / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000516:alpha-Amylases