Cloning, nucleotide sequence, and enzymatic characterization of an alpha-amylase from the ruminal bacterium Butyrivibrio fibrisolvens H17c.
A Butyrivibrio fibrisolvens amylase gene was cloned and expressed by using its own promoter on the recombinant plasmid pBAMY100 in Escherichia coli. The amylase gene consisted of an open reading frame of 2,931 bp encoding a protein of 976 amino acids with a calculated Mr of 106,964. In E. coli(pBAMY100), more than 86% of the active amylase was located in the periplasm, and TnphoA fusion experiments showed that the enzyme had a functional signal peptide. The B. fibrisolvens amylase is a calcium metalloenzyme, and three conserved putative calcium-binding residues were identified. The amylase showed high sequence homology with other alpha-amylases in the three highly conserved regions which constitute the active centers. These and other conserved regions were located in the N-terminal half, and no similarity with any other amylase was detected in the remainder of the protein. Deletion of approximately 40% of the C-terminal portion of the amylase did not result in loss of amylolytic activity. The B. fibrisolvens amylase was identified as an endo-alpha-amylase by hydrolysis of the Phadebas amylase substrate, hydrolysis of gamma-cyclodextrin to maltotriose, maltose, and glucose and the characteristic shape of the blue value and reducing sugar curves. Maltotriose was the major initial hydrolysis product from starch, although extended incubation resulted in its hydrolysis to maltose and glucose.
Rumbak
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Department of Microbiology, University of Cape Town, South Africa.
Rawlings
D E
DE
Lindsey
G G
GG
Woods
D R
DR
D000595:Amino Acid Sequence; D001438:Bacteroidaceae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000737:chemistry; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D000516:alpha-Amylases / Q000235:genetics*
D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D000516:alpha-Amylases