Purification and characterization of a truncated Bacillus subtilis α-amylase produced by Escherichia coli

A Bacillus subtilis amylase gene was inserted into a plasmid which was transferred to Escherichia coli. During cloning, a 3' region encoding 171 carboxy-terminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The tran...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 1996-02, Vol.44 (6), p.746-752
Main Authors: MARCO, J. L, BATAUS, L. A, VALENCIA, F. F, ULHOA, C. J, ASTOLFI-FILHO, S, FELIX, C. R
Format: Article
Language:English
Subjects:
alpha-Amylases - chemistry
alpha-Amylases - genetics
alpha-Amylases - isolation & purification
alpha-Amylases - metabolism
Bacillus subtilis
Bacillus subtilis - enzymology
Biological and medical sciences
Biotechnology
Enzyme Inhibitors - pharmacology
Enzyme Stability
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Genes, Bacterial - genetics
Genetic engineering
Genetic technics
Hot Temperature
Hydrogen-Ion Concentration
Kinetics
Metals - pharmacology
Methods. Procedures. Technologies
Modification of gene expression level
Molecular Weight
Substrate Specificity
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Summary:A Bacillus subtilis amylase gene was inserted into a plasmid which was transferred to Escherichia coli. During cloning, a 3' region encoding 171 carboxy-terminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of the B. subtilis complete alpha-amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with a Km of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50 degrees C, and the purified enzyme was stable at temperatures up to 50 degrees C. While Hg2+, Fe3+ and Al+3 were effective in inhibiting the truncated enzyme, Mn2+ and Co2+ considerably enhanced the activity.
ISSN:0175-7598
1432-0614
DOI:10.1007/s002530050627