Jl. Marco et al., PURIFICATION AND CHARACTERIZATION OF A TRUNCATED BACILLUS-SUBTILIS ALPHA-AMYLASE PRODUCED BY ESCHERICHIA-COLI, Applied microbiology and biotechnology, 44(6), 1996, pp. 746-752
A Bacillus subtilis amylase gene was inserted into a plasmid which was
transferred to Escherichia coli. During cloning, a 3' region encoding
171 carboxyterminal amino acids was replaced by a nucleotide sequence
that encoded 33 amino acid residues not present in the indigenous pro
tein. 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 calculat
ed from the derived amino acid sequences of the B. subtilis complete a
lpha-amylase (57.7 kDa) and the truncated protein (54.1 kDa). This tru
ncated enzyme form hydrolysed starch with a K-m of 3.845 mg/ml. Activi
ty was optimal at pH 6.5 and 50 degrees C, and the purifed enzyme was
stable at temperatures up to 50 degrees C. While Hg2+, Fe3+ and Al+3 w
ere effective in inhibiting the truncated enzyme, Mn2+ and Co2+ consid
erably enhanced the activity.