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F F Valência

Publications and source records attributed to F F Valência.

2 recordsLinked to original sources
PubMed1996

A novel cloning system for direct screening using a suicidal strategy.

The pAC92 plasmid is a direct screening cloning vector which allows positive selection of recombinant clones (re-clones). This new high-copy-number plasmid vector encodes ampicillin resistance and carries the Bacillus subtilis alpha-amylase (alpha-Amy)-encoding gene (amy) containing a multiple cloning site. The pAC92 plasmid confers to Escherichia coli transformants an amylolytic phenotype easily detected by iodine vapor staining. The re-clones are identified by insertional inactivation of alpha-Amy activity. During pAC92 construction, a bacterial growth defect was observed in host cells after some modifications of the promoter region that caused the increase in the amy expression. This suicide characteristic permitted the positive selection of re-clones. A second transformation step was performed to enhance the rate of re-clones per plate.

Bacillus subtilis↗
PubMed1996

Purification and characterization of a truncated Bacillus subtilis alpha-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 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.

Bacillus subtilis↗