Cloning and sequencing of an original gene encoding a maltogenic amylase from Bacillus sp. US149 strain and characterization of the recombinant activity.
A gene encoding maltogenic amylase from acidic Bacillus sp. US149 (maUS149) was cloned, sequenced and over-expressed in Escherichia coli. The nucleotide sequence analysis revealed an open reading frame (ORF) of 1749 bp encoding a protein of 582 residues. The alignment of deduced amino acid sequence revealed a relatively low homology with the already reported maltogenic amylases. In fact, its highest identity, of only 60%, was found with the maltogenic amylase of Thermus sp. IM6501. The recombinant enzyme (MAUS149) was found to be intracellular and was purified to homogeneity from the cell crude extract with a yield of 23%. According to PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of 135 kDa and is composed of two identical subunits of 67.5 kDa each. The maximum activity was obtained at 40 degrees C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose from starch, maltose and glucose from beta-cyclodextrin, and panose from pullulan.
Mabrouk
Sameh Ben
SB
Laboratoire d'Enzymes et de Métabolites, Centre de Biotechnologie de Sfax, BP "K", Sfax, 3038, Tunisia.
Messaoud
Ezzedine Ben
EB
Ayadi
Dorra
D
Jemli
Sonia
S
Roy
Amitava
A
Mezghani
Monia
M
Bejar
Samir
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001426:Bacterial Proteins; D008320:Maltose; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase