Cloning and Sequencing of an Original Gene Encoding a Maltogenic Amylase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. US149 Strain and Characterization of the Recombinant Activity |
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Authors: | Sameh Ben Mabrouk Ezzedine Ben Messaoud Dorra Ayadi Sonia Jemli Amitava Roy Monia Mezghani Samir Bejar |
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Institution: | (1) Laboratoire d’Enzymes et de Métabolites, Centre de Biotechnologie de Sfax, BP “K”, Sfax, 3038, Tunisia |
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Abstract: | 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°C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose
from starch, maltose and glucose from β-cyclodextrin, and panose from pullulan. |
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Keywords: | Cyclodextrinase Inverse PCR Kinetic parameters Maltogenic amylase Purification Sequence homology |
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