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Gene Sequence, Bioinformatics and Enzymatic Characterization of α-Amylase from Saccharomycopsis fibuligera KZ

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Abstract

A fragment coding for a putative extracellular α-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp coding for a 494 amino acid residues long protein with 99% identity to the α-amylase Sfamy from S. fibuligera HUT 7212. The S. fibuligera KZ α-amylase (Sfamy KZ) belongs to typical extracellular fungal α-amylases classified in the glycoside hydrolase family 13, subfamily 1, as supported also by clustering observed in the evolutionary tree. Sfamy KZ, in addition to the essential GH13 α-amylase three-domain arrangement (catalytic TIM barrel plus domains B and C), does not contain any distinct starch-binding domain. Sfamy KZ was expressed as a recombinant protein in Saccharomyces cerevisiae and purified to electrophoretic homogeneity. The enzyme had a molecular mass 53 kDa and contained about 2.5% of carbohydrate. The enzyme exhibited pH and temperature optima in the range of 5–6 and 40–50 °C, respectively. Stable adsorption of the enzyme to starch granules was not detected but a low degradation of raw starch in a concentration-dependent manner was observed.

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Abbreviations

Sfamy:

α-Amylase from Saccharomycopsis fibuligera HUT 7212

Sfamy KZ:

α-amylase from S. fibuligera KZ

ALP1 :

Gene coding for α-amylase from S. fibuligera HUT 7212

LKA1:

α-Amylase 1 from Lipomyces kononenkoae

LKA2:

α-Amylase 2 from Lipomyces kononenkoae

CBM:

Carbohydrate-binding module

SBD:

Starch-binding domain

GH:

Glycoside hydrolase

AMY1:

Barley α-amylase 1 (low pI isozyme)

AMY2:

Barley α-amylase 2 (high pI isozyme)

CAZy:

Carbohydrate-active enzymes

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Acknowledgments

This work was supported in part by the grant No. 2/0114/08 from the Slovak grant agency VEGA.

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Authors and Affiliations

  1. Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51, Bratislava, Slovakia

    Eva Hostinová, Štefan Janeček & Juraj Gašperík

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  1. Eva Hostinová
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  2. Štefan Janeček
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  3. Juraj Gašperík
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Correspondence to Eva Hostinová.

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Hostinová, E., Janeček, Š. & Gašperík, J. Gene Sequence, Bioinformatics and Enzymatic Characterization of α-Amylase from Saccharomycopsis fibuligera KZ. Protein J 29, 355–364 (2010). https://doi.org/10.1007/s10930-010-9260-6

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