Purification and characterization of the extracellular α-amylase activity of the yeast <i>Schwanniomyces alluvius</i>

Moranelli, F.; Yaguchi, Makoto; Calleja, G. B.; Nasim, A.

doi:10.1139/o87-116

Cited by 31 publications

(26 citation statements)

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“…Many known α-amylases including BLA contain structurally essential Ca 2+ to maintain their activity and thermostability. However, these Ca 2+ are often removed by chelating agents such as zeolite and EDTA, and in turn result in the inactivity of enzymes [49,50]. Therefore, Ca-independent enzymes are preferable than Ca-dependent enzymes for industrial applications.…”

Section: Discussionmentioning

confidence: 99%

Close relationship of a novel Flavobacteriaceaeα-amylase with archaeal α-amylases and good potentials for industrial applications

Cheng

et al. 2014

Biotechnol Biofuels

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BackgroundBioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process.ResultsA novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme.ConclusionsThe improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.

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Section: Discussionmentioning

confidence: 99%

Close relationship of a novel Flavobacteriaceaeα-amylase with archaeal α-amylases and good potentials for industrial applications

Cheng

et al. 2014

Biotechnol Biofuels

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“…Among these putative GPI proteins, it was also determined that four genes, SPCC757.12, SPAC23D3.14c, SPCC63.02c, and SPBC16A3.13, are to be classified as -amylase homologs, which catalyze endohydrolysis of -1,4-glucosidic linkages in starch and related oligosaccharides and belong to glucoside hydrolase family 13. 8) Although a large number of studies have described -amylaseproducing yeasts, [9][10][11][12][13][14] S. pombe has no -amylase activity based on the halo assay. 15) We are interested in the function of putative GPIanchored -amylase homologs in S. pombe.…”

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confidence: 99%

An α-Amylase Homologue,aah3, Encodes a GPI-Anchored Membrane Protein Required for Cell Wall Integrity and Morphogenesis inSchizosaccharomyces pombe

Morita

Tanaka

Hosomi

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPI-anchored proteins in S. pombe, four alpha-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3(+) resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3(+) deletion, but the ascal wall of aah3Delta cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Delta cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe.

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“…However, Zn 2+ , Cu 2+ , and Hg 2+ acted as inhibitors of amylase activity, with Cu +2 and Hg 2+ showing a complete inhibition (Table 2). The inhibition by Hg 2+ may indicate the importance of indole group of amino acid residues in enzyme function [4, 24]. The wild-type amylase is inhibited by Hg 2+ , Ag + , Cu 2+ , and Mg 2+ [16] while that from L. kononenkoae CBS 5608 is inhibited by Ag + and Cu 2+ [21].…”

Section: Resultsmentioning

confidence: 99%

Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

et al. 2011

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An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH4+ and inhibited by Cu+2 and Hg+2. Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to Km values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1w, points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.

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Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

Cited by 31 publications

References 0 publications

Close relationship of a novel Flavobacteriaceaeα-amylase with archaeal α-amylases and good potentials for industrial applications

Close relationship of a novel Flavobacteriaceaeα-amylase with archaeal α-amylases and good potentials for industrial applications

An α-Amylase Homologue,aah3, Encodes a GPI-Anchored Membrane Protein Required for Cell Wall Integrity and Morphogenesis inSchizosaccharomyces pombe

Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

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