Biophysical Characterization of a Recombinant α-Amylase from Thermophilic Bacillus sp. strain TS-23

Chi, Meng-Chun; Wu, Tai-Jung; Chuang, Tzu-Ting; Chen, Hsiang-Ling; Lo, Huei-Fen; Lin, Long-Liu

doi:10.1007/s10930-010-9287-8

Cited by 4 publications

(2 citation statements)

References 67 publications

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“…Maximum activity is observed at pH 8 and 70 °C; however, more than 80% of its activity is retained at pH 6.1 to 9.0. A heating rate dependence of the irreversible thermal unfolding kinetics suggests the presence of kinetic barriers during unfolding (Chi et al 2010). The unfolding transitions in urea and GdmCl are non-coincidental with respect to loss of structural and functional properties.…”

Section: Alkaliphilic α-Amylasementioning

confidence: 99%

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Structural and functional adaptation in extremophilic microbial α-amylases

2022

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Maintaining stable native conformation of a protein under a given ecological condition is the prerequisite for survival of organisms. Extremophilic bacteria and archaea have evolved to adapt under extreme conditions of temperature, pH, salt, and pressure. Molecular adaptations of proteins under these conditions are essential for their survival. These organisms have the capability to maintain stable, native conformations of proteins under extreme conditions. The enzymes produced by the extremophiles are also known as extremozyme, which are used in several industries. Stability and functionality of extremozymes under varying temperature, pH, and solvent conditions are the most desirable requirement of industry. α-Amylase is one of the most important enzymes used in food, pharmaceutical, textile, and detergent industries. This enzyme is produced by diverse microorganisms including various extremophiles. Therefore, understanding its stability is important from fundamental as well as an applied point of view. Each class of extremophiles has a distinctive set of dominant non-covalent interactions which are important for their stability. Static information obtained by comparative analysis of amino acid sequence and atomic resolution structure provides information on the prevalence of particular amino acids or a group of non-covalent interactions. Protein folding studies give the information about thermodynamic and kinetic stability in order to understand dynamic aspect of molecular adaptations. In this review, we have summarized information on amino acid sequence, structure, stability, and adaptability of α-amylases from different classes of extremophiles.

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Section: Alkaliphilic α-Amylasementioning

confidence: 99%

“…Both thermal and chemical unfolding transitions are fully irreversible. It shows significant resistance towards thermal and chemical unfolding in the presence of stabilizing cosolvents TMAO and sorbitol in a concentration-dependent manner (Chi et al 2010(Chi et al , 2012.…”

Section: Alkaliphilic α-Amylasementioning

confidence: 99%

Structural and functional adaptation in extremophilic microbial α-amylases

2022

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The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae: biochemical characterization and metal dependence

2014

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Serine/threonine protein phosphatases have been described in many pathogenic bacteria as essential enzymes involved in phosphorylation-dependent signal transduction pathways and frequently associated with the virulence of these organisms. An inspection of Mycoplasma synoviae genome revealed the presence of a gene (prpC) encoding a putative protein phosphatase of the protein phosphatase 2C (PP2C) subfamily. Here, we report a complete biochemical characterization of M. synoviae phosphatase (PrpC) and the particular role of metal ions in the structure-function relationship of this enzyme. PrpC amino acid sequence analysis revealed that all the residues involved in the dinuclear metal center and the putative third metal ion-coordinating residues, conserved in PP2C phosphatases, are present in PrpC. PrpC is a monomeric protein able to dephosphorylate phospho-substrates with Mn(2+) ions' dependence. Thermal stability analysis demonstrated the enzyme stability at mild temperatures and the influence of Mn(2+) ions in this property. Mass spectrometry analysis suggested that three metal ions bind to PrpC, two of which with an apparent high-affinity constant. Mutational analysis of the putative third metal-coordinating residues, Asp122 and Arg164, revealed that these variants exhibited a weaker binding of manganese ions, and that both mutations affected PrpC phosphatase activity. According to these results, PrpC is a metal-dependent protein phosphatase member with an improved stability in the holo form and with Asp122, possibly implicated in the third metal-binding site, essential to catalytic activity.

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Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilicBacillussp. TS-23

Chi

Chen

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.

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Biophysical Characterization of a Recombinant α-Amylase from Thermophilic Bacillus sp. strain TS-23

Cited by 4 publications

References 67 publications

Structural and functional adaptation in extremophilic microbial α-amylases

Structural and functional adaptation in extremophilic microbial α-amylases

The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae: biochemical characterization and metal dependence

Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilicBacillussp. TS-23

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