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<p>The tremendous demand for alpha-amylase in industries has driven the need for thermostable enzymes with greater yields. In this work, we report the biosynthesis of a heat-stable α-amylase derived from the amylolytic bacterium <i>Bacillus stercoris</i> THS-K1, isolated from the Tuva thermal springs in Gujarat, India. The medium components influencing the enzyme production were optimised through the Plackett-Burman Design. Among the experimental variables assessed, starch concentration, pH, and yeast extract had a significant effect on enzyme biosynthesis. The optimal levels of key parameters were systematically optimised using Central Composite Design (CCD) under the Response Surface Methodology (RSM) framework. The optimised culture variables significantly increased enzyme production from 1.1 U/mL to 4.94 ± 0.05 U/mL. The enzyme was precipitated with ammonium sulphate and purified using size exclusion and ion exchange chromatography, achieving a 12.8-fold purification and a 25.14% yield, respectively. Zymographic evaluation revealed that the enzyme’s molecular mass was ~ 25&#xa0;kDa. The most efficient activity of the isolated α-amylase was observed at 50 ± 1&#xa0;°C and pH 7. The purified enzyme exhibited its highest catalytic activity at pH 7.0 and 50 ± 1&#xa0;°C. The gene expression of the enzyme was identified by amylase gene amplification. The PCR-amplified fragment, ~ 1.7&#xa0;kb in length, encoded a polypeptide whose deduced amino acid sequence exhibited a high degree of identity to the α-amylase of <i>Bacillus subtilis</i>. Structural modelling of the AMY-encoded α-amylase suggests the influence of specific amino acids in the enzyme’s catalytic properties or stability. This work will be helpful in further research to produce recombinant α‐amylase.</p> Graphical Abstract <p></p>

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Bioprocess optimisation and molecular characterisation of alpha-amylase from thermotolerant Bacillus stercoris THS-K1

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月15日

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  • Hiral H. Padia,
  • Krushali S. Jain,
  • Vaishnavi C. Goswami,
  • Devayani R. Tipre

摘要

The tremendous demand for alpha-amylase in industries has driven the need for thermostable enzymes with greater yields. In this work, we report the biosynthesis of a heat-stable α-amylase derived from the amylolytic bacterium Bacillus stercoris THS-K1, isolated from the Tuva thermal springs in Gujarat, India. The medium components influencing the enzyme production were optimised through the Plackett-Burman Design. Among the experimental variables assessed, starch concentration, pH, and yeast extract had a significant effect on enzyme biosynthesis. The optimal levels of key parameters were systematically optimised using Central Composite Design (CCD) under the Response Surface Methodology (RSM) framework. The optimised culture variables significantly increased enzyme production from 1.1 U/mL to 4.94 ± 0.05 U/mL. The enzyme was precipitated with ammonium sulphate and purified using size exclusion and ion exchange chromatography, achieving a 12.8-fold purification and a 25.14% yield, respectively. Zymographic evaluation revealed that the enzyme’s molecular mass was ~ 25 kDa. The most efficient activity of the isolated α-amylase was observed at 50 ± 1 °C and pH 7. The purified enzyme exhibited its highest catalytic activity at pH 7.0 and 50 ± 1 °C. The gene expression of the enzyme was identified by amylase gene amplification. The PCR-amplified fragment, ~ 1.7 kb in length, encoded a polypeptide whose deduced amino acid sequence exhibited a high degree of identity to the α-amylase of Bacillus subtilis. Structural modelling of the AMY-encoded α-amylase suggests the influence of specific amino acids in the enzyme’s catalytic properties or stability. This work will be helpful in further research to produce recombinant α‐amylase.

Graphical Abstract