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<p>Chickpea (<i>Cicer arietinum</i>) contains at least four alpha-galactosidase genes (LOC101512744, LOC101513035, LOC101503593, and LOC101502309). The alpha-galactosidase gene (LOC101513035) was isolated from chickpea, cloned as eGFP fusion in pET 15b-eGFP, and overexpressed in <i>E. coli</i> BL21(DE3). The enzyme alpha-galactosidase was purified 32.9 fold using Ni-NTA affinity column with a recovery per cent of 53.7. The enzyme was qualitatively analysed for expression on denaturing and non-denaturing PAGE. The enzyme’s optimal pH and temperature was 5.4 and 46&#xa0;°C, respectively. The Michaelis-Menten constant (<i>K</i><sub>m</sub>) and V<sub>max</sub> for the synthetic substrate <i>p</i>-nitrophenyl-ɑ-D-galactopyranoside (<i>p</i>-NPG) was 0.3535 mM and 107.6 µmol.min<sup>− 1</sup> respectively. Furthermore, 1-deoxygalactonojirimycin (DGJ) a galactose mimic inhibited alpha-galactosidase competitively with a linear curve fit <i>K</i><sub>m</sub> of 6.8 mM and non-linear curve fit <i>K</i><sub>m</sub> 4.4 mM. Higher temperature optima, acidic pH, lower <i>K</i><sub>m</sub>, and higher V<sub>max</sub> collectively open suitable avenues for its applications in diversified process industries.</p>

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Cloning and characterization of a chickpea α-galactosidase befitting industrial bioprocess applications

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月19日

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  • P. M. Vetriselvi,
  • Marcus Samuel,
  • Rex Arunraj

摘要

Chickpea (Cicer arietinum) contains at least four alpha-galactosidase genes (LOC101512744, LOC101513035, LOC101503593, and LOC101502309). The alpha-galactosidase gene (LOC101513035) was isolated from chickpea, cloned as eGFP fusion in pET 15b-eGFP, and overexpressed in E. coli BL21(DE3). The enzyme alpha-galactosidase was purified 32.9 fold using Ni-NTA affinity column with a recovery per cent of 53.7. The enzyme was qualitatively analysed for expression on denaturing and non-denaturing PAGE. The enzyme’s optimal pH and temperature was 5.4 and 46 °C, respectively. The Michaelis-Menten constant (Km) and Vmax for the synthetic substrate p-nitrophenyl-ɑ-D-galactopyranoside (p-NPG) was 0.3535 mM and 107.6 µmol.min− 1 respectively. Furthermore, 1-deoxygalactonojirimycin (DGJ) a galactose mimic inhibited alpha-galactosidase competitively with a linear curve fit Km of 6.8 mM and non-linear curve fit Km 4.4 mM. Higher temperature optima, acidic pH, lower Km, and higher Vmax collectively open suitable avenues for its applications in diversified process industries.