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<p><span>d</span>-allulose, a rare sugar with significant health benefits, has attracted considerable attention due to its prospective uses as a low-calorie sweetener. Enzymes from the ketose 3-epimerase (KEase) family catalyze the epimerization of <span>d</span>-fructose to <span>d</span>-allulose. In this study, a new member of the KEase family was characterized from <i>Leifsonia aquatica</i> (GenBank accession no. WP_021758664.1) and named <i>Leifsonia aquatica</i> <span>l</span>-ribulose 3-epimerase (LaLRE) due to its highest substrate specificity toward <span>l</span>-ribulose. The enzyme was cloned, heterologously expressed in <i>Escherichia coli</i>, and purified. Biochemical characterization revealed that LaLRE is strictly metal-dependent, displaying maximum activity in the presence of Ni<sup>2+</sup>. The optimum pH and temperature were 5.0 and 65&#xa0;°C, respectively. LaLRE demonstrated a broad substrate scope, with significant activity toward <span>d</span>-fructose, making it a promising candidate for industrial <span>d</span>-allulose production. Rational engineering identified Leu114 as a key determinant of substrate specificity. The mutant L114F lost activity towards pentose sugars (<span>l</span>-ribulose and <span>l</span>-xylulose) but retained activity towards hexoses. This finding provides insights into the structural basis of substrate preference among KEases. Under optimal conditions, the wild-type LaLRE achieved a 32.8% conversion of <span>d</span>-fructose (750&#xa0;g/L) to <span>d</span>-allulose. This work underscores the potential of LaLRE in sustainable <span>d</span>-allulose production and its implications for the food industry.</p> Graphical Abstract <p></p>

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Identification and characterization of l-ribulose 3-epimerase from Leifsonia aquatica and its application in d-allulose production

  • 期刊论文

  • 发表日期：

    2026年2月16日

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  • Amol D. Pagar,
  • Julie M. Goddard

摘要

d-allulose, a rare sugar with significant health benefits, has attracted considerable attention due to its prospective uses as a low-calorie sweetener. Enzymes from the ketose 3-epimerase (KEase) family catalyze the epimerization of d-fructose to d-allulose. In this study, a new member of the KEase family was characterized from Leifsonia aquatica (GenBank accession no. WP_021758664.1) and named Leifsonia aquatica l-ribulose 3-epimerase (LaLRE) due to its highest substrate specificity toward l-ribulose. The enzyme was cloned, heterologously expressed in Escherichia coli, and purified. Biochemical characterization revealed that LaLRE is strictly metal-dependent, displaying maximum activity in the presence of Ni2+. The optimum pH and temperature were 5.0 and 65 °C, respectively. LaLRE demonstrated a broad substrate scope, with significant activity toward d-fructose, making it a promising candidate for industrial d-allulose production. Rational engineering identified Leu114 as a key determinant of substrate specificity. The mutant L114F lost activity towards pentose sugars (l-ribulose and l-xylulose) but retained activity towards hexoses. This finding provides insights into the structural basis of substrate preference among KEases. Under optimal conditions, the wild-type LaLRE achieved a 32.8% conversion of d-fructose (750 g/L) to d-allulose. This work underscores the potential of LaLRE in sustainable d-allulose production and its implications for the food industry.

Graphical Abstract