A Novel <scp>d</scp> -Allulose 3-Epimerase Gene from the Metagenome of a Thermal Aquatic Habitat and <scp>d</scp> -Allulose Production by Bacillus subtilis Whole-Cell Catalysis

Abstract

A novel d-allulose 3-epimerase gene (<i>daeM</i>) has been identified from the metagenomic resource of a hot-water reservoir. The enzyme epimerizes d-fructose into d-allulose, a functional sugar of rare abundance in nature. The metagenomic DNA fragment was cloned and expressed in <i>Escherichia coli</i> The purified recombinant protein (DaeM) was found to be metal dependent (Co²⁺ or Mn²⁺). It displayed the maximal levels of catalytic activity in a pH range of 6 to 11 and a temperature range of 75°C to 80°C. The enzyme exhibited remarkably high thermal stability at 60°C and 70°C, with half-life values of 9,900 and 3,240 min, respectively. To the best of our knowledge, this is the highest thermal stability demonstrated by a d-allulose 3-epimerase that has been characterized to date. The enzymatic treatment of 700 mg·ml^-1 d-fructose yielded about 217 mg·ml^-1 d-allulose, under optimal condition. The catalytic product was purified, and its nuclear magnetic resonance (NMR) spectra were found to be indistinguishable from those of standard d-allulose. For biomolecule production, the whole-cell catalysis procedure avoids the tedious process of extraction and purification of enzyme and also offers better biocatalyst stability. Further, it is desirable to employ safe-grade microorganisms for the biosynthesis of a product. The <i>daeM</i> gene was expressed intracellularly in <i>Bacillus subtilis</i> A whole-cell catalysis reaction performed with a reaction volume of 1 liter at 60°C yielded approximately 196 g·liter^-1 d-allulose from 700 g·liter^-1 d-fructose. Further, the whole recombinant cells were able to biosynthesize d-allulose in apple juice, mixed fruit juice, and honey.<b>IMPORTANCE</b> d-Allulose is a noncaloric sugar substitute with antidiabetes and antiobesity potential. With several characteristics of physiological significance, d-allulose has wide-ranging applications in the food and pharmacology industries. The development of a thermostable biocatalyst is an objective of mainstream research aimed at achieving industrial acceptability of the enzyme. Aquatic habitats of extreme temperatures are considered a potential metagenomic resource of heat-tolerant biocatalysts of industrial importance. The present study explored the thermal-spring metagenome of the Tattapani geothermal region, Chhattisgarh, India, discovering a novel d-allulose 3-epimerase gene, <i>daeM</i>, encoding an enzyme of high-level heat stability. The <i>daeM</i> gene was expressed in the microbial cells of a nonpathogenic and safe-grade species, <i>B. subtilis</i>, which was found to be capable of performing d-fructose to d-allulose interconversion via a whole-cell catalysis reaction. The results indicate that DaeM is a potential biocatalyst for commercial production of the rare sugar d-allulose. The study established that extreme environmental niches represent a genomic resource of functional sugar-related biocatalysts.