A novel D-Allulose 3-epimerase gene (daeM) has been identified from the metagenomic resource of the 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 Escherichia coli. The purified recombinant protein (DaeM) was found to be metal-dependent (Co⁺⁺ or Mn⁺⁺). It displayed the maximal levels of catalytic activity in a pH and temperature range of 6 to 11, and 75 °C to 80 °C, respectively. The enzyme exhibited remarkably high thermal stability at 60 °C and 70 °C, with a half-life 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, 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 NMR spectra were found indistinguishable from that 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 more excellent biocatalyst stability. Further, it is desirable to involve safe-grade microorganisms for the biosynthesis of a product. The daeM gene was expressed intracellularly in Bacillus subtilis. Whole-cell catalysis reaction performed in a reaction volume of 1 L at 60 °C yielded approximately 196 g.L^-1 D-Allulose from 700 g.L^-1 D-Fructose. Further, the whole recombinant cells were able to biosynthesize D-Allulose in apple juice, mixed fruit juice, and honey.

IMPORTANCE D-Allulose is a non-caloric sugar substitute with anti-diabetic and anti-obesity potential. With several physiological significance, D-Allulose has wide applications in the food and pharma industries. The development of thermostable biocatalyst is mainstream research for the industrial acceptability of the enzyme. Aquatic habitats of extreme temperatures are considered as a metagenomic resource with thermal tolerant biocatalysts of industrial importance. The present study explored the thermal-spring metagenome of Tattapani geothermal region, Chhattisgarh, India, for the discovery of a novel D-Allulose 3-epimerase gene (daeM), encoding the enzyme of excessive heat-stability. The daeM was expressed in the microbial cells of non-pathogenic and safe-grade species, B. subtilis, which was capable of performing D-Fructose to D-Allulose interconversion via whole-cell catalysis reaction. The results propose DaeM as a potential biocatalyst for commercial production of the rare sugar, D-Allulose. The study established that extreme environmental niches are the genomic resource of functional sugar-related biocatalysts.

从热水库的宏基因组学资源中已经鉴定出一个新颖的D-Allulose 3-epererase基因（daeM）。该酶将D-果糖差向异构化为D-Allulose，D-Allulose是自然界中罕见的功能性糖。宏基因组DNA片段克隆并在大肠杆菌中表达。发现纯化的重组蛋白（DaeM）具有金属依赖性（Co ⁺⁺或Mn ⁺⁺）。它在pH值和温度范围分别为6至11和75°C至80°C时显示出最大的催化活性水平。该酶在60°C和70°C时显示出极高的热稳定性，半衰期分别为9,900分钟和3,240分钟。据我们所知，这是迄今表征的D-阿洛糖3-表异构酶所显示的最高热稳定性。酶处理700 mg.mL ^-1 D-果糖可产生约217 mg.mL ^-1D-阿洛糖，在最佳条件下。纯化了催化产物，发现其NMR谱与标准D-阿洛糖没有区别。对于生物分子生产，全细胞催化过程避免了繁琐的酶提取和纯化过程，还提供了更出色的生物催化剂稳定性。此外，期望涉及用于生物合成产品的安全等级微生物。该DAEM基因在细胞内表达枯草芽孢杆菌。在60°C下以1 L的反应体积进行的全细胞催化反应从700 gL ^-1产生约196 gL ^-1 D-阿洛糖D-果糖。此外，整个重组细胞能够生物合成苹果汁，混合果汁和蜂蜜中的D-阿洛糖。

重要信息D-阿洛糖是一种无热量的糖替代品，具有抗糖尿病和抗肥胖的潜力。具有多种生理意义，D-Allulose在食品和制药行业中具有广泛的应用。热稳定生物催化剂的开发是酶在工业上可接受性的主流研究。极端温度的水生生境被认为是具有工业重要性的耐热生物催化剂的宏基因组资源。本研究探索了印度恰蒂斯加尔邦塔塔帕尼地热区的温泉热基因组，以发现新的D-阿洛糖3-表异构酶基因（daeM），该基因编码具有过度热稳定性的酶。该DAEM在非致病性和安全级物种的微生物细胞中表达，枯草芽孢杆菌，其能够通过全细胞催化反应进行D-果糖到D-阿洛糖的相互转化。结果表明，DaeM可作为商业生产稀有糖D-阿洛糖的潜在生物催化剂。该研究确定，极端的环境生态位是功能性糖相关生物催化剂的基因组资源。