In recent years, there has been a substantial rise in the prevalence of metabolic syndrome, which has emerged as a major global public health issue [1], [2]. The consumption of diets high in sugar and fat has been closely associated with the escalating incidence of metabolic syndrome [3]. Consequently, a growing imperative comes into existence to explore functional sugars that exhibit specific physiological activities, possess low caloric content, and offer potential health benefits [4], [5]. D-mannose, a sugar substitute, has garnered substantial research attention and been found applications in the realms of food, pharmaceuticals, and health products owing to its low-calorie and non-toxic properties [6], [7], [8], [9]. In comparison to D-glucose, D-mannose undergoes slower metabolism within the human body, resulting in a lesser elevation of blood glucose levels. Hence, D-mannose is deemed an ideal alternative sugar for individuals with diabetes or those seeking to regulate their blood sugar levels [10]. Furthermore, D-mannose has been discovered to possess various other physiological activities, including immune function enhancement and positive effects in the treatment of diseases such as cancer, tumors, gastroenteritis, and urinary tract infections [11]. As further research unveils the functional attributes of D-mannose, a surge in demand for high-quality D-mannose is anticipated.

Conventional methods for D-mannose production typically involve the hydrolysis of plant tissues or chemical synthesis using glucose as a starting material. However, these traditional approaches have several drawbacks, such as low yields, complex procedures, and potential risks to the environment and human health [12], [13]. The limitations associated with these methods have spurred researchers to explore biotransformation approaches for more environmentally friendly and efficient D-mannose production [9], [14], [15], [16]. In contrast, biotransformation methods, particularly enzymatic approaches, offer several advantages. They exhibit high conversion rates and specificity, operate under mild reaction conditions, and result in minimal by-product formation. Compared with traditional chemical methods, these biotransformation methods demonstrate superior performance in terms of safety and environmental considerations [17], [18], [19], [20]. Consequently, bioconversion methods, especially enzymatic approaches, hold great promise for the future of D-mannose production [21].

D-mannose isomerase (D-MIase), a pivotal enzyme involved in D-mannose production, plays a crucial role in the reversible molecular conversion between D-fructose and D-mannose. In industrial applications, the extensive utilization of D-MIase has been observed in the production and application of D-mannose. Specifically, industrial D-mannose production favors higher temperatures and mildly acidic conditions as they help minimize non-enzymatic browning and expedite the reaction process [8]. Therefore, discovering novel D-MIases with industrially relevant properties is of utmost importance for enhancing D-mannose production.

The identification of novel D-mannose enzymes is a significant challenge in the field of D-mannose production. Since the initial discovery of D-MIase derived from Pseudomonas saccharophila in 1955 [22], this enzyme has been subsequently identified in other strains, including Xanthomonas rubrilineans [23], Streptomyces aerocolorigenes [24], Mycobacterium smegmatis [25], Agrobacterium radiobacter [26], Thermobifida fusca [27], Pseudomonas syringae [28], Marinomonas mediterranea [29] and Deltaproteobacteria bacterium [14]. Itoh et al. [30] successfully determined the crystal structure of MIase derived from Salmonella and deposited it in the protein database. To the best of our knowledge, the identification of D-MIases from probiotic sources has been relatively limited. Furthermore, conducting comprehensive studies on the structure and function of D-MIase can provide valuable insights into its catalytic mechanism and substrate specificity. This research can serve as a reference and guide for the development of other enzymes with potential applications. It holds great significance in the fields of biocatalysis, enzyme engineering, and enzyme applications.

This study identified and cloned a novel D-mannose isomerase (D-MIase) encoding gene from Bifidobacterium animalis subsp. lactis DSM 10140, belonging to the AGE family. The gene was successfully overexpressed in E. coli BL21 (DE3). The physicochemical properties of the purified enzyme were thoroughly evaluated, and its capability for D-mannose production was assessed. By conducting a comparative analysis of amino acid sequences and kinetic parameters, we successfully elucidated the catalytic mechanism employed by Bifi-MIase. This investigation holds great significance in the realm of environmentally friendly D-mannose production and provides a valuable reference for the development of other functional sugar isomerases with potential applications.