D-Allulose, a promising low-calorie rare sugar substitute for sucrose with multiple physiological functions, is synthesized from D-fructose by D-allulose 3-epimerase (DAEase), whose low catalytic efficiency and poor thermostability restrict industrial-scale production. This study aimed to design and implement a comprehensive experimental course on the green biosynthesis of D-allulose. The course integrates the heterologous expression of DAEase, enzymatic characterization, enzyme immobilization, and catalytic synthesis of D-allulose, enabling students to systematically master the complete experimental workflow from enzyme preparation to product synthesis while cultivating interdisciplinary integration ability, engineering practice awareness, and innovative thinking.

This paper examines the green biosynthesis of D-allulose, concentrating on the expression of D-allulose-3-epimerase, systematic analysis of its enzymatic properties, and the development of effective enzyme immobilization strategies. Furthermore, it designs and implements an integrated experimental course for undergraduates and constructs a modular, progressive experimental teaching system. Through characterization of immobilized enzymes, their catalytic efficiency and thermal stability were evaluated, and the reaction conditions for catalytic synthesis of D-psicose were optimized, achieving a continuous catalytic process for D-psicose synthesis using immobilized enzymes in a flow reactor.

By integrating theoretical knowledge and advanced techniques from microbiology, molecular biology, and enzyme engineering, this paper designs a comprehensive practical chain for the course that spans gene expression, fermentation preparation, enzyme immobilization, and product catalytic synthesis. Moreover, a basic–comprehensive–exploratory capability training structure was formed to improve students’ practical and innovative abilities. The experimental course system allows flexible scheduling and can be adjusted according to different teaching durations. Each independent experimental module can be implemented separately to enhance students’ mastery of the knowledge and operations of a single module. Alternatively, it can be systematically integrated to improve students’ experimental design and data analysis skills, deepening their understanding of the functional sugar synthesis process. Additionally, this course can incorporate various teaching methods such as group discussions and classroom questions to stimulate students’ thinking about the application value of immobilization and the importance of green biosynthesis for human development, adapting to diverse teaching requirements and student backgrounds.

This course emphasizes student-centered learning throughout the teaching implementation process, highlighting students’ full participation in experimental design, hands-on operation, result analysis, and problem-solving. It comprehensively enhances students’ understanding and practical operation abilities throughout the biomanufacturing process, focusing on cultivating interdisciplinary integration skills, engineering practice awareness, and innovative thinking capabilities. The course design closely follows the forefront of the biomanufacturing industry and meets social demands, achieving an effective connection between theoretical teaching and scientific research practice. It not only guides students to establish a sustainable view of research and industry but also lays a solid foundation of talent and technology for research, development, and technical work in the biomanufacturing field. Meanwhile, with the rapid progress of science and technology, this course brings considerable social value, improving public health and promoting medical progress. It not only drives the upgrading of the biopharmaceutical industry but also makes important contributions to sustainable social development.