[Construction of whole-cell catalysts containing sucrose isomerase mutants]

Isomaltulose possesses excellent physiological functions such as inhibiting fat accumulation, improving intestinal flora balance, and preventing diabetes, thus having wide applications in the food industry. Currently, isomaltulose is primarily produced through the catalysis of sucrose isomerase. However, this production method faces challenges such as low enzymatic conversion efficiency of sucrose, cumbersome product separation and extraction steps, and a high proportion of by-products, which have become obstacles to the industrial production development of isomaltulose. Therefore, exploring efficient and economical ways to produce isomaltulose has become a current research focus. This study combined site-directed mutagenesis and surface display technology to mutate key sites of the sucrose isomerase from Klebsiella sp. LX3. With the cell wall protein Pir1 from Yarrowia lipolytica as an anchor protein, the sucrose isomerase mutants were displayed on the yeast surface to construct whole-cell catalysts containing the mutants. Concurrently, its promoter was optimized to improve display efficiency, and the enzymatic properties, conversion products, recycling stability, and display efficiency of the mutants were analyzed. The combined mutant PR424A&S431A' achieved the highest enzyme activity of 2 296.9 U/g dry cell weight (DCW) and an average fluorescence intensity of 1 246, which were 1.64 and 2.13 times of that under the control of the P_hp4d promoter, respectively. Compared with the wild type, the mutant whole-cell catalysts remained unchanged enzymatic properties, improved product conversion rates (the isomaltulose content in the conversion products of PR424A&S431A increased by 5.14%), and consistent number of reuse cycles (20, demonstrating good recycling stability.) This study delves into the properties and application potential of whole-cell catalysts based on sucrose isomerase mutants and explores new ideas and methods for the industrial production of isomaltulose, holding significant theoretical value and practical application significance.