蔗糖异构酶突变体全细胞催化剂的构建

Construction of whole-cell catalysts containing sucrose isomerase mutants

摘要:

异麦芽酮糖具有抑制脂肪累积、改善肠内菌群平衡、预防糖尿病等优良的生理功能，在食品工业领域广泛应用。目前，主要通过蔗糖异构酶催化生产异麦芽酮糖。这种生产方式酶转化蔗糖效率低、产物分离提取步骤繁琐、副产物占比多等问题，已成为异麦芽酮糖的工业化生产发展道路上的阻碍。因此，探索以高效经济的方式生产异麦芽酮糖已经成为当下的一个研究热点。本研究结合定点突变和表面展示这2种技术，对克雷伯氏菌(Klebsiella sp.) LX3来源的蔗糖异构酶的关键位点进行突变，以来自解脂耶氏酵母(Yarrowia lipolytica)的细胞壁蛋白Pir1作为锚定蛋白，将蔗糖异构酶突变体展示于酵母表面，构建蔗糖异构酶突变体全细胞催化剂，同时对其启动子进行优化提高其展示效率，并对突变体酶学性质、转化产物、循环使用稳定性、展示效率进行分析。组合突变体PR424A&S431A’的酶活力最高达到2 296.9 U/g细胞干重(dry cell weight, DCW)，平均荧光强度为1 246，分别是P_hp4d启动子调控下的1.64、2.13倍。对各突变体全细胞催化剂的酶学性质、转化产物及循环使用稳定性进行分析，结果表明各突变体全细胞催化剂的酶学性质与野生型相比均未发生变化；产物转化率较野生型相比均有所提高，其中，突变体PR424A&S431A转化产物中异麦芽酮糖含量提高了5.14%；循环使用次数与野生型相比一致，均为20个循环，具有较好的循环使用稳定性。本研究深入分析了蔗糖异构酶突变体全细胞催化剂的性质和应用潜力，同时也为异麦芽酮糖的工业化生产探索了新的思路和方法，具有重要的理论价值和实际应用意义。

Abstract:

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.