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米曲霉M30011来源的丝氨酸羧肽酶Y的定点突变及共价有机骨架固定化协同提升酶性能的研究
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1.北京工商大学食品质量与安全北京实验室，北京100048;2.北京工商大学北京市食品添加剂工程技术研究中心，北京100048;3.北京工商大学北京食品营养与人类健康高精尖创新中心，北京100048;4.北京工商大学北京市食品风味化学重点实验室，北京100048

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通讯作者:

熊科（1981—），男，博士，教授，博士研究生导师，主要从事食品生物技术与营养安全研究。E-mail：xiongke@btbu.edu.cn

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基金项目:

北京市自然科学基金项目（6242002）。


Site-directed Mutagenesis and Covalent Organic Framework Immobilization of Serine Carboxypeptidase Y from Aspergillus oryzae M30011 for Synergistic Enhancement of Enzyme Performance
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1.Beijing Laboratory for Food Quality and Safety, Beijing Technology and Business University, Beijing 100048, China;2.Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing 100048, China;3.Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100048, China;4.Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business University, Beijing 100048, China

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    摘要:

    【目的】为提高丝氨酸羧肽酶在酶解过程中的稳定性并拓展其工业应用，针对其易失活的问题，通过理性设计与固定化技术开发高效稳定的酶催化剂。【方法】基于米曲霉（Aspergillus oryzae）M30011来源的丝氨酸羧肽酶Y（CPY）设计了单突变酶M517R、I464R、Y271R以及三突变酶M517R/I464R/Y271R，并利用大肠杆菌系统进行原核表达与酶活力检测，随后采用共价有机骨架材料（COFs）固定活性最高的突变酶（COFs@突变酶），系统评估游离突变酶与COFs@突变酶的pH稳定性、温度稳定性、半衰期及重复使用性。此外，以米糠蛋白质为底物，探究其与碱性蛋白酶（AP）联用的酶解效果。【结果】4种突变酶被成功表达并纯化，其中三突变酶M517R/I464R/Y271R的酶活力最高（1 069.54 U/mg），因此用COFs固定化三突变酶M517R/I464R/Y271R（COFs@三突变酶）。与游离三突变酶相比，COFs@三突变酶在较宽的pH与温度范围内具有较强的稳定性，半衰期延长至498 min，且经过6次重复使用后其相对酶活力仍维持在50%左右。酶解试验结果显示，AP与COFs@三突变酶组合酶解米糠蛋白质制得的低聚肽的费歇尔比（F值）为34.01，远超制备阈值（>20），表明该组合在低聚肽制备中具有良好的应用潜力。【结论】通过突变酶设计与固定化技术协同策略显著提升了丝氨酸羧肽酶的工业适用性，为功能性低聚肽生产和工业酶催化效率优化提供了新方向，展现出广阔的应用前景。

    Abstract:

    [Objective] To enhance the stability of serine carboxypeptidase during enzymatic hydrolysis and expand its industrial applicability, the author employed rational design and immobilization technology to develop efficient and stable enzymatic catalysts in view of the easy deactivation of this enzyme. [Method] On the basis of the serine carboxypeptidase Y (CPY) derived from Aspergillus oryzae M30011, single mutant enzymes M517R, I464R, and Y271R and the triple mutant enzyme M517R/I464R/Y271R were designed. These mutant enzymes were then prokaryotically expressed in Escherichia coli and subjected to enzyme activity assays. Subsequently, the mutant enzyme with the highest activity was immobilized with covalent organic framework materials (COFs), yielding the composite designated as COFs@mutant enzyme. The free mutant enzyme and the COFs@mutant enzyme were systematically evaluated in terms of the pH stability, thermal stability, half-life, and reusability. In addition, with rice bran protein as the substrate, the hydrolysis effect of the COFs@mutant enzyme combined with alkaline protease (AP) was investigated. [Result] The four mutant enzymes were successfully expressed and purified, with the triple mutant M517R/I464R/Y271R exhibiting the highest enzyme activity (1 069.54 U/mg). Therefore, the triple mutant enzyme M517R/I464R/Y271R was immobilized with COFs (COFs@triple mutant enzyme).Compared with the free triple mutant enzyme, COFs@triple mutant enzyme demonstrated strong stability across wide ranges of pH and temperature, with a half-life extended to 498 min and the relative enzyme activity of approximately 50% after 6 reuse cycles. The results of the enzymatic hydrolysis experiment showed that the Fischer ratio (F-value) of the oligopeptides prepared from rice bran protein by AP combined with COFs@triple mutant enzyme was 34.01, far exceeding the preparation threshold (>20), indicating the good application potential of this combination in the preparation of oligopeptides. [Conclusion] The synergistic strategy of mutant enzyme design and immobilization technology significantly enhanced the industrial applicability of serine carboxypeptidase, providing a new direction for the production of functional oligopeptides and the optimization of industrial enzyme catalysis efficiency, and demonstrating broad application prospects.

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蒋佳雯,蔡彬,熊科,毛书灿,陈贻强,李松林,张鹏程,杨威,李泊瑞.米曲霉M30011来源的丝氨酸羧肽酶Y的定点突变及共价有机骨架固定化协同提升酶性能的研究[J].食品与生物技术学报,2025,44(8):116-127.

JIANG Jiawen, CAI Bin, XIONG Ke, MAO Shucan, CHEN Yiqiang, LI Songlin, ZHANG Pengcheng, YANG Wei, LI Borui. Site-directed Mutagenesis and Covalent Organic Framework Immobilization of Serine Carboxypeptidase Y from Aspergillus oryzae M30011 for Synergistic Enhancement of Enzyme Performance[J]. Journal of Food Science and Biotechnology,2025,44(8):116-127.

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  • 收稿日期:2024-08-11
  • 最后修改日期:2024-10-01
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  • 在线发布日期: 2025-11-26
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