Site-directed Mutagenesis and Covalent Organic Framework Immobilization of Serine Carboxypeptidase Y from Aspergillus oryzae M30011 for Synergistic Enhancement of Enzyme Performance

Authors

Jiawen JIANG, Beijing Laboratory for Food Quality and Safety , Beijing Technology and Business University , Beijing 100048 , China
Bin CAI, Beijing Engineering and Technology Research Center of Food Additives , Beijing Technology and Business University , Beijing 100048 , China
Ke XIONG, Beijing Engineering and Technology Research Center of Food Additives , Beijing Technology and Business University , Beijing 100048 , ChinaFollow
Shucan MAO, Beijing Advanced Innovation Center for Food Nutrition and Human Health , Beijing Technology and Business University , Beijing 100048 , China
Yiqiang CHEN, Beijing Advanced Innovation Center for Food Nutrition and Human Health , Beijing Technology and Business University , Beijing 100048 , China
Songlin LI, Beijing Engineering and Technology Research Center of Food Additives , Beijing Technology and Business University , Beijing 100048 , China
Pengcheng ZHANG, Beijing Laboratory for Food Quality and Safety , Beijing Technology and Business University , Beijing 100048 , China
Wei YANG, Beijing Key Laboratory of Flavor Chemistry , Beijing Technology and Business University , Beijing 100048 , China
Borui LI, Beijing Advanced Innovation Center for Food Nutrition and Human Health , Beijing Technology and Business University , Beijing 100048 , China

Corresponding Author(s)

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

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 M 517R,I464R,and Y 271R and the triple mutant enzyme M 517R/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 M 517R/I464R/Y271R exhibiting the highest enzyme activity (1 069.54 U/mg ).Therefore,the triple mutant enzyme M 517R/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.

Publication Date

8-15-2025

First Page

116

Last Page

127

DOI

10.12441/spyswjs.20240811002

Recommended Citation

JIANG, Jiawen; CAI, Bin; XIONG, Ke; MAO, Shucan; CHEN, Yiqiang; LI, Songlin; ZHANG, Pengcheng; YANG, Wei; and LI, Borui (2025) "Site-directed Mutagenesis and Covalent Organic Framework Immobilization of Serine Carboxypeptidase Y from Aspergillus oryzae M30011 for Synergistic Enhancement of Enzyme Performance," Journal of Food Science and Biotechnology: Vol. 44: Iss. 8, Article 13.
DOI: 10.12441/spyswjs.20240811002
Available at: https://spsw.spyswjs.cnjournals.com/journal/vol44/iss8/13

References

［1］ 吴静，闵柔，邬敏辰，等.羧肽酶研究进展 ［J］.食品与生物技术学报，2012，31（8）：793-801.WU J，MIN R，WU M C，et al.Research progresses on the carboxypeptidase ［J］.Journal of Food Science and Biotechnology，2012，31（8）：793-801.（in Chinese ）
［2］ 于晓婷，陈晓艺，李苗，等.海藻酸钙 /硅胶 /羧甲基壳聚糖复合微球固定化柚苷酶的研究 ［J］.食品与生物技术学报，2023，42（1）：93-101.YU X T，CHEN X Y，LI M，et al.Study on immobilization of naringinase on calcium alginate/silica gel/carboxymethyl chitosan composite microspheres ［J］.Journal of Food Science and Biotechnology，2023，42（1）：93-101.（in Chinese ）
［3］ 晁昊.基于共价有机骨架胶囊材料的酶原位封装方法研究及其分析应用 ［D］.北京：北京化工大学，2022.
［4］ ZHONG C，MA W D，HE Y T，et al.Controllable synthesis of hollow microtubular covalent organic frameworks as an enzyme-immobilized platform for enhancing catalytic activity ［J］.ACS Applied Materials & Interfaces，2021，13（44）：52417 -52424.
［5］ FENG M C，NIU Z R，XING C Y，et al.Covalent organic framework based crosslinked porous microcapsules for enzymatic catalysis ［J］.Angewandte Chemie International Edition，2023，62（33）：e202306621.
［6］ WANG L，LIANG H H，XU M L，et al.Ratiometric electrochemical biosensing based on double-enzymes loaded on two-dimensional dual-pore COFETTA-TPAL［J］.Sensors and Actuators B：Chemical，2019，298：126859.
［7］ XU S J，WANG Y Y，LI W，et al.Covalent organic framework incorporated chiral polymer monoliths for capillary electrochromatography ［J］.Journal of Chromatography A，2019，1602：481-488.
［8］ 刘海梅，王芸，裴继伟，等.活性炭法制备高 F值栉孔扇贝酶解液的工艺条件 ［J］.食品与发酵工业，2020，46（22）：167-172.LIU H M，WANG Y，PEI J W，et al.Process conditions of enzymatic hydrolysis of scallop Chlamys farreri with high Fischer ratio by activated carbon method ［J］.Food and Fermentation Industries，2020，46（22）：167-172.（in Chinese）
［9］ 李小勇，李洪军.高F值低聚肽研究进展 ［J］.粮食与油脂，2006，19（6）：9-11.LI X Y，LI H J.Research progress in high Fischer ratio oilgo-peptide ［J］.Cereals & Oils，2006，19（6）：9-11.（in Chinese）
［10］ 李婷婷，田亚平，周楠迪，等.超声振荡改性玉米醇溶蛋白及高 F值寡肽制备 ［J］.食品与生物技术学报，2020，39（11）：104-111.LI T T，TIAN Y P，ZHOU N D，et al.Modification of zein by ultrasound oscillation and preparation of oligopeptides with a high Fischer ’s ratio［J］.Journal of Food Science and Biotechnology，2020，39（11）：104-111.（in Chinese ）
［11］ 尤孝鹏，陈智仙.运动营养食品的营养素、功能及酵母蛋白在其中的应用前景 ［J］.食品工业科技，2024，45（8）：366-371.YOU X P，CHEN Z X.Nutrients，functions and application prospects of yeast protein in sports nutrition foods［J］.Science and Technology of Food Industry，2024，45（8）：366-371.（in Chinese ）
［12］ 秦于思，程明，陈平华，等.基于乳源性蛋白制备高 F值寡肽的研究进展 ［J］.乳业科学与技术，2020，43（6）：31-35.QIN Y S，CHENG M，CHEN P H，et al.Recent progress in preparation of oligopeptides with high F value from milk proteins ［J］.Journal of Dairy Science and Technology，2020，43（6）：31-35.（in Chinese ）
［13］ 韩昊天，李晓彤，吴澎，等.花生多肽研究进展 ［J］.中国粮油学报，2019，34（10）：140-146.HAN H T，LI X T，WU P，et al.Research progress of peanut peptide ［J］.Journal of the Chinese Cereals and Oils Association，2019，34（10）：140-146.（in Chinese ）
［14］ 周德庆，李娜，王珊珊，等.水产加工副产物源抗氧化肽的 研 究 现 状 与 展 望 ［J］.水 产 学 报，2019，43（1）：188-196.ZHOU D Q，LI N，WANG S S，et al.Research progress of antioxidant peptides from aquatic by-product ［J］.Journal of Fisheries of China，2019，43（1）：188-196.（in Chinese ）
［15］ JIANG X Y，DING H Y，LIU Q，et al.Effects of peanut meal extracts fermented by Bacillus natto on the growth performance，learning and memory skills and gut microbiota modulation in mice ［J］.British Journal of Nutrition，2020，123（4）：383-393.
［16］ ROHIMAH A，SETIAWAN B，PALUPI E，et al.Comparison of peanut and black oncom biscuit：nutritional characteristics and aflatoxin evaluation with the potential health benefits ［J］.Annals of Agricultural Sciences，2021，66（1）：87-92.
［17］ 杨华青，侯威，赵磊，等.玉米黄粉二步酶解制备高 F值寡肽的工艺优化 ［J］.食品科学技术学报，2019，37（6）：100-107.YANG H Q，HOU W，ZHAO L，et al.Optimization on preparation of high Fischer ratio oligopeptides by two-step enzymolysis from corn yellow powder ［J］.Journal of Food Science and Technology，2019，37（6）：100-107.（in Chinese）
［18］ ZHEN H M，LIU J L，XIONG K，et al.Engineering a carboxypeptidase from Aspergillus oryzae M30011 to improve the terminal-specific enzymatic hydrolysis of aromatic amino acids ［J］.Process Biochemistry，2023，126：186-199.
［19］ SHARIF M K，BUTT M S，ANJUM F M，et al.Rice bran：a novel functional ingredient ［J］.Critical Reviews in Food Science and Nutrition，2014，54（6）：807-816.
［20］ MUGISHA S，杨套伟，徐美娟，等.定点突变提高过氧化氢酶热稳定性和催化效率 ［J］.食品与生物技术学报，2020，39（3）：104-111.SAMSON M，YANG T W，XU M J，et al.Enhanced thermo-stability and catalytic efficiency of catalase by site-directed mutagenesis ［J］.Journal of Food Science and Biotechnology，2020，39（3）：104-111.（in Chinese ）
［21］ ROSANO G L，MORALES E S，CECCARELLI E A.New tools for recombinant protein production in Escherichia coli：a 5-year update ［J］.Protein Science，2019，28（8）：1412 -1422.
［22］ 刘丽琴，陈婷婷，李少伟，等.大肠杆菌表达系统在基因工程疫苗研发中的应用与策略优化 ［J］.中国新药杂志，2020，29（21）：2434 -2442.LIU L Q，CHEN T T，LI S W，et al.Escherichia coli expression system applied in the development of recombinant human vaccines and its potential improvement ［J］.Chinese Journal of New Drugs，2020，29（21）：2434 -2442.（in Chinese ）
［23］ AFSHARI O，SALEHI Z.β-Cyclodextrin and APTES functionalized magnetic iron oxide nanoparticles for improved lipase immobilization，transesterification efficiency，and reusability ［J］.Journal of Industrial and Engineering Chemistry，2025，144：476-486.
［24］ TIAN P J，YANG M X，MUHMOOD A，et al.In situ immobilization multi-enzyme biocatalytic system on covalent organic frameworks for efficient conversion of lignocellulose to glucose ［J］.Environmental Technology & Innovation，2024，36：103796.
［25］ AL-HARBI S A，ALMULAIKY Y Q.Cu-trimesic acid-based metal organic frameworks for improved β-galactosidase immobilization：enhanced stability，reusability and lactose hydrolysis ［J］.Journal of Molecular Liquids，2023，392：123456.［26］ 陶静，丁冬各，陈荫，等.带鱼高 F值寡肽的制备工艺及活性［J］.水产学报，2018，42（10）：1648 -1660.TAO J，DING D G，CHEN Y，et al.Preparation and bioactivity evaluation of high Fischer ratio oligo-peptide from Trichiurus japonicus ［J］.Journal of Fisheries of China，2018，42（10）：1648 -1660.（in Chinese ）
［27］ 王秋萍，余新威，童晓倩，等.固定化酶水解鱿鱼碎肉制备高 F值寡肽的工艺优化 ［J］.中国食品学报，2017，17（3）：113-119.WANG Q P，YU X W，TONG X Q，et al.Process optimization of high Fischer ratio oligo-peptide from hydrolysates of squid minced meat by immobilized enzyme［J］.Journal of Chinese Institute of Food Science and Technology，2017，17（3）：113-119.（in Chinese ）
［28］ YANDRI Y，SUHARTATI T，SATRIA H，et al.Increasing stability of α-amylase obtained from Bacillus subtilis ITBCCB 148 by immobilization with chitosan ［J］.Mediterranean Journal of Chemistry，2020，10（2）：155-161.
［29］ 张华伟，樊帅，杨兆勇，等.来源于 Anoxybacillus sp.SK3-4的普鲁兰酶 PulASK 固定化及酶学性质研究 ［J］.食品与生物技术学报，2023，42（4）：24-30.ZHANGH W，FANS，YANGZ Y，et al.Immobilization of pullulanase PulASK from Anoxybacillus sp.SK3-4 and its enzymatic properties ［J］.Journal of Food Science and Biotechnology，2023，42（4）：24-30.（in Chinese ）
［30］ SAHIN B，OZBEY-UNAL B，DIZGE N，et al.Optimization of immobilized urease enzyme on porous polymer for enhancing the stability，reusability and enzymatic kinetics using response surface methodology ［J］.Colloids and Surfaces B：Biointerfaces，2024，240：113986.