Directional Immobilization of Mutant β-Agarase Aga 50D onto Magnetic Nanoparticles and Its Thermal Stability

Authors

Qiaoling XIE, School of Food Science and Technology , Jiangnan University , Wuxi 214122 , China
Jie LONG, School of Food Science and Technology , Jiangnan University , Wuxi 214122 , China
Long CHEN, School of Food Science and Technology , Jiangnan University , Wuxi 214122 , China
Chao QIU, School of Food Science and Technology , Jiangnan University , Wuxi 214122 , China
Xing ZHOU, School of Food Science and Technology , Jiangnan University , Wuxi 214122 , China
Zhengyu JIN, School of Food Science and Technology , Jiangnan University , Wuxi 214122 , China ;State Key Laboratory of Food Science and Resources , Jiangnan University , Wuxi 214122 , ChinaFollow

Corresponding Author(s)

金征宇（1960—），男，博士，教授，博士研究生导师，主要从事食品科学与工程领域碳水化合物资源开发与利用研究。E-mail：fpcenter@jiangnan.edu.cn

Abstract

[Objective ] This study aims to achieve site-directed immobilization of β-agarase at targeted residues,thereby enhancing its thermal stability and operational performance.[Method ] Computer-aided design was employed to selectively immobilize the mutant β-agarase Aga 50D via sulfhydryl groups introduced at specific cysteine residues,enabling binding to the carrier.[Result ] The immobilization conditions were optimized as follows:reaction duration of 12 h,an enzyme loading level (enzyme-to-carrier mass ratio ) of 1∶50,and a crosslinker-to-carrier mass ratio of 10∶50.The immobilization efficiency ranged from 40% to 75%,with most mutants retaining the relative enzyme activity > 80%.Compared with the wild-type free enzyme,the site-directed immobilized mutant β-agarase Aga 50D exhibited superior thermal stability at 40 ℃ and 45 ℃.After four reuse cycles,the enzyme activity retention rate of site-directed immobilized mutant β-agarase Aga 50D remained above 30%.Notably,while the wild-type free enzyme completely lost its activity in organic reagents,the enzyme activity retention rate of most site-directed immobilized mutant β-agarase Aga50D remained above 70%.[Conclusion ] The site-directed immobilized mutant β-agarase Aga 50D demonstrated high enzyme activity and enhanced thermal/operational stability,showing significant promise for industrial applications.

Publication Date

10-15-2025

First Page

28

Last Page

38

DOI

10.12441/spyswjs.20230410001

Recommended Citation

XIE, Qiaoling; LONG, Jie; CHEN, Long; QIU, Chao; ZHOU, Xing; and JIN, Zhengyu (2025) "Directional Immobilization of Mutant β-Agarase Aga 50D onto Magnetic Nanoparticles and Its Thermal Stability," Journal of Food Science and Biotechnology: Vol. 44: Iss. 10, Article 4.
DOI: 10.12441/spyswjs.20230410001
Available at: https://spsw.spyswjs.cnjournals.com/journal/vol44/iss10/4

References

［1］ SELVAM K，GOVARTHANAN M，SENBAGAM D，et al.Activity and stability of bacterial cellulase immobilized on magnetic nanoparticles ［J］.Chinese Journal of Catalysis，2016，37（11）：1891 -1898.［2］ CHEN X Y，LI H J，QIAO X N，et al.Agarose oligosaccharide- silver nanoparticle- antimicrobial peptide- composite for wound dressing ［J］.Carbohydrate Polymers，2021，269：118258.
［3］ HONG S J，LEE J H，KIM E J，et al.Toxicological evaluation of neoagarooligosaccharides prepared by enzymatic hydrolysis of agar ［J］.Regulatory Toxicology and Pharmacology，2017，90：9-21.
［4］ LEE D G，JANG M K，LEE O H，et al.Over-production of a glycoside hydrolase family 50 β-agarase from Agarivorans sp.JA-1 in Bacillus subtilis and the whitening effect of its product ［J］.Biotechnology Letters，2008，30（5）：911-918.
［5］ ENOKI T，OKUDA S，KUDO Y，et al.Oligosaccharides from agar inhibit pro-inflammatory mediator release by inducing heme oxygenase 1［J］.Bioscience，Biotechnology，and Biochemistry，2010，74（4）：766-770.
［6］ MA C，YANG K，WANG Y F，et al.Anti-aging effect of agar oligosaccharide on male Drosophila melanogaster and its preliminary mechanism ［J］.Marine Drugs，2019，17（11）：632.
［7］ LEE M R，KIM J E，JIN Y J，et al.Anti-obesity effects of agar （Gelidium amansii ）-derived oligosaccharides in high-fat diet-treated C 57BL/6N mice due to differential regulations of lipogenesis and lipolysis ［J］.Bioscience，Biotechnology，and Biochemistry，2022，86（12）：1648 -1657.
［8］ XU S Y，KAN J，HU Z，et al.Quantification of neoagaro-oligosaccharide production through enzymatic hydrolysis and its anti-oxidant activities ［J］.Molecules，2018，23（6）：1354.
［9］ NGUYEN H H，LEE S H，LEE U J，et al.Immobilized enzymes in biosensor applications ［J］.Materials，2019，12（1）：121.
［10］ LI X X，LI D M，WANG W F，et al.Immobilization of SMG 1-F278N lipase onto a novel epoxy resin：characterization and its application in synthesis of partialglycerides ［J］.Journal of Molecular Catalysis B：Enzymatic，2016，133：154-160.
［11］ LIBURDI K，BENUCCI I，PALUMBO F，et al.Lysozyme immobilized on chitosan beads：kinetic characterization and antimicrobial activity in white wines［J］.Food Control，2016，63：46-52.
［12］ BASSO A，SERBAN S.Industrial applications of immobilized enzymes：a review［J］.Molecular Catalysis，2019，479：110607.
［13］ 聂铭甫，李由然，石贵阳.基于磁性壳聚糖复合材料对耐高温酶的固定化研究 ［J］.中国生物工程杂志，2023，43（Supp l.1）：83-94.NIE M F，LI Y R，SHI G Y.Immobilization of high temperature resistant enzymes based on magnetic chitosan composites ［J］.China Biotechnology，2023，43（Suppl.1）：83-94.（in Chinese ）
［14］ KANBAR B，OZDEMIR E.Thermal stability of carbonic anhydrase immobilized within polyurethane foam ［J］.Biotechnology Progress，2010，26（5）：1474 -1480.
［15］ 祖昕，杜凯，曹方，等.有机溶剂处理提高固定化脂肪酶活性及稳定性 ［J］.食品与生物技术学报，2016，35（1）：89-94.ZU X，DU K，CAO F，et al.Treatment of organic solvents to improve activity and stability of immobilized lipases［J］.Journal of Food Science and Biotechnology，2016，35（1）：89-94.（in Chinese ）
［16］ 汝成业，张娟，堵国成，等.包埋固定化酶降解牛奶αS1-酪蛋白过敏性研究 ［J］.食品与生物技术学报，2016，35（4）：387-392.RU C Y，ZHANG J，DU G C，et al.Allergenicity investigation of hydrolyzed αS1-casein in bovine milk by immobilized protease ［J］.Journal of Food Science and Biotechnology，2016，35（4）：387-392.（in Chinese ）
［17］ 张宇军，曹慧，徐斐，等.枯草芽孢杆菌中性蛋白酶的固定化研究 ［J］.食品与生物技术学报，2019，38（10）：98-104.ZHANG Y J，CAO H，XU F，et al.Studies on immobilization of neutral protease from Bacillus subtilis［J］.Journal of Food Science and Biotechnology，2019，38（10）：98-104.（in Chinese ）
［18］ 张玉然，辛瑜，杨海麟，等.黄嘌呤氧化酶酶学性质及共价交联固定化［J］.食品与生物技术学报，2014，33（1）：16-21.ZHANG Y R，XIN Y，YANG H L，et al.Enzymatic properties of xanthine oxidase and its covalent immobilization on different supports ［J］.Journal of Food Science and Biotechnology，2014，33（1）：16-21.（in Chinese ）
［19］ FARMAKES J，SCHUSTER I，OVERBY A，et al.Enzyme immobilization on graphite oxide （GO） surface via one-pot synthesis of GO/metal-organic framework composites for large-substrate biocatalysis ［J］.ACS Applied Materials & Interfaces，2020，12（20）：23119 -23126.
［20］ PAGOLU R，SINGH R，SHANMUGAM R，et al.Site-directed lysine modification of xylanase for oriented immobilization onto silicon dioxide nanoparticles ［J］.Bioresource Technology，2021，331：125063.
［21］ 李海新，梁家铭，李蓉，等.琼胶寡糖的抗氧化活性研究［J］.广州化工，2018，46（20）：66-68.LI H X，LIANG J M，LI R，et al.Study on antioxidant activity of agar-oligosaccharide ［J］.Guangzhou Chemical Industry，2018，46（20）：66-68.（in Chinese ）
［22］ 陈亮，张萍波，蒋平平，等.基于二硫键的自修复水性聚氨酯的制备与性能研究 ［J］.中国塑料，2022，36（6）：46-53.CHEN L，ZHANG P B，JIANG P P，et al.Preparation and properties of self-healing waterborne polyurethane based on disulfide bonds ［J］.China Plastics，2022，36（6）：46-53.（in Chinese ）
［23］ MATEO C，PALOMO J M，FERNANDEZ-LORENTE G，et al.Improvement of enzyme activity，stability and selectivity via immobilization techniques ［J］.Enzyme and Microbial Technology，2007，40（6）：1451 -1463.