Food and Fermentation Industries >

2024 , Vol. 50 >Issue 8: 161 - 168

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.038179

Preparation and enzymatic properties of papain immobilized by nano-magnetic spheres

  • LIANG Jie ,
  • LIN Guorong ,
  • ZHOU Fengchao ,
  • YANG Lei ,
  • ZOU Hanxun ,
  • LIU Tao ,
  • ZHANG Li ,
  • GUO Tianzhang ,
  • CAI Lifeng
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  • 1(College of Environmental and Biological Engineering, Putian University, Putian 351100, China)
    2(Key Laboratory of Ecology-toxicological Effects & Rubber Research Institute, Putian 351100, China)
    3(Key Laboratory of Ecological Environment and Information Atlas, Fujian Provincial University, Putian 351100, China)
    4(Fujian Province Sailor Foods Co.Ltd., Putian 351100, China)

Received date: 2023-12-06

  Revised date: 2024-01-22

  Online published: 2024-06-11

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Abstract

To realize the immobilization of papain (PAP), core-shell magnetic nanoparticles Fe3O4@PMMA were prepared by uniform coating of polymethylmethacrylate (PMMA) on the surface by suspension polymerization.Furthermore, the surface of PMMA microsphere was deoxidized to form carboxyl group, following by grafted on Polyethyleneimine (PEI).To construct a hydrophilic three-dimensional spatial structure Fe3O4@PMMA-PEI 1800 with magnetic response and amino rich surface, the PAP was physically absorbed on PEI-1800 chain and was crosslinked by glutaraldehyde (GA).The PAP was then encapsulated and fixed on the surface of the nano-magnetic ball.The morphology and physicochemical properties of the carrier were analyzed, and the enzymatic properties of the immobilized enzyme Fe3O4@PMMA-PEI 1800-PAP were evaluated.Results indicated that the specific surface area of Fe3O4@PMMA-PEI 1800 was 151.30 m2/g and the pore volume was 0.27 cm3/g.The optimum temperature of Fe3O4@PMMA-PEI 1800-PAP was 60 ℃, with an optimum pH at 6.0, the relative enzyme activity above 80% was maintained in the range of 40~80 ℃, and it can be maintained above 66.84% at 90 ℃.Fe3O4@PMMA-PEI 1800-PAP showed good thermal stability and 80% and 36.63% enzyme activity was retained after incubation at 60 ℃ and 90 ℃ for 2 h, respectively.The relative enzyme activity of Fe3O4@PMMA-PEI 1800-PAP was maintain over 80% during the pH range from 5.0 to 9.0.After 7 times of reuse and elution, the activity of Fe3O4@PMMA-PEI 1800-PAP enzyme remained above 70%.

Key words： magnetic nanospheres; immobilization; papain(PAP); grafting; polyethyleneimide(PEI); crosslinked; enzymatic propertities

Cite this article

LIANG Jie , LIN Guorong , ZHOU Fengchao , YANG Lei , ZOU Hanxun , LIU Tao , ZHANG Li , GUO Tianzhang , CAI Lifeng . Preparation and enzymatic properties of papain immobilized by nano-magnetic spheres[J]. Food and Fermentation Industries, 2024 , 50(8) : 161 -168 . DOI: 10.13995/j.cnki.11-1802/ts.038179

References

[1] 朱恒伟. 多级双水相萃取木瓜蛋白酶的研究[D].海口:海南大学, 2023.
ZHU H W.Study on multi-stage aqueous two phase extraction of papain[D].Haikou:Hainan University, 2023.
[2] 孙树远, 陈琪, 何静, 等.木瓜蛋白酶与超高压技术嫩化驼肉的研究[J].中国食品学报, 2023, 23(4):239-250.
SUN S Y, CHEN Q, HE J, et al.Tenderization and comparison of camel meat by papain and ultra-high pressure technology[J].Journal of Chinese Institute of Food Science and Technology, 2023, 23(4):239-250.
[3] BOTINESTEAN C, HOSSAIN M, MULLEN A M, et al.The influence of the interaction of sous-vide cooking time and papain concentration on tenderness and technological characteristics of meat products[J].Meat Science, 2021, 177:108491.
[4] TACIAS-PASCACIO V G, CASTAÑEDA-VALBUENA D, MORELLON-STERLING R, et al.Bioactive peptides from fisheries residues:A review of use of papain in proteolysis reactions[J].International Journal of Biological Macromolecules, 2021, 184:415-428.
[5] 陈振斌, 周永山, 张霞云.Fe3O4磁性纳米颗粒的RAFT试剂化研究[J].兰州理工大学学报, 2020, 46(5):27-33.
CHEN Z B, ZHOU Y S, ZHANG X Y.Functionalization of Fe3O4 magnetic nanoparticles using RAFT reagent[J].Journal of Lanzhou University of Technology, 2020, 46(5):27-33.
[6] HAN C R, ZHAI W L, WANG Y L,et al.A SERS aptasensor for rapiddetection of aflatoxin B1 in coix seed usingsatellite structured Fe3O4@Au nanocomposites[J].Food Control, 2022, 142:109228.
[7] QIU X, WANG S S, MIAO S S, et al.Co-immobilization of laccase and ABTS onto amino-functionalized ionic liquid-modified magnetic chitosan nanoparticles for pollutants removal[J].Journal of Hazardous Materials, 2021, 401:123353.
[8] 毛梦雷, 孟令玎, 高蕊, 等.多孔框架材料固定化酶研究进展[J].化工进展, 2023, 42(5):2516-2535.
MAO M L, MENG L D, GAO R, et al.Research progress on enzyme immobilization on porous framework materials[J].Chemical Industry and Engineering Progress, 2023, 42(5):2516-2535.
[9] FU M H, XING J F, GE Z Q.Preparation of laccase-loaded magnetic nanoflowers and their recycling for efficient degradation of bisphenol A[J].The Science of the Total Environment, 2019, 651:2857-2865.
[10] 赵伟, 戴培邦, 田磊.水相沉淀聚合法制备聚甲基丙烯酸甲酯的研究[J].塑料工业, 2022, 50(S1):44-48.
ZHAO W, DAI P, TIAN L.Study on Preparation of Poly(methylmethacrylate) by Aqueous Precipitation Polymerization[J].China Plastics Industry, 2022, 50(S1):44-48.
[11] 肖鹭, 周锦涛, 刘振, 等.固定化纳米酶的研究进展[J].食品科学, 2023, 44(11):205-213.
XIAO L, ZHOU J, LIU Z, et al.Research Progress on Immobilized Nanoenzymes[J].Food Science, 2023, 44(11):205-213.
[12] 陈孝鹏, 沈炜, 王亚军.固定化细胞催化合成6-氰基-(3R,5R)-二羟基乙酸叔丁酯工艺研究[J].化学反应工程与工艺, 2017, 33(3):236-242.
CHEN X P, SHEN W, WANG Y J J.Biocatalytic process of t-Butyl 6-cyano-(3R,5R)-dihydroxylhexanoate by immobilized cells[J].Chemical Reaction Engineering and Technology, 2017, 33(3):236-242.
[13] ZAAK H, KORNECKI J F, SIAR E H, et al.Coimmobilization of enzymes in bilayers using PEI as a glue to reuse the most stable enzyme:Preventing PEI release during inactivated enzyme desorption[J].Process Biochemistry, 2017, 61:95-101.
[14] VIRGEN-ORTÍZ J J, DOS SANTOS J C S, BERENGUER-MURCIA Á B, et al.Polyethylenimine:A very useful ionic polymer in the design of immobilized enzyme biocatalysts[J].Journal of Materials Chemistry B, 2017, 5(36):7461-7749.
[15] 肖云燕. PVAC磁性微球表面构建分子笼封装固定化酶及应用. 保定: 河北大学, 2022.
XIAO Y Y. Construction of molecular cage on the surface of PVAC magnetic microspheres to encapsulate immobilized enzyme and its application.Baoding: Hebei University, 2022.
[16] 夏玉佩. 磁性微球表面交联封装固定化酶及其催化性能研究. 保定: 河北大学, 2019.
XIA Y P. Study on immobilized enzyme encapsulated by cross-linking on the surface of magnetic microspheres and its catalytic performance.Baoding: Hebei University, 2019.
[17] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry, 1976, 72(1):248-254.
[18] 高进. 木瓜蛋白酶加工过程中的性质及性质研究[D].天津:天津科技大学, 2018.
GAO J.Study on the properties and structure of papain in processing[D].Tianjin:Tianjin University of Science and Technology, 2018.
[19] 李有花. 木瓜蛋白酶的定向固定化研究[D].无锡:江南大学, 2021:37-46.
LI Y H.Study on the directional immobilization of papain[D].Wuxi:Jiangnan University, 2021.
[20] 王秀奇, 秦淑媛, 高天慧, 等.基础生物化学实验(第二版)[M].北京:高等教育出版社, 2004:200-230.
WANG X Q, QIN S Y, GAO T H, et al.Basic biochemistry experiments(second edition) [M].Beijing:Higher Education Press, 2004:200-230.
[21] 李群艳, 孙路瑶, 韩寿德, 等.分级介孔SiO2空心微球氨基修饰及木瓜蛋白酶固定化[J].高校化学工程学报, 2022, 36(5):708-714.
LI Y Q, SUN L Y, HAN D S, et al.Amino modification of hierarchical mesoporous SiO2 hollow microspheres and their application in papain immobilization[J].Journal of Chemical Engineering of Chinese Universities, 2022, 36(5):708-714.
[22] LEE H R, CHUNG M, KIM M I, et al.Preparation of glutaraldehydetreated lipase-inorgnaic hybrid nanoflowers and their catalytic performance as immobilized enzymes[J] Enzyme and Microbial Technology, 2017,105:24-29.
[23] 江维婷, 李翠苹, 姜晓辉, 等.Fe/Fe3C4/Fe3O4@C磁性微球的制备及吸波性能研究[J].材料工程, 2022, 50(11): 81-91.
JIANG W T, LI C P, JIANG X H.Preparation and absorbing properties of Fe/Fe3C4/Fe3O4@C magnetic microspheres[J].Journal of Materials Engineering, 2022, 50(11): 81-91.
[24] 桑冀蒙, 李学平, 赵瑾, 等.P(AA-co-MPC)修饰超顺磁性纳米粒子的制备与表征[J].材料工程, 2019, 47(8):82-89.
SANG J M, LI X P, ZHAO J.Preparation and characterization of superaramagnetic Fe3O4nanoparticles modified by P(AA-co-MPC)[J].Journal of Materials Engineering, 2019, 47(8):82-89.
[25] 王志琰, 毋伟, 张魁, 等.Pickering乳液法原位制备载药磁性SiO2空心球及缓释性能[J].北京化工大学学报(自然科学版), 2010, 37(3):110-114.
WANG Z Y, WU W, ZHANG K, et al.In situ preparation and characterizations of drug-loaded magnetic hollow silica microspheres by a pickering emulsion based route[J].Journal of Beijing University of Chemical Technology(Natural Science Edition), 2010, 37(3):110-114.
[26] DING Q, LIU D F, GUO D W, et al.Shape-controlled fabrication of magnetite silver hybrid nanoparticles with high performance magnetic hyperthermia[J].Biomaterials, 2017, 124(04):35-46.
[27] 高丰琴, 刘洋, 张引莉, 等.羧基功能化Fe3O4固定化酶反应器的构筑及性能研究[J].化学学报, 2023, 81(4):338-344.
GAO F Q, LIU Y, ZHANG Y L, et al.Study on construction and performance of immobilized enzyme reactors by carboxyl-functionalized Fe3O4[J].Acta Chimica Sinica, 2023, 81(4):338-344.
[28] 李玲慧. Fe3O4@TiO2磁性纳米粒子的制备及吸附重金属性能研究[D].北京:北京化工大学, 2017.
LI L L.Preparation of Fe3O4@TiO2magnetic nanoparticles and study on the adsorption of heavy metals[D].Beijing:Beijing University of Chemical Technology, 2017.
[29] BAO S Y, TANG L H, LI K, et al.Highly selective removal of Zn(II) ion from hot-dipgalvanizing pickling waste with amino-functionalized Fe3O4@SiO2 magnetic nano-adsorbent[J].Journal of colloid and interface science, 2016, 462:235-242.
[30] WANG L L, LOU Z, DENG J N, et al.Ethanol gas detection using a yolk-shell(core-shell) α- Fe2O3nanospheres as sensing material[J].ACS Applied Materials & Interfaces, 2015, 7(23):13098-13104.
[31] 苏二正, 李明亮, 魏东芝.木瓜蛋白酶的原位固定化及理化性质研究[J].南京林业大学学报(自然科学版), 2014, 38(6):99-104.
SU E Z, LI M L, WEI D Z.Study on in situ immobilization and characterization of papain[J].Journal of Nanjing Forestry University(Natural Sciences Edition), 2014, 38(6):99-104.
[32] 瞿叶辉. 罗非鱼副产物酶解工艺优化及壳聚糖埃洛石微球固定化木瓜蛋白酶的研究. 广州: 广东工业大学, 2016.
QU Y H. Optimization of enzymatic hydrolysis of tilapia byproducts and study on immobilization of papain on chitosan halloysite microspheres.Guangzhou: Guangdong University of Technology, 2016.
[33] 胡艺凡, 李正军, 刘长霞.基于磁性Fe3O4粒子的固定化过氧化氢酶的制备及催化性质研究[J].北京化工大学学报(自然科学版),2023, 50(2):53-62.
HU Y F, LI Z J, LIU C X.Preparation and catalytic properties of catalase immobilized on magnetic Fe3O4particles[J].Journal of Beijing University of Chemical Technology(Natural Science Edition), 2023, 50(2):53-62.
[34] MOSAFA L, MOGHADAM M, SHAHEDI M.Papain enzyme supported on magnetic nanoparticles:Preparation, characterization and application in the fruit juice clarification[J].Chinese Journal of Catalysis, 2013,34:1897-1904.
[35] 汤燕明, 孙丽霞, 郑燕宁, 等.氮掺杂多孔炭材料固定化木瓜蛋白酶研究[J].食品科技, 2019,44(8):261-267.
TANG Y M, SUN L X, ZHENG Y N, et al.Study on immobilized papain by N-drops porous carbon material[J].Food Science and Technology, 2019, 44(8):261-267.
[36] 丁利君, 瞿叶辉, 刘丹.壳聚糖埃洛石纳米管微球的制备及其对木瓜蛋白酶的固定化[J].食品科学, 2017, 38(10):123-129.
DING L J, QU Y H, LIU D.Preparation of chitosan-halloysites nanotube microspheres and their application in immobilization of Papain[J].Food Science, 2017,38(10):123-129.
[37] 韦美苹, 庞玉凤, 陈正毅, 等.磁性可再生性固定化酶制备及其在微波辅助蛋白酶解中的应用[J].桂林理工大学学报, 2014, 34(4):748-754.
WEI M P, PANG Y F, CHEN Z Y.Preparation of regenerated immobilized enzyme reactor and application in microwave assisted digestion[J].Journal of Guilin University of Technology, 2014, 34(4):748-754.
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