Lipase is an enzyme with diverse functions and broad applications. However, due to the easy loss of enzyme activity and difficulty of being separated and recovered, free lipase is usually not suitable for practical application. In order to improve the poor stability and reusability of free lipase in application, in this study, Candida parapsilosis was first used to produce lipase. Then, polyamidoamine dendrimers grafted magnetic poly(methyl methacrylate) microspheres [Fe3O4@poly(methyl methacrylate)/polyamidoamine,Fe3O4@PMMA/PAMAM] were synthesized and characterized in various ways. Subsequently, Fe3O4@PMMA/PAMAM was used as the support for lipase immobilization. The optimal immobilization conditions were determined as follows: 0.6 mL glutaraldehyde, 5 h, pH 8.0, and 35 ℃. Under these conditions, the immobilized lipase exhibited an activity of 864 U/g and the activity recovery of immobilized lipase was 74.29%. Compared with the free lipase in fermentation broth, the thermal stability and pH stability of immobilized lipase were enhanced significantly. After repeated use for 10 times, immobilized lipase still maintained 72.23% of its initial activity. After 30 d of storage at 4 ℃, the immobilized lipase still retained 71.44% of its initial activity. It was indicated that Fe3O4@PMMA/PAMAM immobilized lipase had good enzyme activity, stability and reusability.
[1] CHEN J, SUN B Z, SUN C R, et al.Immobilization of lipase AYS on UiO-66-NH2 metal-organic framework nanoparticles as a recyclable biocatalyst for ester hydrolysis and kinetic resolution[J].Separation and Purification Technology, 2020, 251:117398.
[2] YUAN X, OU J, ZHANG P L, et al.PEG-modified lipase immobilized onto NH2-MIL-53 MOF for efficient resolution of 4-fluoromandelic acid enantiomers[J].International Journal of Biological Macromolecules, 2020, 165:1793-1802.
[3] KHOZEYMEH NEZHAD M, AGHAEI H.Tosylated cloisite as a new heterofunctional carrier for covalent immobilization of lipase and its utilization for production of biodiesel from waste frying oil[J].Renewable Energy, 2021, 164:876-888.
[4] MAHMOUD M E, EL-SHARKAWY R M, IBRAHIM G A A.A novel bionanocomposite from doped lipase enzyme into magnetic graphene oxide-immobilized-cellulose for efficient removal of methylene blue and malachite green dyes[J].Journal of Molecular Liquids, 2022, 368(15):120676.
[5] GHIDE M K, LI K, WANG J H, et al.Immobilization of Rhizomucor miehei lipase on magnetic multiwalled carbon nanotubes towards the synthesis of structured lipids rich in Sn-2 palmitic acid and Sn-1, 3 oleic acid (OPO) for infant formula use[J].Food Chemistry, 2022, 390:133171.
[6] CAO X Y, XU H, LI F S, et al.One-step direct transesterification of wet yeast for biodiesel production catalyzed by magnetic nanoparticle-immobilized lipase[J].Renewable Energy, 2021, 171:11-21.
[7] HERNÁNDEZ-CORROTO E, SÁNCHEZ-MILLA M, SÁNCHEZ-NIEVES J, et al.Immobilization of thermolysin enzyme on dendronized silica supports.Evaluation of its feasibility on multiple protein hydrolysis cycles[J].International Journal of Biological Macromolecules, 2020, 165:2338-2348.
[8] SOOZANIPOUR A, TAHERI-KAFRANI A, RAZMJOU A, et al.Hyaluronidase enzyme conjugated polyamidoamine dendrimer:An efficient and stable nanobiocatalyst for enzymatic degradation of hyaluronic acid[J].Journal of Molecular Liquids, 2022, 349:118111.
[9] PEIMAN S, BAHARFAR R, MALEKI B.Immobilization of trypsin onto polyamidoamine dendrimer functionalized iron oxide nanoparticles and its catalytic behavior towards spirooxindole-pyran derivatives in aqueous media[J].Materials Today Communications, 2021, 26:101759.
[10] 马红丽, 付晓平, 郑雯, 等.费希尔曲霉脂肪酶在毕赤酵母中的优化表达及高密度发酵[J].微生物学通报, 2020, 47(7):2140-2150.
MA H L, FU X P, ZHENG W, et al.Optimized expression and high-density fermentation of Aspergillus fischeri lipase in Pichia pastoris[J].Microbiology China, 2020, 47(7):2140-2150.
[11] 高大伟, 宋晓蕾, 邹鑫, 等.洗毛用头状丝孢酵母产脂肪酶发酵条件研究[J].盐城工学院学报(自然科学版), 2014, 27(1):1-4;13.
GAO D W, SONG X L, ZOU X, et al.Optimization of protease production fermentation conditions by Trichosporon capitatum[J].Journal of Yancheng Institute of Technology (Natural Science Edition), 2014, 27(1):1-4;13.
[12] 李晓雪. 产脂肪酶耐冷菌的分离及其对油脂降解吸附的条件优化[D].哈尔滨:哈尔滨工业大学, 2020.
LI X X.Isolation of lipase-producing psychrotrophs and optimization of conditions for adsorption of oil degradation[D].Harbin:Harbin Institute of Technology, 2020.
[13] 杨宇童. 产脂肪酶菌株的分离鉴定及其产酶条件的优化[D].广州:华南农业大学, 2018.
YANG Y T.Screening and identification of lipase producing bacteria and optimization of enzyme producing conditions[D].Guangzhou:South China Agricultural University, 2018.
[14] 马志亚. 磁性分离载体的制备、表面功能化修饰及蛋白质分离纯化应用研究[D].北京:中国科学院大学, 2005.
MA Z Y.Study on preparation, surface functional modification and protein separation and purification of magnetic separation carrier[D].Beijing:University of Chinese Academy of Sciences, 2005.
[15] LINSHA V, ABOO SHUHAILATH K A, MAHESH K V, et al.Biocatalytic conversion efficiency of steapsin lipase immobilized on hierarchically porous biomorphic aerogel supports[J].ACS Sustainable Chemistry & Engineering, 2016, 4(9):4692-4703.
[16] ZHAO P P, TIAN L, LI X E, et al.Effect of the structure and length of flexible chains on dendrimers grafted Fe3O4@SiO2/PAMAM magnetic nanocarriers for lipase immobilization[J].ACS Sustainable Chemistry & Engineering, 2016, 4(12):6382-6390.
[17] WANG J H, LI K, HE Y J, et al.Lipase immobilized on a novel rigid-flexible dendrimer-grafted hierarchically porous magnetic microspheres for effective resolution of (R, S)-1-phenylethanol[J].ACS Applied Materials & Interfaces, 2020, 12(4):4906-4916.
[18] SUN X T, YANG L R, XING H F, et al.Synthesis of polyethylenimine-functionalized poly(glycidyl methacrylate) magnetic microspheres and their excellent Cr(VI) ion removal properties[J].Chemical Engineering Journal, 2013, 234:338-345.
[19] SUN X T, YANG L R, LI Q, et al.Amino-functionalized magnetic cellulose nanocomposite as adsorbent for removal of Cr(VI):Synthesis and adsorption studies[J].Chemical Engineering Journal, 2014, 241:175-183.
[20] SUN X T, YANG L R, LI Q, et al.Polyethylenimine-functionalized poly(vinyl alcohol) magnetic microspheres as a novel adsorbent for rapid removal of Cr(VI) from aqueous solution[J].Chemical Engineering Journal, 2015, 262:101-108.
[21] ABDULMALEK S A, LI K, WANG J H, et al.Enhanced performance of Rhizopus oryzae lipase immobilized onto a hybrid-nanocomposite matrix and its application for biodiesel production under the assistance of ultrasonic technique[J].Fuel Processing Technology, 2022, 232:107274.
[22] CHEN X L, XUE S, LIN Y L, et al.Immobilization of porcine pancreatic lipase onto a metal-organic framework, PPL@MOF:A new platform for efficient ligand discovery from natural herbs[J].Analytica Chimica Acta, 2020, 1099:94-102.
[23] GUO H, LEI B S, YU J W, et al.Immobilization of lipase by dialdehyde cellulose crosslinked magnetic nanoparticles[J].International Journal of Biological Macromolecules, 2021, 185:287-296.
[24] SONI S, DWIVEDEE B P, BANERJEE U C.Tailoring a stable and recyclable nanobiocatalyst by immobilization of surfactant treated Burkholderia cepacia lipase on polyaniline nanofibers for biocatalytic application[J].International Journal of Biological Macromolecules, 2020, 161:573-586.
[25] PARANDI E, SAFARIPOUR M, ABDELLATTIF M H, et al.Biodiesel production from waste cooking oil using a novel biocatalyst of lipase enzyme immobilized magnetic nanocomposite[J].Fuel, 2022, 313:123057.
[26] BILGIN SIMSEK E, SALOGLU D.Exploring the structural and catalytic features of lipase enzymes immobilized on g-C3N4:A novel platform for biocatalytic and photocatalytic reactions[J].Journal of Molecular Liquids, 2021, 337:116612.
[27] YU D Y, LI Z Y, ZHOU X N, et al.Study on the modification of magnetic graphene oxide and the effect of immobilized lipase[J].International Journal of Biological Macromolecules, 2022, 216:498-509.
[28] AMERI A, SHAKIBAIE M, KHOOBI M, et al.Optimization of immobilization conditions of Bacillus atrophaeus FSHM2 lipase on maleic copolymer coated amine-modified graphene oxide nanosheets and its application for valeric acid esterification[J].International Journal of Biological Macromolecules, 2020, 162:1790-1806.
[29] OZYILMAZ E, ASCIOGLU S, YILMAZ M.Calix[4]arene tetracarboxylic acid-treated lipase immobilized onto metal-organic framework:Biocatalyst for ester hydrolysis and kinetic resolution[J].International Journal of Biological Macromolecules, 2021, 175:79-86.
[30] AGHAEI H, YASINIAN A, TAGHIZADEH A.Covalent immobilization of lipase from Candida rugosa on epoxy-activated cloisite 30B as a new heterofunctional carrier and its application in the synthesis of banana flavor and production of biodiesel[J].International Journal of Biological Macromolecules, 2021, 178:569-579.
