Abstract
Entrapment of enzymes into a cross-linked network is an effective way to enable their recycling. In order to obtain a satisfied recovery of enzyme activity, a mild encapsulating condition is highly required due to the delicate nature of enzymes. Herein, a facile and mild visible light-induced inverse emulsion polymerization technique was developed for in situ entrapment of enzyme. In this method, poly (ethylene glycol) diacrylate (PEGDA) was dissolved in phosphate buffer saline and used as disperse phase, while continuous phase was composed of liquid paraffin, photoinitiators (isopropylthioxanthone and ethyl 4-dimethylaminobenzoate) and emulsifier (Span 80 and Tween 80). Under the irradiation of visible light, PEGDA could be cross-linkedly polymerized and formed microparticles with diameter ranged from 0.75 to 6.5 μm. When the glutaraldehyde cross-linked papain was added into disperse phase, it could be in situ entrapped into the microparticles after the visible light-induced inverse emulsion polymerization. The immobilized papain exhibited higher activity in a wide range of temperature and pH than free papain. Moreover, the immobilized papain could maintain 60% of its initial activity even after ten cycles of usage. This simple and mild strategy to in situ entrapment of enzymes has potential application in fields such as biocatalyst, biosensor and drug delivery.
Similar content being viewed by others
References
Li J, Ma J, Jiang Y, Jiang T, Wang Y, Chen Y, Liu S (2016) Immobilizing enzymes in regular-sized gelatin microspheres through a membrane emulsification method. J Mater Sci 51(13):6357–6369. doi:10.1007/s10853-016-9932-5
Sheldon RA (2007) Enzyme immobilization: the quest for optimum performance. Adv Synth Catal 349(8–9):1289–1307
Tielmann P, Kierkels H, Zonta A, Ilie A, Reetz MT (2014) Increasing the activity and enantioselectivity of lipases by sol–gel immobilization: further advancements of practical interest. Nanoscale 6(12):6220–6228
Zhu X, Ma Y, Zhao C, Lin Z, Zhang L, Chen R, Yang W (2014) A mild strategy to encapsulate enzyme into hydrogel layer grafted on polymeric substrate. Langmuir 30(50):15229–15237
Grotzky A, Altamura E, Adamcik J, Carrara P, Stano P, Mavelli F, Nauser T, Mezzenga R, Schlüter AD, Walde P (2013) Structure and enzymatic properties of molecular dendronized polymer-enzyme conjugates and their entrapment inside giant vesicles. Langmuir 29(34):10831–10840. doi:10.1021/la401867c
Schachschal S, Adler H-J, Pich A, Wetzel S, Matura A, van Pee K-H (2011) Encapsulation of enzymes in microgels by polymerization/cross-linking in aqueous droplets. Colloid Polym Sci 289(5):693–698. doi:10.1007/s00396-011-2392-1
Si Tamaru, Kiyonaka S, Hamachi I (2005) Three distinct read-out modes for enzyme activity can operate in a semi-wet supramolecular hydrogel. Chem-A Eur J 11(24):7294–7304
Ren C, Zhang J, Chen M, Yang Z (2014) Self-assembling small molecules for the detection of important analytes. Chem Soc Rev 43(21):7257–7266
Cantone S, Ferrario V, Corici L, Ebert C, Fattor D, Spizzo P, Gardossi L (2013) Efficient immobilisation of industrial biocatalysts: criteria and constraints for the selection of organic polymeric carriers and immobilisation methods. Chem Soc Rev 42(15):6262–6276. doi:10.1039/C3CS35464D
Cosnier S (1999) Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review. Biosens Bioelectron 14(5):443–456
Alnaief M, Alzaitoun MA, García-González CA, Smirnova I (2011) Preparation of biodegradable nanoporous microspherical aerogel based on alginate. Carbohyd Polym 84(3):1011–1018. doi:10.1016/j.carbpol.2010.12.060
Kabanov AV, Vinogradov SV (2009) Nanogels as pharmaceutical carriers: finite networks of infinite capabilities. Angew Chem Int Ed 48(30):5418–5429
Donini C, Robinson D, Colombo P, Giordano F, Peppas N (2002) Preparation of poly (methacrylic acid-g-poly (ethylene glycol)) nanospheres from methacrylic monomers for pharmaceutical applications. Int J Pharm 245(1):83–91
Zhang X, Malhotra S, Molina M, Haag R (2015) Micro-and nanogels with labile crosslinks–from synthesis to biomedical applications. Chem Soc Rev 44(7):1948–1973
Bao S, Wu D, Su T, Wu Q, Wang Q (2015) Microgels formed by enzyme-mediated polymerization in reverse micelles with tunable activity and high stability. RSC Adv 5(55):44342–44345. doi:10.1039/C5RA02162F
Fouassier JP, Allonas X, Lalevee J, Visconti M (2000) Radical polymerization activity and mechanistic approach in a new three-component photoinitiating system. J Polym Sci Pol Chem 38(24):4531–4541. doi:10.1002/1099-0518(20001215)38:24<4531:AID-POLA220>3.0.CO;2-U
Zhang L, Ma Y, Zhao C, Zhu X, Chen R, Yang W (2015) Synthesis of pH-responsive hydrogel thin films grafted on PCL substrates for protein delivery. J Mater Chem B 3(39):7673–7681
Zhao C, Lin Z, Yin H, Ma Y, Xu F, Yang W (2014) PEG molecular net-cloth grafted on polymeric substrates and Its bio-merits. Sci Rep 4:4982. doi:10.1038/srep04982
Zhang H, Wu C, Zhang Y, White CJB, Xue Y, Nie H, Zhu L (2010) Elaboration, characterization and study of a novel affinity membrane made from electrospun hybrid chitosan/nylon-6 nanofibers for papain purification. J Mater Sci 45(9):2296–2304. doi:10.1007/s10853-009-4191-3
Yoo G, Bong J-H, Kim S, Jose J, Pyun J-C (2014) Microarray based on autodisplayed Ro proteins for medical diagnosis of systemic lupus erythematosus (SLE). Biosens Bioelectron 57:213–218. doi:10.1016/j.bios.2014.02.018
Vasconcellos FC, Goulart GA, Beppu MM (2011) Production and characterization of chitosan microparticles containing papain for controlled release applications. Powder Technol 205(1):65–70
Müller C, Perera G, König V, Bernkop-Schnürch A (2014) Development and in vivo evaluation of papain-functionalized nanoparticles. Eur J Pharm Biopharm 87(1):125–131. doi:10.1016/j.ejpb.2013.12.012
Sahoo B, Sahu SK, Bhattacharya D, Dhara D, Pramanik P (2013) A novel approach for efficient immobilization and stabilization of papain on magnetic gold nanocomposites. Colloid Surface B 101:280–289
Homaei A (2015) Enhanced activity and stability of papain immobilized on CNBr-activated sepharose. Int J Biol Macromol 75:373–377. doi:10.1016/j.ijbiomac.2015.01.055
Mahmoud KA, Lam E, Hrapovic S, Luong JH (2013) Preparation of well-dispersed gold/magnetite nanoparticles embedded on cellulose nanocrystals for efficient immobilization of papain enzyme. ACS Appl Mater Interfaces 5(11):4978–4985
Yang Y-C, Deka JR, Wu C-E, Tsai C-H, Saikia D, Kao H-M (2017) Cage like ordered carboxylic acid functionalized mesoporous silica with enlarged pores for enzyme adsorption. J Mater Sci 52(11):6322–6340. doi:10.1007/s10853-017-0864-5
Miyamoto D, Watanabe J, Ishihara K (2004) Effect of water-soluble phospholipid polymers conjugated with papain on the enzymatic stability. Biomaterials 25(1):71–76. doi:10.1016/S0142-9612(03)00474-5
Barbosa O, Torres R, Ortiz C, Berenguer-Murcia A, Rodrigues RC, Fernandez-Lafuente R (2013) Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromol 14(8):2433–2462
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1):248–254. doi:10.1016/0003-2697(76)90527-3
Guo Y, Wang Z, Qu W, Shao H, Jiang X (2011) Colorimetric detection of mercury, lead and copper ions simultaneously using protein-functionalized gold nanoparticles. Biosens Bioelectron 26(10):4064–4069
Nitsawang S, Hatti-Kaul R, Kanasawud P (2006) Purification of papain from Carica papaya latex: aqueous two-phase extraction versus two-step salt precipitation. Enzyme Microb Tech 39(5):1103–1107
Bai H, Huang Z, Yang W (2009) Visible light-induced living surface grafting polymerization for the potential biological applications. J Polym Sci Pol Chem 47(24):6852–6862
Graillat C, Pichot C, Guyot A, El Aasser M (1986) Inverse emulsion polymerization of acrylamide. I. Contribution to the study of some mechanistic aspects. J Polym Sci Pol Chem 24(3):427–449
Benda D, Šňupárek J, Čermák V (2001) Oxygen inhibition and the influence of pH on the inverse emulsion polymerization of the acrylic monomers. Eur Polym J 37(6):1247–1253
Wang S, Chen K, Li L, Guo X (2013) Binding between proteins and cationic spherical polyelectrolyte brushes: effect of pH, ionic strength, and stoichiometry. Biomacromol 14(3):818–827. doi:10.1021/bm301865g
Birner-Grünberger R, Scholze H, Faber K, Hermetter A (2004) Identification of various lipolytic enzymes in crude porcine pancreatic lipase preparations using covalent fluorescent inhibitors. Biotechnol Bioeng 85(2):147–154
Zhou Y-J, Hu C-L, Wang N, Zhang W-W, Yu X-Q (2013) Purification of porcine pancreatic lipase by aqueous two-phase systems of polyethylene glycol and potassium phosphate. J Chromatogr B 926:77–82
Wan X, Liu T, Hu J, Liu S (2013) Photo-degradable, protein-polyelectrolyte complex-coated, mesoporous silica nanoparticles for controlled co-release of protein and model drugs. Macromol Rapid Comm 34(4):341–347
Shakya AK, Sami H, Srivastava A, Kumar A (2010) Stability of responsive polymer–protein bioconjugates. Prog Polym Sci 35(4):459–486
Bhardwaj A, Lee J, Glauner K, Ganapathi S, Bhattacharyya D, Butterfield DA (1996) Biofunctional membranes: an EPR study of active site structure and stability of papain non-covalently immobilized on the surface of modified poly (ether) sulfone membranes through the avidin-biotin linkage. J Membrane Sci 119(2):241–252
Fernandez-Lafuente R, Rosell C, Rodriguez V, Guisan J (1995) Strategies for enzyme stabilization by intramolecular crosslinking with bifunctional reagents. Enzyme Microb Technol 17(6):517–523
Mateo C, Abian O, Fernandez-Lafuente R, Guisan JM (2000) Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment ☆. Enzyme Microb Technol 26(7):509–515
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 51521062, 51103009, 51473015, 51273012) and the Fundamental Research Funds for the Central Universities and Beijing Natural Science Foundation (Grant No. 2162035).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare no conflict of interest exist.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Wang, G., Chen, D., Zhang, L. et al. A mild route to entrap papain into cross-linked PEG microparticles via visible light-induced inverse emulsion polymerization. J Mater Sci 53, 880–891 (2018). https://doi.org/10.1007/s10853-017-1484-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10853-017-1484-9