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Review Article

Practical insights on enzyme stabilization

, , , &
Pages 335-350 | Received 09 Nov 2016, Accepted 17 May 2017, Published online: 01 Aug 2017

References

  • Kumar VSD, Sangwan P. Industrial enzymes trends: scope and relevance. 1st ed. Chapter: 10. In: Beniwal V, Sharma AK, editors. Global market scenario of industrial enzymes. India: Nova Science Publishers; 2014. p.173–196.
  • Beilen JB, Li Z. Enzyme technology: an overview. Curr Opin Biotechnol. 2002;13:338–344.
  • Iyer PV, Ananthanarayan L. Enzyme stability and stabilization—aqueous and non- aqueous environment. Process Biochem. 2008;43:1019–1032.
  • Ó’Fágáin C. Enzyme stabilization—recent experimental progress. Enzyme Microb Technol. 2003;33:137–49.
  • Kirk O, Borchert TV, Fuglsang CC. Industrial enzyme applications. Curr Opin Biotechnol. 2002;13:345–351.
  • Gianfreda L, Scarfi MR. Enzyme stabilization: state of the art. Mol Cell Biochem. 1991;100:97–128.
  • Horwitz J, Bova MP, Ding L, et al. crystallin: function and structure. Eye. 1999;13:403–408.
  • Katyal N, Deep S. Revisiting the conundrum of trehalose stabilization. Phys Chem Chem Phys. 2014;16:26746–26761.
  • Chang BS, Yeung B. Physical stability of protein pharmaceuticals. formulation and process development strategies for manufacturing biopharmaceuticals. California: John Wiley & Sons, Inc. 2010. p. 69–104.
  • Robic S. Mathematics, thermodynamics, and modeling to address ten common misconceptions about protein structure, folding, and stability. CBE Life Sci Educ. 2010;9:189–195.
  • Burioni R, Cassi D, Cecconi F, et al. Topological thermal instability and length of proteins. Proteins. 2004;55:529–535.
  • Bommarius AS, Paye MF. Stabilizing biocatalysts. Chem Soc Rev. 2013;42:6534–6565.
  • Saptarshi SR, Duschl A, Lopata AL. Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle. J Nanobiotechnology. 2013;11:26–21.
  • Salloum DS, Schlenoff JB. Protein adsorption modalities on polyelectrolyte multilayers. Biomacromolecules. 2004;5:1089–1096.
  • Jeong S. Analytical methods and formulation factors to enhance protein stability in solution. Arch Pharm Res. 2012;35:1871–1886.
  • Sinha N, Mohan S, Lipschultz CA, et al. Differences in electrostatic properties at antibody–antigen binding sites: implications for specificity and cross-reactivity. Biophys J. 2002;83:2946–2968.
  • Andrade JD, Hlady V. Protein adsorption materials biocompatibility: a tutorial review. Berlin Heidelberg: Springer, 1986. p. 1–63.
  • van der Veen M, Norde W, Stuart MC. Electrostatic interactions in protein adsorption probed by comparing lysozyme and succinylated lysozyme. Colloids Surf B. 2004;35:33–40.
  • Yano YF, Uruga T, Tanida H, et al. Protein salting out observed at an air − water interface. J Phys Chem Lett. 2011;2:995–999.
  • Dill KA. Dominant forces in protein folding. Biochemistry. 1990;29:7133–7155.
  • Shen DZ, Xue YH, Kang Q, et al. Monitoring of adsorption of lysozyme onto quartz using an electrode-separated piezoelectric sensor. Microchem J. 1998;60:1–7.
  • Dobson CM. Protein folding and misfolding. Nature. 2003;426:884–890.
  • Demirjian DC, Morı´s-Varas F, Cassidy CS. Enzymes from extremophiles. Curr Opin Chem Biol. 2001;5:144–151.
  • Bergquist PL, Morgan HW, Saul D. Selected enzymes from extreme thermophiles with applications in biotechnology. Curr Biotechnol. 2014;3:45–59.
  • Haki GD, Rakshit SK. Developments in industrially important thermostable enzymes: a review. Bioresour Technol. 2003;89:17–34.
  • Andrade CMMC Pereira Jr, N, Antranikian G. Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes. Rev Microbiol. 1999;30:287–298.
  • Bornscheuer UT, Huisman GW, Kazlauskas RJ, et al. Engineering the third wave of biocatalysis. Nature. 2012;485:185–194.
  • Davids T, Schmidt M, Böttcher D, et al. Strategies for the discovery and engineering of enzymes for biocatalysis. Curr Opin Chem Biol. 2013;17:215–220.
  • Behrens GA, Hummel A, Padhi SK, et al. Discovery and protein engineering of biocatalysts for organic synthesis. Adv Synth Catal. 2011;353:2191–2215.
  • Lutz S. Beyond directed evolution—semi-rational protein engineering and design. Curr Opin Biotechnol. 2010;21:734–743.
  • Bommarius AS, Blum JK, Abrahamson MJ. Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst. Curr Opin Chem Biol. 2011;15:194–200.
  • Böttcher D, Bornscheuer UT. Protein engineering of microbial enzymes. Curr Opin Microbiol. 2010;13:274–282.
  • Woodley JM. Protein engineering of enzymes for process applications. Curr Opin Chem Biol. 2013;17:310–316.
  • Otten LG, Hollmann F, Arends IWCE. Enzyme engineering for enantioselectivity: from trial-and-error to rational design? Trends Biotechnol. 2010;28:46–54.
  • Brode PF, Erwin CR, Rauch DS, et al. Subtilisin BPN‘Variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity. Biochemistry. 1996;35:3162–3169.
  • Beer HD, Wohlfahrt G, McCarthy JEG, et al. Analysis of the catalyic mechanism of a fungal lipase using computer-aided design and structural mutants. Protein Eng Des Sel. 1996;9:507–517.
  • Martinelle M, Holmquist M, Clausen IG, et al. The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase. Protein Eng Des Sel. 1996;9:519–524.
  • Pedersen S, Lange NK, Nissen AM. Novel industrial enzyme applications. Ann Ny Acad Sci. 1995;750:376–390.
  • Jegannathan KR, Nielsen PH. Environmental assessment of enzyme use in industrial production – a literature review. J Clean Prod. 2013;42:228–240.
  • Koivula A, Reinikainen T, Ruohonen L, et al. The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169. Protein Eng. 1996;9:691–699.
  • Davis BG. Chemical modification of biocatalysts. Curr Opin Biotechnol. 2003;14:379–386.
  • Vinogradov AA, Kudryashova EV, Grinberg VY, et al. The chemical modification of α-chymotrypsin with both hydrophobic and hydrophilic compounds stabilizes the enzyme against denaturation in water–organic media. Protein Eng. 2001;14:683–689.
  • Ueji S-i, Tanaka H, Hanaoka T, et al. Effects of chemical modification of lipase on its enantioselectivity in organic solvents. Chem Lett. 2001;30:1066–1067.
  • Díaz-Rodríguez A, Davis BG. Chemical modification in the creation of novel biocatalysts. Curr Opin Chem Biol. 2011;15:211–219.
  • Bund RK, Singhal RS. Chemical modification of cellulase by maleic anhydride and N-bromosuccinimide for improved detergent stability. J Surfact Deterg. 2002;5:1–4.
  • DeSantis G, Jones JB. Chemical modification of enzymes for enhanced functionality. Curr Opin Biotechnol. 1999;10:324–330.
  • Quiocho FA, Richards FM. Intermolecular cross linking of a protein in the crystalline state: carboxypeptidase-A. Proc Natl Acad Sci USA. 1964;52:833–839.
  • Govardhan CP. Crosslinking of enzymes for improved stability and performance. Curr Opin Biotechnol. 1999;10:331–335.
  • Silva CJ, Sousa F, Guebitz G, Cavaco-Paulo A. Chemical modifications on proteins using glutaraldehyde. Food Technol Biotechnol. 2004;42:51–16.
  • Mayolo-Deloisa K, González-González M, Simental-Martínez J, et al. Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity. J Mol Recognit. 2015;28:173–179.
  • López-Cruz JI, Viniegra-González G, Hernández-Arana A. Thermostability of native and pegylated myceliophthora thermophila laccase in aqueous and mixed solvents. Bioconjugate Chem. 2006;17:1093–1098.
  • Homaei AA, Sariri R, Vianello F, et al. Enzyme immobilization: an update. J Chem Biol. 2013;6:185–205.
  • Asgher M, Shahid M, Kamal S, et al. Recent trends and valorization of immobilization strategies and ligninolytic enzymes by industrial biotechnology. J Mol Catal B Enzym. 2014;101:56–66.
  • Ritter DW, Newton JM, McShane MJ. Modification of PEGylated enzyme with glutaraldehyde can enhance stability while avoiding intermolecular crosslinking. RSC Adv. 2014;4:28036–28040.
  • Ansari SA, Husain Q. Potential applications of enzymes immobilized on/in nano materials: a review. Biotechnol Adv. 2012;30:512–523.
  • Cowan DA, Fernandez-Lafuente R. Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzyme Microb Technol. 2011;49:326–346.
  • Taylor RF. A comparison of various commercially-available liquid chromatographic supports for immobilization of enzymes and immunoglobulins. Anal Chim Acta. 1985;172:241–248.
  • Palomo JM. Lipases enantioselectivity alteration by immobilization techniques. Curr Bioact Comp. 2008;4:126–138.
  • Kamori M, Hori T, Yamashita Y, et al. Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and enantioselectivity of the immobilized lipase. J Mol Catal B Enzym. 2000;9:269–274.
  • Sangeetha K, Emilia Abraham T. Preparation and characterization of cross-linked enzyme aggregates (CLEA) of Subtilisin for controlled release applications. Int J Biol Macromol. 2008;43:314–319.
  • Talekar S, Ghodake V, Ghotage T, et al. Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase. Bioresour Technol. 2012;123:542–547.
  • Stepankova V, Bidmanova S, Koudelakova T, et al. Strategies for stabilization of enzymes in organic solvents. ACS Catal. 2013;3:2823–2836.
  • Balcão VM, Vila MMDC. Structural and functional stabilization of protein entities: state-of-the-art. Adv Drug Deliv Rev. 2015;93:25–41.
  • Hudson EP, Eppler RK, Clark DS. Biocatalysis in semi-aqueous and nearly anhydrous conditions. Curr Opin Biotechnol. 2005;16:637–643.
  • Scharnagl C, Reif M, Friedrich J. Stability of proteins: temperature, pressure and the role of the solvent. Biochim Biophys Acta. 2005;1749:187–213.
  • Miyawaki O. Hydration state change of proteins upon unfolding in sugar solutions. Biochim Biophys Acta. 2007;1774:928–935.
  • Kumar V, Chari R, Sharma VK, et al. Modulation of the thermodynamic stability of proteins by polyols: significance of polyol hydrophobicity and impact on the chemical potential of water. Int J Plast. 2011;413:19–28.
  • Saenger W. Structure and dynamics of water surrounding biomolecules. Annu Rev Biophys Biophys Chem. 1987;16:93–114.
  • Arakawa T, Prestrelski SJ, Kenney WC, et al. Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev. 1993;10:1–28.
  • Wong YH, Tayyab S. Protein stabilizing potential of simulated honey sugar cocktail under various denaturation conditions. Process Biochem. 2012;47:1933–1943.
  • Carpenter JF, Crowe JH. The mechanism of cryoprotection of proteins by solutes. Cryobiology. 1988;25:244–255.
  • Haque I, Singh R, Moosavi-Movahedi AA, et al. Effect of polyol osmolytes on ΔgD, the Gibbs energy of stabilisation of proteins at different pH values. Biophys Chem. 2005;117:1–12.
  • Haque I, Singh R, Ahmad F, et al. Testing polyols’ compatibility with Gibbs energy of stabilization of proteins under conditions in which they behave as compatible osmolytes. FEBS Lett. 2005;579:3891–3898.
  • Ebel C, Eisenberg H, Ghirlando R. Probing protein-sugar interactions. Biophys J. 2000;78:385–393.
  • Timasheff SN, Arakawa T. Mechanism of protein precipitation and stabilization by co-solvents. J Cryst Growth. 1988;90:39–46.
  • Sanchez-Ruiz JM. Protein kinetic stability. Biophys Chem. 2010;148:1–15.
  • Becktel WJ, Schellman JA. Protein stability curves. Biopolymers. 1987;26:1859–1877. Peptide Science Section.
  • Miyawaki O, Tatsuno M. Thermodynamic analysis of alcohol effect on thermal stability of proteins. J Biosci Bioeng. 2011;111:198–203.
  • Linhananta A, Hadizadeh S, Plotkin SS. An effective solvent theory connecting the underlying mechanisms of osmolytes and denaturants for protein stability. Biophys J. 2011;100:459–468.
  • Kristjánsson MM, Kinsella JE. Protein and enzyme stability: structural, thermodynamic, and experimental aspects. In: John EK, editor. Advances in food and nutrition research. Netherlands: Academic Press; 1991. p. 237–316.
  • Lavelle L, Fresco JR. Stabilization of nucleic acid triplexes by high concentrations of sodium and ammonium salts follows the Hofmeister series1. Biophys Chem. 2003;105:681–699.
  • Kamiyama T, Sadahide Y, Nogusa Y, et al. Polyol-induced molten globule of cytochrome c: an evidence for stabilization by hydrophobic interaction. Biochim Biophys Acta. 1999;1434:44–57.
  • Timasheff SN, Arakawa T. Mechanism of protein precipitation and stabilization by co-solvents. J Cryst Growth. 1988;90:39–46.
  • Kherb J, Flores SC, Cremer PS. Role of carboxylate side chains in the cation hofmeister series. J Phys Chem B. 2012;116:7389–7397.
  • Zhang Y, Cremer PS. The inverse and direct Hofmeister series for lysozyme. Proc Natl Acad Sci USA. 2009;106:15249–15253.
  • Sedlák E, Stagg L, Wittung-Stafshede P. Effect of Hofmeister ions on protein thermal stability: Roles of ion hydration and peptide groups? Arch Biochem Biophys. 2008;479:69–73.
  • Gao W-W, Zhang F-X, Zhang G-X, et al. Key factors affecting the activity and stability of enzymes in ionic liquids and novel applications in biocatalysis. Biochem Eng J. 2015;99:67–84.
  • Zhao H. Ionic liquids as (Co-)solvents for hydrolytic enzymes. Ionic liquids in biotransformations and organocatalysis. Hoboken (NJ): John Wiley & Sons, Inc., 2012. p. 151–227.
  • Weingartner H, Cabrele C, Herrmann C. How ionic liquids can help to stabilize native proteins. Phys Chem Chem Phys. 2012;14:415–426.
  • Yang Z. Hofmeister effects: an explanation for the impact of ionic liquids on biocatalysis. J Biotechnol. 2009;144:12–22.
  • Zhao H. Protein stabilization and enzyme activation in ionic liquids: specific ion effects. J Chem Technol Biotechnol. 2016;91:25–50.
  • Shulgin IL, Ruckenstein E. Preferential hydration and solubility of proteins in aqueous solutions of polyethylene glycol. Biophys Chem. 2006;120:188–198.
  • Liang Y, Yuan X, Zeng G, et al. Effects of surfactants on enzyme-containing reversed micellar system. Sci China Chem. 2011;54:715–723.
  • Brown ED, Yada RY, Marangoni AG. The dependence of the lipolytic activity of Rhizopus arrhizus lipase on surfactant concentration in Aerosol-OT/isooctane reverse micelles and its relationship to enzyme structure. Biochim Biophys Acta. 1993;1161:66–72.
  • Simpson RJ. Stabilization of Proteins for Storage. Cold Spring Harb Protoc. 2010;2010:pdb.top79.
  • Hwang ET, Gu MB. Enzyme stabilization by nano/microsized hybrid materials. Eng Life Sci. 2013;13:49–61.
  • Jia H, Zhu G, Wang P. Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility. Biotechnol Bioeng. 2003;84:406–414.
  • Betancor L, Luckarift HR. Bioinspired enzyme encapsulation for biocatalysis. Trends Biotechnol. 2008;26:566–572.
  • Poulsen N, Sumper M, Kröger N. Biosilica formation in diatoms: characterization of native silaffin-2 and its role in silica morphogenesis. Proc Natl Acad Sci USA. 2003;100:12075–12080.
  • Kim J, Grate JW, Wang P. Nanobiocatalysis and its potential applications. Trends Biotechnol. 2008;26:639–646.
  • Kim J, Grate JW. Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Lett. 2003;3:1219–1222.
  • Kim J, Jia H, Lee C-w, et al. Single enzyme nanoparticles in nanoporous silica: a hierarchical approach to enzyme stabilization and immobilization. Enzyme Microb Technol. 2006;39:474–480.
  • Yang Z, Si S, Zhang C. Magnetic single-enzyme nanoparticles with high activity and stability. Biochem Biophys Res Commun. 2008;29:169–175.
  • Colletier J-P, Chaize B, Winterhalter M, et al. Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer. BMC Biotechnol. 2002;2:9.
  • Zhou X-M, Entwistle A, Zhang H, et al. Self-assembly of amyloid fibrils that display active enzymes. ChemCatChem. 2014;6:1961–1968.
  • Zhou X-M, Shimanovich U, Herling TW, et al. Enzymatically active microgels from self-assembling protein nanofibrils for microflow chemistry. ACS Nano. 2015;9:5772–5781.
  • Song Y, Shimanovich U, Michaels TCT, et al. Fabrication of fibrillosomes from droplets stabilized by protein nanofibrils at all-aqueous interfaces. Nat Commun. 2016;7:12934.
  • Li AB, Kluge JA, Guziewicz NA, et al. Silk-based stabilization of biomacromolecules. J Control Release. 2015;219:416–430.
  • Montes T, Grazú V, López-Gallego F, et al. Genetic modification of the penicillin G acylase surface to improve its reversible immobilization on ionic exchangers. Appl Environ Microbiol. 2007;73:312–319.
  • Fernandez-Lafuente R, Rosell CM, Caanan-Haden L, et al. Facile synthesis of artificial enzyme nano-environments via solid-phase chemistry of immobilized derivatives: dramatic stabilization of penicillin acylase versus organic solvents. Enzyme Microb Technol. 1999;24:96–103.
  • Abian O, Mateo C, Fernández-Lorente G, et al. Stabilization of immobilized enzymes against water-soluble organic cosolvents and generation of hyper-hydrophilic micro-environments surrounding enzyme molecules. Biocatal Biotransformation. 2001;19:489–503.
  • Hassani L, Ranjbar B, Khajeh K, et al. Horseradish peroxidase thermostabilization: the combinatorial effects of the surface modification and the polyols. Enzyme Microb Technol. 2006;38:118–125.
  • Chan L, Cross HF, She JK, et al. Covalent attachment of proteins to solid supports and surfaces via sortase-mediated ligation. PLoS One. 2007;2:e1164.
  • La´szló K, Szava A, Ma´ria Simon L. Stabilization of various α-chymotrypsin forms in aqueous-organic media by additives. J Mol Catal B Enzym. 2001;16:141–146.
  • Montes T, Grazu V, López-Gallego F, et al. Chemical modification of protein surfaces to improve their reversible enzyme immobilization on ionic exchangers. Biomacromolecules. 2006;7:3052–3058.
  • Araújo R, Casal M, Cavaco-Paulo A. Application of enzymes for textile fibres processing. Biocatal Biotransformation. 2008;26:332–349.
  • Chand N, Nateri AS, Sajedi RH, et al. Enzymatic desizing of cotton fabric using a Ca2+-independent α-amylase with acidic pH profile. J Mol Catal B Enzym. 2012;83:46–50.
  • Levene R, Cohen Y, Barkai D. Applying proteases to confer improved shrink resistance to wool. J Soc Dyers Colour. 1996;112:6–10.
  • Freddi G, Mossotti R, Innocenti R. Degumming of silk fabric with several proteases. J Biotechnol. 2003;106:101–112.
  • Shen J, Rushforth M, Cavaco-Paulo A, et al. Development and industrialisation of enzymatic shrink-resist process based on modified proteases for wool machine washability. Enzyme Microb Technol. 2007;40:1656–1661.
  • Araújo R, Silva C, Machado R, et al. Proteolytic enzyme engineering: a tool for wool. Biomacromolecules. 2009;10:1655–1661.
  • Hajighasemi M, Nocek BP, Tchigvintsev A, et al. Biochemical and structural insights into enzymatic depolymerization of polylactic acid and other polyesters by microbial carboxylesterases. Biomacromolecules. 2016;17:2027–2039.
  • Yang Y, Yu Y, Zhang Y, et al. Lipase/esterase-catalyzed ring-opening polymerization: a green polyester synthesis technique. Process Biochem. 2011;46:1900–1908.
  • Silva CM, Carneiro F, O’Neill A, et al. Cutinase—a new tool for biomodification of synthetic fibers. J Polym Sci A Polym Chem. 2005;43:2448–2450.
  • Ronkvist ÅM, Xie W, Lu W, et al. Cutinase-catalyzed hydrolysis of poly(ethylene terephthalate). Macromolecules. 2009;42:5128–5138.
  • Matamá T, Araújo R, Gübitz GM, et al. Functionalization of cellulose acetate fibers with engineered cutinases. Biotechnol Progress. 2010;26:636–643.
  • Araújo R, Silva C, O’Neill A, et al. Tailoring cutinase activity towards polyethylene terephthalate and polyamide 6,6 fibers. J Biotechnol. 2007;128:849–857.
  • Galli C, Gentili P, Jolivalt C, et al. How is the reactivity of laccase affected by single-point mutations? Engineering laccase for improved activity towards sterically demanding substrates. Appl Microbiol Biotechnol. 2011;91:123–131.
  • Zhang Z-G, Yi Z-L, Pei X-Q, et al. Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis. Bioresour Technol. 2010;101:9272–9278.
  • Singh RK, Zhang Y-W, Nguyen N-P-T, et al. Covalent immobilization of β-1,4-glucosidase from Agaricus arvensis onto functionalized silicon oxide nanoparticles. Appl Microbiol Biotechnol. 2011;89:337–344.
  • Cristóvão RO, Silvério SC, Tavares APM, et al. Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization. World J Microbiol Biotechnol. 2012;28:2827–2838.
  • Silva CJSM Zhang Q, Shen J, et al. Immobilization of proteases with a water soluble–insoluble reversible polymer for treatment of wool. Enzyme Microb Technol. 2006;39:634–640.
  • Silva C, Silva CJ, Zille A, et al. Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres. Enzyme Microb Technol. 2007;41:867–875.
  • Miletić N, Nastasović A, Loos K. Immobilization of biocatalysts for enzymatic polymerizations: possibilities, advantages, applications. Bioresour Technol. 2012;115:126–135.
  • Stavila E, Arsyi RZ, Petrovic DM, et al. Fusarium solani pisi cutinase-catalyzed synthesis of polyamides. Eur Polymer J. 2013;49:834–842.
  • Zhao H, Olubajo O, Song Z, et al. Effect of kosmotropicity of ionic liquids on the enzyme stability in aqueous solutions. Bioorg Chem. 2006;34:15–25.
  • Yoshimoto M. Stabilization of enzymes through encapsulation in liposomes. In: Minteer DS, editor. Enzyme stabilization and immobilization: methods and protocols. Totowa, NJ: Humana Press; 2011. p. 9–18.
  • Yang K, Xu N-S, Su WW. Co-immobilized enzymes in magnetic chitosan beads for improved hydrolysis of macromolecular substrates under a time-varying magnetic field. J Biotechnol. 2010;148:119–127.
  • Martins M, Azoia N, Silva C, et al. Stabilization of enzymes in micro-emulsions for ultrasound processes. Biochem Eng J. 2015;93:115–118.
  • Lu S, Wang X, Lu Q, et al. Stabilization of enzymes in silk films. Biomacromolecules. 2009;10:1032–1042.
  • Demura M, Asakura T, Kuroo T. Immobilization of biocatalysts with bombyx mori silk fibroin by several kinds of physical treatment and its application to glucose sensors. Biosensors. 1989;4:361–372.
  • Shaw KL, Grimsley GR, Yakovlev GI, et al. The effect of net charge on the solubility, activity, and stability of ribonuclease Sa. Protein Sci Soc. 2001;10:1206–1215.

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