Abstract
Methods for the preparation of carrier-free insoluble enzymes are reviewed. The technology of cross-linked enzyme aggregates has now been applied to a range of synthetically useful activities. Fusion proteins are also gaining momentum because they allow a relatively selective aggregation or even a specific self-assembly of the desired enzyme activity into insoluble particles in the absence of potentially denaturing chemicals required for precipitation and cross-linking. Recycling of insoluble protein particles for multiple rounds of batchwise reaction has been demonstrated in selected biotransformations. However, for application in a fully continuous biocatalytic process, low resistance to mechanical stress and high compressibility are issues for consideration on carrier-free enzyme particles.
Similar content being viewed by others
References
Aytar BS, Bakir U (2008) Preparation of cross-linked tyrosinase aggregates. Process Biochem 43:125–131
Bayley H, Braha O, Cheley S, Gu L-Q (2004) Engineered nanopores. In: Niemeyer CM, Mirkin CA (eds) Nanobiotechnology. Wiley-VCH, Weinheim, pp 93–112
Betancor L, Luckarift HR (2008) Bioinspired enzyme encapsulation for biocatalysis. Trends Biotechnol 26:566–572
Blecha A, Zarschler K, Sjollema KA, Veenhuis M, Rödel G (2005) Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells. Microb Cell Fact 4:28
Bommarius AS, Riebel BR (2005) Biocatalysis. Wiley-VCH, Weinheim
Buchholz K, Kasche V, Bornscheuer UT (2005) Biocatalysts and enzyme technology. Wiley-VCH, Weinheim
Cabana H, Jones JP, Agathos SN (2007) Preparation and characterization of cross-linked laccase aggregates and their application to the elimination of endocrine disrupting chemicals. J Biotechnol 132:23–31
Cabirol FL, Tan PL, Tay B, Cheng S, Hanefeld U, Sheldon RA (2008) Linum usitatissimum hydroxynitrile lyase cross-linked enzyme aggregates: a recyclable enantioselective catalyst. Adv Synth Catal 350:2329–2338
Cao L (2005) Carrier-bound immobilized enzymes: principles, applications and design. Wiley-VCH, Weinheim
Cao L, van Rantwijk F, Sheldon RA (2000) Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase. Org Lett 2:1361–1364
Cao L, van Langen L, Sheldon RA (2003) Immobilised enzymes: carrier-bound or carrier-free? Curr Opin Biotechnol 14:387–394
Carette N, Engelkamp H, Akpa E, Pierre SJ, Cameron NR, Christianen PCM et al (2006) A virus-based biocatalyst. Nat Nanotechnol 2:226–229
Chen J, Zhang J, Han B, Li Z, Li J, Feng X (2006) Synthesis of cross-linked enzyme aggregates (CLEAs) in CO2-expanded micellar solutions. Colloids Surf B 48:72–76
Chien L-J, Lee C-K (2007) Biosilicification of dual-fusion enzyme immobilized on magnetic nanoparticle. Biotechnol Bioeng 100:223–230
Dalal S, Sharma A, Gupta MN (2007) A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities. Chem Cent J 1:16
Fujita Y, Mie M, Kobatake E (2009) Construction of nanoscale protein particle using temperature-sensitive elastin-like peptide and polyaspartic acid chain. Biomaterials 30:3450–3457
Graff A, Benito SM, Verbert C, Meier W (2004) Polymer nanocontainers. In: Niemeyer CM, Mirkin CA (eds) Nanobiotechnology. Wiley-VCH, Weinheim, pp 168–184
Grage K, Rehm BHA (2008) In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner. Bioconjug Chem 19:254–262
Haering D, Schreier P (1998) Novel biocatalysts by chemical modification of known enzymes: cross-linked microcrystals of the semisynthetic peroxidase seleno-subtilisin. Angew Chem Int Ed 37:2471–2473
Hampp N, Oesterhelt D (2004) Bacteriorhodopsin and its potential in technical applications. In: Niemeyer CM, Mirkin CA (eds) Nanobiotechnology. Wiley-VCH, Weinheim, pp 146–167
Hanefeld U, Gardossi L, Magner E (2009) Understanding enzyme immobilisation. Chem Soc Rev 38:453–468
Hara P, Hanefeld U, Kanerva LT (2007) Sol–gels and cross-linked aggregates of lipase PS from Burkholderia cepacia and their application in dry organic solvents. J Mol Catal B 50:80–86
Heyman A, Levy I, Altman A, Shoseyov O (2007) SP1 as a novel scaffold building block for self-assembly nanofabrication of submicron enzymatic structures. Nano Lett 7:1575–1579
Hickey AM, Marle L, McCreedy T, Watts P, Greenway GM, Littlechild JA (2007) Immobilization of thermophilic enzymes in miniaturized flow reactors. Biochem Soc Trans 35:1621–1623
Hickey AM, Ngamsom B, Wiles C, Greenway GM, Watts P, Littlechild JA (2009) A microreactor for the study of biotransformations by a cross-linked γ-lactamase enzyme. Biotechnology J 4:510–516
Jekel M, Buhr A, Willke T, Vorlop K-D (1998) Neuartige Geleinschlußimmobilisate (LentiKats) in der Biotechnologie. Chem lngenieur Technik 70:438–441
Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674
Khare SK, Vaidya S, Gupta MN (1991) Entrapment of proteins by aggregation within sephadex beads. Appl Biochem Biotechnol 27:205–216
Kim MI, Kim J, Lee J, Jia H, Na HB, Youn JK, Kwak JH, Dohnalkova A, Grate JW, Wang P, Hyeon T, Park HG, Chang HN (2007) Cross-linked enzyme aggregates in hierarchically-ordered mesoporous silica: a simple and effective method for enzyme stabilization. Biotechnol Bioeng 96:210–218
Kröger N, Lorenz S, Brunner E, Sumper M (2002) Self-assembly of highly phosphorylated silaffins and their function in biosilica morphogenesis. Science 298:584–586
Kubáč D, Kaplan O, Elišáková V, Pátek M, Vejvoda V, Slámová K, Tóthová A, Lemaire M, Gallienne E, Lutz-Wahl S, Fischer L, Kuzma M, Pelantová H, van Pelt S, Bolte J, Křen V, Martínková L (2008) Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4. J Mol Catal B 50:107–113
Lim DW, Nettles DL, Setton LA, Chilkoti A (2007) Rapid cross-linking of elastin-like polypeptides with hydroxymethylphosphines in aqueous solution. Biomacromolecules 8:1463–1470
Littlechild JA, Guy J, Connelly S, Mallett L, Waddell S, Rye CA, Line K, Isupov M (2007) Natural methods of protein stabilization: thermostable biocatalysts. Biochem Soc Trans 35:1558–1563
López-Gallego F, Betancor L, Hidalgo A, Alonso N, Fernández-Lafuente R, Guisán JM (2005) Co-aggregation of enzymes and polyethyleneimine: a simple method to prepare stable and immobilized derivatives of glutaryl acylase. Biomacromolecules 6:1839–1842
López-Serrano P, Cao L, van Rantwijk F, Sheldon RA (2002) Cross-linked enzyme aggregates with enhanced activity: application to lipases. Biotechnol Lett 24:1379–1383
Lu J, Tappel RC, Nomura CT (2009) Mini-review: biosynthesis of poly(hydroxyalkanoates). J Macromol Sci C 49:226–248
Luckarift HR, Spain JC, Naik RR, Stone MO (2004) Enzyme immobilization in a biomimetic silica support. Nat Biotechnol 22:211–213
Madison LL, Huisman GW (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63:21–53
Margolin AL (1996) Novel crystalline catalysts. Trends Biotechnol 14:223–230
Marner WD, Shaikh AS, Muller SJ, Keasling JD (2009) Enzyme immobilization via silaffin-mediated autoencapsulation in a biosilica support. Biotechnol Prog 25:417–423
Mateo C, Palomo JM, van Langen LM, van Rantwijk F, Sheldon RA (2004) A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates. Biotechnol Bioeng 86:273–276
Mateo C, Chmura A, Rustler S, van Rantwijk F, Stolz A, Sheldon RA (2006) Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase–nitrilase bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activity. Tetrahedron 17:320–323
Meyer DE, Chilkoti A (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 17:1112–1115
Meyer DE, Trabbic-Carlson K, Chilkoti A (2001) Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin. Biotechnol Prog 17:720–728
Moldes C, García P, García JL, Prieto MA (2004) In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF. Appl Environ Microbiol 70:3205–3212
Nahálka J (2008) Physiological aggregation of maltodextrin phosphorylase from Pyrococcus furiosus and its application in a process of batch starch degradation to α-d-glucose-1-phosphate. J Ind Microbiol Biotechnol 35:219–223
Nahálka J, Nidetzky B (2007) Fusion to a pull-down domain: a novel approach of producing Trigonopsis variabilis d-amino acid oxidase as insoluble enzyme aggregates. Biotechnol Bioeng 97:454–461
Nahálka J, Pätoprstý V (2009) Enzymatic synthesis of sialylation substrates powered by a novel polyphosphate kinase (PPK3). Org Biomol Chem 7:1778–1780
Nahálka J, Vikartovská A, Hrabárová E (2008) A cross-linked inclusion body process for sialic acid synthesis. J Biotechnol 134:146–153
Nahálka J, Mislovičová D, Kavcová H (2009) Targeting lectin activity into inclusion bodies for the characterisation of glycoproteins. Mol Biosyst 5:819–821
Naik RR, Tomczak MM, Luckarift HR, Spain JC, Stone MO (2004) Entrapment of enzymes and nanoparticles using biomimetically synthesized silica. Chem Commun 1684–1685
Neumann L, Spinozzi F, Sinibaldi R, Rustichelli F, Pötter M, Steinbüchel A (2008) Binding of the major phasin, PhaP1, from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules. J Bacteriol 190:2911–2919
Peters V, Rehm BHA (2006) In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase. Appl Environ Microbiol 72:1777–1783
Poulsen N, Berne C, Spain J, Kröger N (2007) Silica immobilization of an enzyme through genetic engineering of the diatom Thalassiosira pseudonana. Angew Chem 119:1875–1878
Quiocho FA, Richards FM (1964) Intermolecular cross-linking of a protein in the crystalline state: carboxypeptidase-A. PNAS 52:833–839
Rajan A, Abraham TE (2008) Studies on crystallization and cross-linking of lipase for biocatalysis. Bioprocess Biosyst Eng 31:87–94
Rajendhran J, Gunasekaran P (2007) Application of cross-linked enzyme aggregates of Bacillus badius penicillin G acylase for the production of 6-aminopenicillanic acid. Lett Appl Microbiol 44:43–49
Rehm BHA (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15–33
Roy JJ, Abraham TE (2006) Preparation and characterization of cross-linked enzyme crystals of laccase. J Mol Catal B 38:31–36
Schäffer C, Novotny R, Küpcü S, Zayni S, Scheberl A, Friedmann J, Sleytr UB, Messner P (2007) Novel biocatalysts based on S-layer self-assembly of Geobacillus stearothermophilus NRS 2004/3a: a nanobiotechnological approach. Small 3:1549–1559
Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom APG, van Rantwijk F, van der Wielen LAM, Sheldon RA (2004) Preparation, optimization and structures of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 87:754–762
Schuster B, Pum D, Sleytr UB (2008) S-layer stabilized lipid membranes (review). Biointerphases 3:FA3-11
Schwarz A, Thomsen MS, Nidetzky B (2009) Enzymatic synthesis of β-glucosylglycerol using a continuous-flow microreactor containing thermostable β-glycoside hydrolase CelB immobilized on coated microchannel walls. Biotechnol Bioeng 103:865–872
Shah S, Sharma A, Gupta MN (2006) Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder. Anal Biochem 351:207–213
Sheldon RA (2007a) Enzyme immobilization: the quest for optimum performance. Adv Synth Catal 349:1289–1307
Sheldon RA (2007b) Cross-linked enzyme aggregates (CLEAs): stable and recyclable biocatalysts. Biochem Soc Trans 35:1583–1587
Sleytr UB, Egelseer E-M, Pum D, Schuster B (2004) S-layers. In: Niemeyer CM, Mirkin CA (eds) Nanobiotechnology. Wiley-VCH, Weinheim, pp 77–92
Sleytr UB, Huber C, Ilk N, Pum D, Schuster B, Egelseer EM (2007) S-layers as a tool kit for nanobiotechnological applications. Microbiol Lett 267:131–144
Tang J, Badelt-Lichtblau H, Ebner A, Preiner J, Kraxberger B, Gruber HJ, Sleytr UB, Ilk N, Hinterdorfer P (2008) Fabrication of highly ordered gold nanoparticle arrays templated by crystalline lattices of bacterial S-layer protein. ChemPhysChem 9:2317–2320
Thomsen MS, Nidetzky B (2009) Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes. Biotechnol J 4:98–107
Tschiggerl H, Breitwieser A, de Roo G, Verwoerd T, Schäffer C, Sleytr UB (2008) Exploitation of the S-layer self-assembly system for site directed immobilization of enzymes demonstrated for an extremophilic laminarinase from Pyrococcus furiosus. J Biotechnol 133:403–411
Vafiadi C, Topakas E, Christakopoulos P (2008) Preparation of multipurpose cross-linked enzyme aggregates and their application to production of alkyl ferulates. J Mol Catal B 54:35–41
Villaverde A, Carrió MM (2003) Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett 25:1385–1395
Wang W-X, Pelah D, Alergand T, Shoseyov O, Altman A (2002) Characterization of SP1, a stress-responsive, boiling-soluble, homo-oligomeric protein from aspen. Plant Physiol 130:865–875
Wang W, Dgany O, Wolf SG, Levy I, Algom R, Pouny Y et al (2006) Aspen SP1, an exceptional thermal, protease and detergent-resistant self-assembled nano-particle. Biotechnol Bioeng 95:161–168
Wilson L, Illanes A, Pessela BCC, Abian O, Fernández-Lafuente R, Guisán JM (2004) Encapsulation of cross-linked penicillin G acylase aggregates in lentikats: evaluation of a novel biocatalyst in organic media. Biotechnol Bioeng 86(5):558–562
Yu HW, Chen H, Wang X, Yang YY, Ching CB (2006) Cross-linked enzyme aggregates (CLEAs) with controlled particles: application to Candida rugosa lipase. J Mol Catal B 43:124–127
Zhao L, Zheng L, Gao G, Jia F, Cao S (2008) Resolution of N-(2-ethyl-6-methylphenyl) alanine via cross-linked aggregates of Pseudomonas sp. lipase. J Mol Catal B 54:7–12
Acknowledgments
B.N. and J.N. acknowledge support from a project for scientific and technological co-operation between Austria and Slovakia (SK-AT-0024-08).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Roessl, U., Nahálka, J. & Nidetzky, B. Carrier-free immobilized enzymes for biocatalysis. Biotechnol Lett 32, 341–350 (2010). https://doi.org/10.1007/s10529-009-0173-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-009-0173-4