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Cross-Linked Enzyme Aggregates for Applications in Aqueous and Nonaqueous Media

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Enzyme Stabilization and Immobilization

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1504))

Abstract

Extensive cross-linking of a precipitate of a protein by a cross-linking reagent (glutaraldehyde has been most commonly used) creates an insoluble enzyme preparation called cross-linked enzyme aggregates (CLEAs). CLEAs show high stability and performance in conventional aqueous as well as nonaqueous media. These are also stable at fairly high temperatures. CLEAs with more than one kind of enzyme activity can be prepared, and such CLEAs are called combi-CLEAs or multipurpose CLEAs. Extent of cross-linking often influences their morphology, stability, activity, and enantioselectivity.

Prof. Finn Wold, while at University of Minnesota, St. Paul, USA, introduced bifunctional reagents (more frequently called cross-linking reagents) to protein chemistry. Consequently several subsequent developments including CLEA design were possible. Prof. Wold was one of the early mentors of one of the authors (Munishwar N. Gupta). This chapter is dedicated to the memory of Prof. Finn Wold who was a great scientist and a great human being.

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References

  1. Wold F (1967) Bifunctional reagents. Methods Enzymol 11:617–640

    Article  CAS  Google Scholar 

  2. Wold F (1972) Bifunctional reagents. Methods Enzymol 25:623–651

    Article  CAS  PubMed  Google Scholar 

  3. Broun GB (1976) Chemically aggregated enzymes. Methods Enzymol 44:263–280

    Article  CAS  PubMed  Google Scholar 

  4. Gupta MN (1993) Applications of crosslinking techniques to enzyme/protein stabilization and bioconjugate preparation. In: Himmel ME, Georgiou G (eds) Biocatalyst design for stability and specificity. ACS Symposium Series Am. Chem. Soc, Washington, DC, pp 307–324

    Chapter  Google Scholar 

  5. 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

    Article  CAS  PubMed  Google Scholar 

  6. Cao L, van Langen LM, van Rantwijk F, Sheldon RA (2001) Crosslinked aggregates of penicillin acylase. Robust catalyst for the synthesis of ß-lactam antibiotics. J Mol Catal B Enzym 11:665–670

    Article  CAS  Google Scholar 

  7. Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom AP, van Rantwijk F, van der Wielen LA, Sheldon RA (2004) Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 87:754–762

    Article  CAS  PubMed  Google Scholar 

  8. Sheldon RA, Schoevaart R, van Langen LM (2006) Cross-linked enzyme aggregates. In: Guisan JM (ed) Immobilization of enzymes and cells. Humana Press, Totowa, NJ, p 43

    Google Scholar 

  9. van Langen LM, Selassa RP, van Rantwijk F, Sheldon RA (2005) Cross-linked aggregates of (R)-oxynitrilase: a stable, recyclable biocatalyst for enantioselective hydrocyanation. Org Lett 7:327–329

    Article  PubMed  Google Scholar 

  10. Majumder AB, Mondal K, Singh TP, Gupta MN (2008) Designing cross-linked lipase aggregates for optimum performance as biocatalysts. Biocatal Biotransform 26:235–242

    Article  CAS  Google Scholar 

  11. Dalal S, Sharma A, Gupta MN (2007) A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities. Chem Cent J 1:16

    Article  PubMed  PubMed Central  Google Scholar 

  12. 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

    Article  CAS  PubMed  Google Scholar 

  13. Sheldon RA (2006) Immobilization of enzymes as cross-linked enzyme aggregates: a simple method for improving performance. In: Patel RN (ed) Biocatalysis in the pharmaceutical and biotechnology industries. CRC Press, Boca Raton, NY, pp 350–362

    Google Scholar 

  14. Illanes A, Wilson L, Caballero E, Fernández-Lafuente R, Guisan JM (2006) Cross-linked penicillin acylase aggregates for synthesis of β-lactam antibiotics in organic medium. Appl Biochem Biotechnol 133:189–202

    Article  CAS  PubMed  Google Scholar 

  15. Sheldon RA, Schoevaart R, van Landen LM (2005) Cross-linked enzyme aggregates (CLEAs): a novel and versatile method for enzyme immobilization (a review). Biocatal Biotransform 23:141–147

    Article  CAS  Google Scholar 

  16. Ruiz Toral A, de Los Rios AP, Hernandez FJ, Janssen MHA, Schoevaart R, van Rantwijk F, Sheldon RA (2007) Cross-linked Candida antarctica lipase B is active in denaturing ionic liquids. Enzyme Microb Technol 40:1095–1099

    Article  Google Scholar 

  17. Shah S, Gupta MN (2007) Kinetic resolution of (±)–1-phenylethanol in [Bmim][PF6] using high activity preparations of lipases. Bioorg Med Chem Lett 17:921–924

    Article  CAS  PubMed  Google Scholar 

  18. Roy I, Gupta MN (2004) Preparation of highly active alpha-chymotrypsin for catalysis in organic media. Bioorg Med Chem Lett 14:2191–2193

    Article  CAS  PubMed  Google Scholar 

  19. Solanki K, Gupta MN (2008) Optimizing biocatalyst design for obtaining high transesterification activity by a-chymotrypsin in non-aqueous media. Chem Cent J 2:1–7

    Article  Google Scholar 

  20. Majumder AB, Gupta MN (2011) Increasing catalytic efficiency of Candida rugosa lipase for the synthesis of tert-alkyl butyrates in low water media. Biocatal Biotrasform 29:238–245

    Article  CAS  Google Scholar 

  21. Solanki K, Gupta MN, Halling PJ (2012) Examining structure-activity correlations of some high activity enzyme preparations for low water media. Bioresour Technol 115:147–151

    Article  CAS  PubMed  Google Scholar 

  22. Hobbs HR, Kondor B, Stephenson P, Sheldon RA, Thomas NR, Poliakoff M (2006) Continuous kinetic resolution catalysed by cross-linked enzyme aggregates, “CLEAs”, in supercritical CO2. Green Chem 8:816–821

    Article  CAS  Google Scholar 

  23. Mateo B, 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 Asymm 17:320–323

    Article  CAS  Google Scholar 

  24. St. Clair NL, Navia MA (1992) Cross-linked enzyme crystal as robust biocatalysts. J Am Chem Soc 114:7314–7316

    Article  CAS  Google Scholar 

  25. Kumari V, Shah S, Gupta MN (2007) Preparation of biodiesel by lipase-catalyzed transesterification of high free fatty acid containing oil from Madhuca indica. Energ Fuel 21:368–372

    Article  CAS  Google Scholar 

  26. Ribero MH, Rabaca M (2011) Cross-linked enzyme aggregates of naringinase: novel biocatalysts for naringin hydrolysis. Enzym Res 2011:851272

    Google Scholar 

  27. Yan J, Gui X, Wang G, Yan Y (2012) Improving stability and activity of cross-linked enzyme aggregates based on polyethyleneimine in hydrolysis of fish oil for enrichment of polyunsaturated fatty acids. Appl Biochem Biotechnol 166:925–932

    Article  CAS  PubMed  Google Scholar 

  28. Cui JD, Zhang S, Sun LM (2012) Cross-linked enzyme aggregates of phenylalanine ammonia lyase: novel biocatalysts for synthesis of L-phenylalanine. Appl Biochem Biotechnol 167:835–844

    Article  CAS  PubMed  Google Scholar 

  29. Wang M, Jia C, Qi W, Yu Q, Peng X, Su R, He Z (2011) Porous CLEAs of papain: application to enzymatic hydrolysis of macromolecules. Bioresour Technol 102:3541–3545

    Article  CAS  PubMed  Google Scholar 

  30. Hormigo D, García-Hidalgo J, Acebal C, de la Mata I, Arroyo M (2012) Preparation and characterization of cross-linked enzyme aggregates (CLEAs) of recombinant poly-3-hydroxybutyrate depolymerase from Streptomyces exfoliatus. Bioresour Technol 115:177–182

    Article  CAS  PubMed  Google Scholar 

  31. Majumder AB, Gupta MN (2010) Stabilization of Candida rugosa lipase during transacetylation with vinyl acetate. Bioresour Technol 101:2877–2879

    Article  CAS  PubMed  Google Scholar 

  32. Guauque TMP, Foresti ML, Ferreira ML (2013) Cross-linked enzyme aggregates (CLEAs) of selected lipases: a procedure for the proper calculation of their recovered activity. AMB Express 3:25

    Article  Google Scholar 

  33. Li L, Li G, Cao LC, Ren GH, Kong W, Wang SD, Guo GS, Liu YH (2015) Characterization of cross-linked enzyme aggregates of a novel beta galactosidase, a potential catalyst for the synthesis of galacto-oligosaccharides. J Agric Food Chem 63:894–901

    Article  CAS  PubMed  Google Scholar 

  34. Wilson L, Illanes A, Abian O, Pessela BCC, Fernandez-Lafuente R, Guisán JM (2004) Co-aggregation of penicillin G acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media. Biomacromolecules 5:852–857

    Article  CAS  PubMed  Google Scholar 

  35. 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. Biotech Bioeng 96:210–218

    Article  CAS  Google Scholar 

  36. Hilal N, Nigmatullin R, Alpatova A (2004) Immobilization of cross-linked lipase aggregates within microporous polymeric membranes. J Memb Sci 238:131–141

    Article  CAS  Google Scholar 

  37. Mukherjee J, Gupta MN (2015) Paradigm shifts in our view on inclusion bodies. Curr Biochem Eng 2:1–9

    CAS  Google Scholar 

  38. Mateo C, Palomo JM, van Langen LM, Rantwijik FV, Sheldon RA (2004) A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates. Biotechnol Bioeng 86:273–276

    Article  CAS  PubMed  Google Scholar 

  39. Bell G, Halling PJ, Moore BD, Partridge J, Rees DG (1995) Biocatalyst behavior in low-water systems. Trends Biotechnol 13:468–473

    Article  CAS  Google Scholar 

  40. Tyagi R, Batra R, Gupta MN (1999) Amorphous enzyme aggregates: stability towards heat and aqueous-organic cosolvent mixtures. Enzyme Microb Technol 24:348–353

    Article  CAS  Google Scholar 

  41. López-Gallego F, Betancor L, Hidalgo A, Alonso N, Fernandez-Láfuente R, Guisán JM (2005) Co-aggregation of enzymes and polyethyleneimine: a simple method to prepare stable and immobilized derivatives of glutaryl acylase. Biomacromolecules 6:1639–1842

    Article  Google Scholar 

  42. Vaidya A, Fischer L (2006) Stabilization of new imprint property of glucose oxidase in pure aqueous medium by cross-linked-imprinting approach. In: Guisan JM (ed) Immobilization of enzymes and cells. Humana Press, NJ, pp 175–183

    Chapter  Google Scholar 

  43. Dalal S, Kapoor M, Gupta MN (2007) Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions. J Mol Catal B Enzymatic 44:128–132

    Article  CAS  Google Scholar 

  44. Arsenault A, Cabana H, Peter Jones J (2011) Laccase-based CLEAs: Chitosan as a novel cross-linking agent. Enzym Res 2011:376015

    Article  Google Scholar 

  45. Fairhead M, Thony-Meyer L (2010) Cross-linking and immobilization of different proteins with recombinant Verrucomicrobium spinosum tyrosinase. J Biotechnol 150:546–551

    Article  CAS  PubMed  Google Scholar 

  46. Garcia-Garcia MI, Sola-Carvajal A, Sanchez-Carron G, Carcia-Carmona F, Sanchez-Ferrer A (2011) New stabilized FastPrep-CLEAs for sialic acid synthesis. Bioresour Technol 102:6186–6191

    Article  CAS  PubMed  Google Scholar 

  47. Chen J, Zhang J, Han B, Li Z, Li J, Feng X (2006) Synthesis of crosslinked enzyme aggregates (CLEAs) in CO2 expanded micellar solutions. Colloids Surf B Biointerfaces 48:72–76

    Article  CAS  PubMed  Google Scholar 

  48. Cui JD, Cui LL, Zhang SP, Zhang YF, Su ZG, Ma GH (2014) Hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase from Rhodotorula glutinis. PLoS One 9:e97221

    Article  PubMed  PubMed Central  Google Scholar 

  49. Cui JD, Li LL, Bian HJ (2013) Immobilization of cross-linked phenylalanine ammonia lyase aggregates in microporous silica gel. PLoS One 8:e80581

    Article  PubMed  PubMed Central  Google Scholar 

  50. Talekar S, Ghodake V, Ghotage T, Rathod P, Deshmukh P, Nadar S, Mulla M, Ladole M (2012) Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase. Bioresour Technol 123:542–547

    Article  CAS  PubMed  Google Scholar 

  51. Jiang Y, Shi L, Huang Y, Gao J, Zhang X, Zhou L (2014) Preparation of robust biocatalyst based on cross-linked enzyme aggregates entrapped in three-dimensionally ordered macroporous silica. ACS Appl Mater Interfaces 6:2622–2628

    Article  CAS  PubMed  Google Scholar 

  52. Ning C, Su E, Tian Y, Wei D (2014) Combined cross-linked enzyme aggregates (combi-CLEAs) for efficient integration of a ketoreductase and a cofactor regeneration system. J Biotechnol 184:7–10

    Article  CAS  PubMed  Google Scholar 

  53. Jung DH, Jung JH, Seo DH, Ha SJ, Kweon DK, Park CS (2013) One-pot bioconversion of sucrose to trehalose using enzymatic sequential reactions in combined cross-linked enzyme aggregates. Bioresour Technol 130:801–804

    Article  CAS  PubMed  Google Scholar 

  54. Ba S, Peter-Jones J, Cabana H (2014) Hybrid bioreactor (HBR) of hollow fibre microfilter membrane and cross-linked laccase aggregates eliminate aromatic pharmaceuticals in waste waters. J Hazard Mater 280:662–670

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by funds obtained from Department of Science and Technology [Grant No.: SR/SO/BB-68/2010] and Department of Biotechnology [Grant No.: BT/PR14103/BRB/10/808/2010], both Government of India organizations. Finally, we thank past members of our research group; Dr. Kalyani Mondal, Dr. Shweta Shah, Dr. Abir Majumder, Dr. Sohel Dalal, and Veena Singh, whose work has been described/quoted in this chapter.

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Authors and Affiliations

  1. Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab, India

    Ipsita Roy

  2. Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110 016, India

    Joyeeta Mukherjee

  3. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110 016, India

    Munishwar N. Gupta

Authors
  1. Ipsita Roy
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  2. Joyeeta Mukherjee
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  3. Munishwar N. Gupta
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Corresponding author

Correspondence to Munishwar N. Gupta .

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Editors and Affiliations

  1. Department of Chemistry, and Department of Materials Science and Engineering, University of Utah, Salt Lake City, Utah, USA

    Shelley D. Minteer

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Roy, I., Mukherjee, J., Gupta, M.N. (2017). Cross-Linked Enzyme Aggregates for Applications in Aqueous and Nonaqueous Media. In: Minteer, S. (eds) Enzyme Stabilization and Immobilization. Methods in Molecular Biology, vol 1504. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6499-4_9

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  • DOI: https://doi.org/10.1007/978-1-4939-6499-4_9

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