Advertisement

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

  • Ipsita Roy
  • Joyeeta Mukherjee
  • Munishwar N. GuptaEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Keywords

Cross-linking agents Glutaraldehyde Cross-linked enzyme aggregates Immobilization Thermal stability Enantioselectivity 

Notes

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.

References

  1. 1.
    Wold F (1967) Bifunctional reagents. Methods Enzymol 11:617–640CrossRefGoogle Scholar
  2. 2.
    Wold F (1972) Bifunctional reagents. Methods Enzymol 25:623–651CrossRefPubMedGoogle Scholar
  3. 3.
    Broun GB (1976) Chemically aggregated enzymes. Methods Enzymol 44:263–280CrossRefPubMedGoogle Scholar
  4. 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 307–324CrossRefGoogle Scholar
  5. 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 2:1361–1364CrossRefPubMedGoogle Scholar
  6. 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 11:665–670CrossRefGoogle Scholar
  7. 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 87:754–762CrossRefPubMedGoogle Scholar
  8. 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 43Google Scholar
  9. 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 7:327–329CrossRefPubMedGoogle Scholar
  10. 10.
    Majumder AB, Mondal K, Singh TP, Gupta MN (2008) Designing cross-linked lipase aggregates for optimum performance as biocatalysts. Biocatal Biotransform 26:235–242CrossRefGoogle Scholar
  11. 11.
    Dalal S, Sharma A, Gupta MN (2007) A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities. Chem Cent J 1:16CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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 351:207–213CrossRefPubMedGoogle Scholar
  13. 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 350–362Google Scholar
  14. 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 133:189–202CrossRefPubMedGoogle Scholar
  15. 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 23:141–147CrossRefGoogle Scholar
  16. 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 40:1095–1099CrossRefGoogle Scholar
  17. 17.
    Shah S, Gupta MN (2007) Kinetic resolution of (±)–1-phenylethanol in [Bmim][PF6] using high activity preparations of lipases. Bioorg Med Chem Lett 17:921–924CrossRefPubMedGoogle Scholar
  18. 18.
    Roy I, Gupta MN (2004) Preparation of highly active alpha-chymotrypsin for catalysis in organic media. Bioorg Med Chem Lett 14:2191–2193CrossRefPubMedGoogle Scholar
  19. 19.
    Solanki K, Gupta MN (2008) Optimizing biocatalyst design for obtaining high transesterification activity by a-chymotrypsin in non-aqueous media. Chem Cent J 2:1–7CrossRefGoogle Scholar
  20. 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 29:238–245CrossRefGoogle Scholar
  21. 21.
    Solanki K, Gupta MN, Halling PJ (2012) Examining structure-activity correlations of some high activity enzyme preparations for low water media. Bioresour Technol 115:147–151CrossRefPubMedGoogle Scholar
  22. 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 8:816–821CrossRefGoogle Scholar
  23. 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 17:320–323CrossRefGoogle Scholar
  24. 24.
    St. Clair NL, Navia MA (1992) Cross-linked enzyme crystal as robust biocatalysts. J Am Chem Soc 114:7314–7316CrossRefGoogle Scholar
  25. 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 21:368–372CrossRefGoogle Scholar
  26. 26.
    Ribero MH, Rabaca M (2011) Cross-linked enzyme aggregates of naringinase: novel biocatalysts for naringin hydrolysis. Enzym Res 2011:851272Google Scholar
  27. 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 166:925–932CrossRefPubMedGoogle Scholar
  28. 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 167:835–844CrossRefPubMedGoogle Scholar
  29. 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 102:3541–3545CrossRefPubMedGoogle Scholar
  30. 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 115:177–182CrossRefPubMedGoogle Scholar
  31. 31.
    Majumder AB, Gupta MN (2010) Stabilization of Candida rugosa lipase during transacetylation with vinyl acetate. Bioresour Technol 101:2877–2879CrossRefPubMedGoogle Scholar
  32. 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 3:25CrossRefGoogle Scholar
  33. 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 63:894–901CrossRefPubMedGoogle Scholar
  34. 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 5:852–857CrossRefPubMedGoogle Scholar
  35. 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 96:210–218CrossRefGoogle Scholar
  36. 36.
    Hilal N, Nigmatullin R, Alpatova A (2004) Immobilization of cross-linked lipase aggregates within microporous polymeric membranes. J Memb Sci 238:131–141CrossRefGoogle Scholar
  37. 37.
    Mukherjee J, Gupta MN (2015) Paradigm shifts in our view on inclusion bodies. Curr Biochem Eng 2:1–9Google Scholar
  38. 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 86:273–276CrossRefPubMedGoogle Scholar
  39. 39.
    Bell G, Halling PJ, Moore BD, Partridge J, Rees DG (1995) Biocatalyst behavior in low-water systems. Trends Biotechnol 13:468–473CrossRefGoogle Scholar
  40. 40.
    Tyagi R, Batra R, Gupta MN (1999) Amorphous enzyme aggregates: stability towards heat and aqueous-organic cosolvent mixtures. Enzyme Microb Technol 24:348–353CrossRefGoogle Scholar
  41. 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 6:1639–1842CrossRefGoogle Scholar
  42. 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 175–183CrossRefGoogle Scholar
  43. 43.
    Dalal S, Kapoor M, Gupta MN (2007) Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions. J Mol Catal B Enzymatic 44:128–132CrossRefGoogle Scholar
  44. 44.
    Arsenault A, Cabana H, Peter Jones J (2011) Laccase-based CLEAs: Chitosan as a novel cross-linking agent. Enzym Res 2011:376015CrossRefGoogle Scholar
  45. 45.
    Fairhead M, Thony-Meyer L (2010) Cross-linking and immobilization of different proteins with recombinant Verrucomicrobium spinosum tyrosinase. J Biotechnol 150:546–551CrossRefPubMedGoogle Scholar
  46. 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 102:6186–6191CrossRefPubMedGoogle Scholar
  47. 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 48:72–76CrossRefPubMedGoogle Scholar
  48. 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:e97221CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Cui JD, Li LL, Bian HJ (2013) Immobilization of cross-linked phenylalanine ammonia lyase aggregates in microporous silica gel. PLoS One 8:e80581CrossRefPubMedPubMedCentralGoogle Scholar
  50. 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 123:542–547CrossRefPubMedGoogle Scholar
  51. 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 6:2622–2628CrossRefPubMedGoogle Scholar
  52. 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 184:7–10CrossRefPubMedGoogle Scholar
  53. 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 130:801–804CrossRefPubMedGoogle Scholar
  54. 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 280:662–670CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Ipsita Roy
    • 1
  • Joyeeta Mukherjee
    • 2
  • Munishwar N. Gupta
    • 3
    Email author
  1. 1.Department of BiotechnologyNational Institute of Pharmaceutical Education and Research (NIPER)PunjabIndia
  2. 2.Chemistry DepartmentIndian Institute of Technology DelhiNew DelhiIndia
  3. 3.Department of Biochemical Engineering and BiotechnologyIndian Institute of Technology DelhiNew DelhiIndia

Personalised recommendations