Immobilization of Purified Fungal Laccase on Cost Effective Green Coconut Fiber and Study of its Physical and Kinetic Characteristics in Both Free and Immobilized Form

Author(s): Priyanka Ghosh, Uma Ghosh*.

Journal Name: Current Biotechnology

Volume 8 , Issue 1 , 2019

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Abstract:

Background: Laccases are important enzymes that have numerous applications in different biotechnological sectors.

Objective: The aim was to purify laccase from Aspergillus flavus PUF5, successfully immobilize it on coconut fiber and characterize different physical and kinetic properties under both free and immobilize conditions.

Methods: Laccase from A. flavus PUF5 was purified using ammonium sulfate precipitation, followed by DEAE column chromatography and gel filtration using Sephadex G100. The molecular weight was determined through SDS-PAGE (12%). It was immobilized on pretreated coconut fiber through crosslinking by glutaraldehyde (4% v/v). Physical and kinetic parameters like optimum temperature, pH, thermostability, the effect of additives, activation energy, Km and Vmax for free and immobilized laccase were also analyzed. Recycling stability of the immobilized laccase was further determined.

Results: The extracellular laccase (65 kDa) was purified up to homogeneity and was immobilized on acid-pretreated coconut fiber by 4% (v/v) glutaraldehyde solution at 30°C, pH 5.0. Activation energy (Ea) of free and immobilized laccase for oxidation of guaiacol was found to be 24.69 and 32.76 kJ mol-1 respectively. Immobilized laccase showed higher melting temperature (Tm) of (82.5°C) than free enzyme (73°C). Km and Vmax for free and immobilized laccase were found to be 0.67 mM, 0.70 mM and 280 U/mg, 336 U/mg respectively when guaiacol was used as substrate. Additionally, in immobilized condition laccase retained ˃80% of its initial activity after use till six repeated cycles.

Conclusion: The purified laccase enzyme and the cheap immobilization seem to be a prospective process for different biotechnological and industrial applications.

Keywords: Laccase, Aspergillus flavus PUF5, purification, coconut fiber, glutaraldehyde, immobilization, characterization.

[1]
Giardina P, Faraco V, Pezzella C, Piscitelli A, Vanhulle S, Sannia G. Laccases: a never-ending story. Cell Mol Life Sci 2010; 67(3): 369-85.
[http://dx.doi.org/10.1007/s00018-009-0169-1] [PMID: 19844659]
[2]
Hu X, Zhao X, Hwang HM. Comparative study of immobilized Trametes versicolor laccase on nanoparticles and kaolinite. Chemosphere 2007; 66(9): 1618-26.
[http://dx.doi.org/10.1016/j.chemosphere.2006.08.004] [PMID: 16979219]
[3]
Fernando Bautista L, Morales G, Sanz R. Immobilization strategies for laccase from Trametes versicolor on mesostructured silica materials and the application to the degradation of naphthalene. Bioresour Technol 2010; 101(22): 8541-8.
[http://dx.doi.org/10.1016/j.biortech.2010.06.042] [PMID: 20599376]
[4]
Maryšková M, Ardao I, García-González CA, Martinová L, Rotková J, Ševců A. Polyamide 6/chitosan nanofibers as support for the immobilization of Trametes versicolor laccase for the elimination of endocrine disrupting chemicals. Enzyme Microb Technol 2016; 89(1): 31-8.
[http://dx.doi.org/10.1016/j.enzmictec.2016.03.001] [PMID: 27233125]
[5]
Alberts JF, Gelderblom WC, Botha A, van Zyl WH. Degradation of aflatoxin B(1) by fungal laccase enzymes. Int J Food Microbiol 2009; 135(1): 47-52.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2009.07.022] [PMID: 19683355]
[6]
Plagemann R, Jonas L, Kragl U. Ceramic honeycomb as support for covalent immobilization of laccase from Trametes versicolor and transformation of nuclear fast red. Appl Microbiol Biotechnol 2011; 90(1): 313-20.
[http://dx.doi.org/10.1007/s00253-010-3038-9] [PMID: 21181152]
[7]
Jiang DS, Long SY, Huang J, Xiao HY, Zhou JY. Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochem Eng J 2005; 25(1): 15-23.
[http://dx.doi.org/10.1016/j.bej.2005.03.007]
[8]
Desai SS, Tennali GB, Channur N, Anup AC, Deshpande G, Murtuza BPA. Isolation of laccase producing fungi and partial characterization of laccase. Biotechnol Bioinf Bioeng 2011; 1(4): 543-9.
[9]
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193(1): 265-75.
[PMID: 14907713]
[10]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-5.
[http://dx.doi.org/10.1038/227680a0] [PMID: 5432063]
[11]
Singh AK, Chhatpar HS. Purification and characterization of chitinase from Paenibacillus sp. D1. Appl Biochem Biotechnol 2011; 164(1): 77-88.
[http://dx.doi.org/10.1007/s12010-010-9116-8] [PMID: 21049291]
[12]
Díaz R, Saparrat MC, Jurado M, et al. Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production. Appl Microbiol Biotechnol 2010; 88(1): 133-42.
[http://dx.doi.org/10.1007/s00253-010-2723-z] [PMID: 20607234]
[13]
Adamafio NA, Sarpong NS, Mensah CA, Obodai M. Extracel-lular laccase from Pleurotus ostreatus strain EM-1: Thermal stability and response to metal ions. Asian J Biochem 2012; 7(3): 143-50.
[http://dx.doi.org/10.3923/ajb.2012.143.150]
[14]
Nitheranont T, Watanabe A, Asada Y. Extracellular laccase produced by an edible basidiomycetous mushroom, Grifola frondosa: purification and characterization. Biosci Biotechnol Biochem 2011; 75(3): 538-43.
[http://dx.doi.org/10.1271/bbb.100790] [PMID: 21389619]
[15]
Asgher M, Iqbal HMN, Asad MJ. Kinetic characterization of purified laccase produced from Trametes versicolor IBL-04 in solid state bio-processing of corncobs. BioResources 2012; 7(1): 1171-88.
[16]
Xu L, Wang H, Ng T. A laccase with HIV-1 reverse transcriptase inhibitory activity from the broth of mycelial culture of the mushroom Lentinus tigrinus. J Biomed Biotechnol 2012.2012536725
[http://dx.doi.org/10.1155/2012/536725] [PMID: 22536022]
[17]
Chairin T, Nitheranont T, Watanabe A, Asada Y, Khanongnuch C, Lumyong S. Purification and characterization of the extracellular laccase produced by Trametes polyzona WR710-1 under solid-state fermentation. J Basic Microbiol 2014; 54(1): 35-43.
[http://dx.doi.org/10.1002/jobm.201200456] [PMID: 23775771]
[18]
Mainak M, Rintu B. Purification and biochemical characterization of a newly pro-duced yellow laccase from Lentinus squarrosulus MR13. 3 Biotech 2015; 5(3): 227-36.
[19]
Schilling JS, Tewalt JP, Duncan SM. Synergy between pretreatment lignocellulose modifications and saccharification efficiency in two brown rot fungal systems. Appl Microbiol Biotechnol 2009; 84(3): 465-75.
[http://dx.doi.org/10.1007/s00253-009-1979-7] [PMID: 19343340]
[20]
Jordaan J, Mathye S, Simpson C, Brady D. Improved chemical and physical stability of laccase after spherezyme immobilization. Enzyme Microb Technol 2009; 45: 43-5.
[http://dx.doi.org/10.1016/j.enzmictec.2009.08.017]
[21]
Soden DM, O’Callaghan J, Dobson AD. Molecular cloning of a laccase isozyme gene in the heterologous Pichia pastoris host. Microbiology 2001; 148: 4003-14.
[http://dx.doi.org/10.1099/00221287-148-12-4003] [PMID: 12480904]
[22]
Baldrian P. Fungal laccases - occurrence and properties. FEMS Microbiol Rev 2006; 30(2): 215-42.
[http://dx.doi.org/10.1111/j.1574-4976.2005.00010.x] [PMID: 16472305]
[23]
Patrick F, Mtui G, Mshandete AM, Johansson G, Kivaisi A. Purification and characterization of a laccase from the basidiomycete Funalia trogii (Berk.) isolated in Tanzania. Afr J Biochem Res 2009; 3(5): 250-8.
[24]
Tinoco R, Pickard MA, Vazquez-Duhalt R. Kinetic differences of purified laccases from six Pleurotus ostreatus strains. Lett Appl Microbiol 2001; 32(5): 331-5.
[http://dx.doi.org/10.1046/j.1472-765X.2001.00913.x] [PMID: 11328500]
[25]
Sangeetha K, Morris VB, Abraham TE. Stability and catalytic properties of encapsulated subtilisin in xerogels of alkoxisilanes. Appl Catal A Gen 2008; 341(1-2): 168-73.
[http://dx.doi.org/10.1016/j.apcata.2008.02.043]
[26]
Al-Adhami AJH, Bryjak J, Greb-Markiewicz B, Peczyn’ska-Czoch W. Immobilization of wood-rotting fungi laccases on modified cellulose and acrylic carriers. Process Biochem 2002; 37: 1387-94.
[http://dx.doi.org/10.1016/S0032-9592(02)00023-7]
[27]
Mirzadeh SS, Khezri SM, Rezaei S, Forootanfar H, Mahvi AH, Faramarzi MA. Decolorization of two synthetic dyes using the purified lac-case of Paraconiothyrium variabile immobilized on porous silica beads. J Environmental health Sci and Engineering 2014; 12(6)
[28]
Attala MM, Kheiralla ZH, Hamed ER, Youssry AA, Aty AA. Characterization and purification of the crude Trematosphaeria mangrovei laccase enzyme. Egypt Pharmaceut J 2015; 11(2): 93-100.
[29]
Nagai M, Sato T, Watanabe H, Saito K, Kawata M, Enei H. Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes, and decolorization of chemically different dyes. Appl Microbiol Biotechnol 2002; 60(3): 327-35.
[http://dx.doi.org/10.1007/s00253-002-1109-2] [PMID: 12436315]
[30]
Gianfreda L, Xu F, Bollag JM. Laccases: a useful group of oxidoreductive enzymes. Bioremediat J 1999; 3(1): 1-25.
[http://dx.doi.org/10.1080/10889869991219163]
[31]
More SS, Renuka PS, Pruthvi K, Swetha M, Malini S, Veena SM. Isolation, purification, and characterization of fungal laccase from Pleurotus sp. Enzyme Res 2011.2011248735
[http://dx.doi.org/10.4061/2011/248735] [PMID: 21977312]
[32]
Chakroun H, Mechichi T, Martinez MJ, Dhouib A, Sayadi S. Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: application on bioremediation of phenolic compounds. Process Biochem 2010; 45(4): 507-13.
[http://dx.doi.org/10.1016/j.procbio.2009.11.009]
[33]
Johannes C, Majcherczyk A. Laccase activity tests and laccase inhibitors. J Biotechnol 2000; 78(2): 193-9.
[http://dx.doi.org/10.1016/S0168-1656(00)00208-X] [PMID: 10725542]


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Article Details

VOLUME: 8
ISSUE: 1
Year: 2019
Page: [3 - 14]
Pages: 12
DOI: 10.2174/2211550108666190201151816

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