Malays. Appl. Biol. (2010) 39(2): 25-30
EXPRESSION AND CHARACTERISATION OF A RECOMBINANT ENDOGLUCANASE A (EglA) FROM Aspergillus niger
NURULERMILA MINOR1, ABDUL MUNIR ABDUL MURAD1, NOR MUHAMMAD MAHADI2, ROSLI MD ILLIAS3 and FARAH DIBA ABU BAKAR1*
1 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan. E-mail:
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2 Malaysia Genome Institute, Ministry of Science, Technology and Innovation, MTDC-UKM Technology Centre, Bangi 43600, Selangor Darul Ehsan.
3 Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81300 Skudai, Johor Darul Takzim
ABSTRACT
Fungi are efficient producers of very active cellulases. Aspergillus species can efficiently produce cellulolytic enzymes, which comprise three types of enzymes acting synergistically towards cellulose degradation, known as endoglucanase, exoglucanase, and ?-glucosidase. EglA is the major component of cellulose degradation by A. niger from glycosyl hydrolase family 12. Endoglucanase is characterised by its activity toward substituted cellulose derivatives, such as carboxymethylcellulose (CMC). The aim of this study is to express and characterise a recombinant EglA from A. niger. The EglA cDNA was successfully cloned into pET32b expression vector with the addition of linkers BamHI and HindIII at 5’ and 3’ end of the sequence, respectively. The protein was expressed in the Escherichia coli Origami DE3 expression system using optimised conditions of 20oC, 130 r.p.m. agitation, and 24 hours induction with 0.02 mM isopropyl-?-D-1- thiogalactopyranoside (IPTG). The EglA was then purified via Immobilised Metal-ion Affinity Chromatography (IMAC) and protein characterisation showed that it is a protein with a molecular weight of 44 kDa including hexa-histidine N- and C-terminal tags, having a pI of 4.62, with a specific activity of 65.01 U/mg towards CMC. The enzyme has an optimum temperature and pH of 50oC and 4 respectively. Temperature and pH stability are 30-50oC and pH 3-7, with more than 95% and 60% residual activities respectively.
ABSTRAK
Kulat merupakan salah satu mikroorganisma pengeluar selulase yang sangat aktif. Spesies Aspergillus menghasilkan enzim selulolitik yang terdiri daripada tiga jenis enzim bertindak secara sinergistik terhadap penguraian selulosa, iaitu endoglukanase, eksoglukanase, dan ?-glukosidase. EglA merupakan komponen utama penguraian selulosa oleh A. niger dan dikategorikan di dalam famili 12 glikosil hidrolase. Endoglukanase dicirikan melalui kebolehannya untuk menguraikan terbitan selulosa, seperti karboksimetilselulosa (CMC). Tujuan utama kajian ini adalah untuk mengekspres dan mencirikan EglA rekombinan daripada A. niger. cDNA EglA telah berjaya diklonkan ke dalam vektor pengekspresan pET32b dengan penambahan tapak penyesuai BamHI dan HindIII pada hujung 5’ dan 3’ jujukan. Protein tersebut diekspreskan menggunakan sistem pengekspresan Escherichia coli Origami DE3 dengan menggunakan keadaan yang telah dioptimumkan iaitu pada suhu 20oC, kadar goncangan 130 r.p.m., dan diaruh selama 24 jam dengan 0.02 mM isopropil-?-D-1-thiogalaktopiranosida (IPTG). Protein EglA kemudiannya telah berjaya ditulenkan dengan menggunakan kaedah Kromatografi Keafinan Ion Logam Terpegun (IMAC) dan hasil pencirian menunjukkan bahawa protein ini mempunyai berat molekul bersaiz 44 kDa termasuk penanda heksa-histidina penghujung-N dan penghujung-C, mempunyai pI 4.62, dengan aktiviti spesifik sebanyak 65.01 U/mg terhadap CMC. Suhu dan pH optimum enzim ini adalah 50oC dan 4, masing-masing. Manakala, kestabilan suhu dan pH pula adalah 30-50oC dan pH 3-7 dengan aktiviti residu sebanyak lebih daripada 95% dan 60%, masing-masing.
REFERRENCES
Alinda, A.H., Ester, D., Marc, V.M., Peter, J.I. & Leo, H. 2002. EglC, a New Endoglucanase from Aspergillus niger with Major Activity towards Xyloglucan. Applied And Environmental Microbiology, 68: 1556–1560.
Bhat, M.K. 2000. Cellulases and related enzymes in biotechnology. Biotechnology Advances, 18: 355- 383.
Blanco, A., Diaz, P., Martinez, J., Vidal, T., Torres, Parry, N.J., Beever, D.E., Owen, E., Nerinckx, W., A.L. & Pastor, F. 1998. Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterization of the enzyme. Performance in paper manufacture from cereal straw. Applied Microbiology Biotechnology, 50: 48–54.
Davies, G.J., Mackenzie, L., Varrot, A., Dauter, M., Brzozowski, A.M., Schulein, M. & Whiters, S.G. 1998. Snapshots along an enzymatic reaction coordinate: analysis of a retaining â-glycoside hydrolase. Biochemistry, 37: 11707-11713.
Doris, Q.H.X. 2010. Pembangunan selulase rekombinan kulat untuk pencuraian selulosa tandan kosong kelapa sawit. UKM MSc. Thesis Dissertation.
Harman, G.F. & Kubick, C.P. 1998. In Trichoderma and Gliocladium Enzymes, Biological Control and Commercial Applications; Taylor and Francis Ltd: London, U.K. 2:3.
Henrissat, B. and Davies, G. 1997. Structural and sequence-based classification of glycoside hydrolase. Current Opinion in Structural Biology, 7: 637- 644.
Howard, R.L., Abotsi, E., Jansen van Rensburg, E.L. & Howard, S. 2003. Lignocellulose biotechnology: issues of bioconversion and enzyme production. African Journal of Biotechnology, 2 (12): 602-619.
Khademi, S., Zhang, D., Swanson, S.M., Wartenberg, A., Witteb, K. & Meyera, E.F. 2002. Determination of the structure of an endoglucanase from Aspergillus niger and its mode of inhibition by palladium chloride. Biological Crystallography, 58: 660-667.
Lutzen, N.W., Nielsen, M.H., Oxiboell, K.M., Schiilein, M. & Olessen, B.S. 1983. Cellulase and their application in the conversion of lignocellulose to fermentable sugar. Philosophical Transactions of the Royal Societ, 300: 283-291.
Nishimura, A., Morita, M., Nishimura, Y. & Sugino, Y. 1990. A rapid and highly efficient method for preparation of competent cell Escherichia coli cells. Nucleic Acids Research 18(20): 6169.
Claeyssens, M., van Beeumen, J. & Bhat, M.K. 2002. Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus. Archieves of Biochemistry and Biophysics, 404: 243-253.
Ramirez, M.G., Avelizapa, L.I.R., Avelizapa, N.G.R. & Camarillo, R.C. 2004. Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases. Journal of Microbiological Methods 56: 213-219.
Vasina, J.A. & Baneyx, F. 1997. Expression of aggregation prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expression and Purification, 9: 211-218.
Weickert, M.J., Doherty, D.H., Best, E.A. & Olins, P.O. 1996. Optimization of heterologous protein production in Escherichis coli. Current Opinion Biotechnology, 7: 494-499.
Westra, D.F., Welling, G.W., Koedijk, D.G., Scheffer, A.J., The, T.H. & Welling-Wester, S. 2001. Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA. Journal of Chromatography B: Biomedical Sciences and Applications, 760: 129- 136.
Whitaker, J.R., Voragen, A.G.J. & Wong, D.W.S. 2003. Handbook of Food Enzymology; Marcel Dekker, Inc: New York. 61:761-762.
