Sains Malaysiana 42(6)(2013): 715–724

Pengenalpastian dan Pencirian Gen Trichoderma virens UKM1 Mengekod Enzim

Terlibat dalam Pencuraian Kitin Krustasea

(Identification and Characterisation of Trichoderma virens UKM1 Genes Encoding

for Enzymes Involved in the Degradation of Crustacean Chitin)

Abdul Munir Abdul Murad*, Rafidah Badrun, Sakina Shahabudin, Shazilah Kamaruddin, Madihah Ahmad Zairun, Farahayu Khairuddin, Zamri Zainal & Farah Diba Abu Bakar

Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi

Universiti Kebangsaaan Malaysia, 43600 Bangi, Selangor D.E. Malaysia

Nor Muhammad Mahadi

Malaysia Genome Institute, Jalan Bangi Lama, 43000 Kajang, Selangor, Malaysia

Rosli Md. Illias

Jabatan Kejuruteraan Bioproses, Fakulti Kejuruteraan Kimia dan Kejuruteraan Sumber Asli,

Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

Diserahkan: 14 Mei 2012/Diterima: 13 September 2012

ABSTRAK

Kitin merupakan polisakarida struktur yang dapat dicurai oleh enzim kitinolisis kepada pelbagai terbitan yang boleh digunakan dalam bidang perubatan, pertanian dan rawatan air. Pengenalpastian dan pencirian gen-gen Trichoderma virens UKM1 mengekod enzim terlibat dalam pencuraian kitin krustasea telah dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) dan analisis pengekspresan gen menggunakan mikroatur DNA. Sebanyak tiga perpustakaan cDNA T. virens UKM1 yang masing-masing diaruh oleh kitin, glukosamina dan kitosan telah dibina. Sejumlah 1536 klon cDNA telah dijujuk dan sebanyak 1033 ESTs berkualiti telah dijana. Seterusnya, perbezaan pengekspresan gen apabila pertumbuhan kulat diaruh dengan kehadiran kitin krustasea dan tanpa kitin pada hari ketiga dan kelima telah ditentukan. Sebanyak 1824 klon cDNA telah dititik ke atas slaid kaca dan dihibrid bersama dengan cDNA terlabel Cy3 atau Cy5 yang disintesis daripada mRNA yang dipencil daripada kulat yang ditumbuhkan dalam medium mengandungi kitin krustasea atau glukosa (kawalan). Sebanyak 91 dan 61 gen, masing-masing bagi hari ketiga dan kelima didapati terekspres melebihi dua gandaan apabila kulat menggunakan kitin krustasea sebagai sumber karbon. Beberapa gen mengekod kitinase seperti ech1 dan cht3 (endokitinase), nag1 (eksokitinase) dan nagB(glukosamina 6-P-deaminase) didapati terekspres dengan tinggi pada kedua-dua hari. Selain daripada itu, gen mengekod protein hidrofobin, protease serina dan beberapa protein hipotetik juga terekspres dengan tinggi dengan kehadiran kitin krustasea. Protein-protein ini dijangka memainkan peranan penting dalam membantu pencuraian kitin krustasea.

Kata kunci: Kitin; kitinase; mikroatur DNA; penanda jujukan terekspres; Trichoderma virens

ABSTRACT

Chitin is a structural polysaccharide which can be degraded by chitinolytic enzymes to various derivatives that can be utilised in medicine, agriculture and water treatment. The identification and characterisation of Trichoderma virens UKM1 genes encoding for enzymes involved in crustacean chitin degradation were carried out by generating expressed sequence tags (ESTs) and analysing gene expression via DNA microarray. Three cDNA libraries of T. virens UKM1 induced with chitin, glucosamine and chitosan, respectively, were constructed. A total of 1536 cDNA clones were sequenced and 1033 of high-quality ESTs were generated. Subsequently, differences in gene expression between cells grown in the presence and absence of crustacean chitin on the third and fifth days were determined. A total of 1824 cDNA clones were spotted on glass slides and co-hybridised with Cy3- or Cy5-labeled cDNA, synthesised from mRNA isolated from cells grown in medium containing crustacean chitin or glucose (control). A total of 91 and 61 genes were expressed by more than two-fold on the third and fifth day, respectively, when the fungus used crustacean chitin as the carbon source. Several genes encoding for chitinase such as ech1 and cht3 (endochitinases), nag1 (exochitinase) and nagB(glucosamine 6-P-deaminase) were found highly expressed on both days. In addition, genes encoding for hydrophobin, serine protease and several hypothetical proteins were also expressed at high levels when cells were exposed to crustacean chitin. These proteins may play significant role in the degradation of crustacean chitin.

Keywords: Chitin; chitinase; DNA microarray; expressed sequence tag; Trichoderma virens

RUJUKAN

Al-Rashed, S.A.A., Bakar, F.D.A., Said, M., Hassan, O., Rabu, A., Illias, R.M. & Murad, A.M.A. 2010. Expression and characterization of the recombinant Trichoderma virens endochitinase Cht2. African Journal of Microbiology Research 4: 1758-1767.

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402.

Berger, J., Hautaniemi, S., Jarvinen, A., Edgren, H., Mitra, S. & Astola, J. 2004. Optimized LOWESS normalization parameter selection for DNA microarray data. BMC Bioinformatics 5: 194.

George, R.A. 2001. StackPACK clustering system. Briefings in Bioinformatics 2: 394-397.

Harman, G.E., Howell, C.R., Viterbo, A., Chet, I. & Lorito, M. 2004. Trichoderma species - opportunistic, avirulent plant symbionts. Nature Reviews Microbiology 2: 43-56.

Karlsson, M. & Stenlid, J. 2008. Comparative evolutionary histories of the fungal chitinase gene family reveal non-random size expansions and contractions due to adaptive natural selection. Evolution Bioinformatics 4: 47-60.

Kendrick, A. & Ratledge, C. 1992. Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme. European Journal of Biochemistry 209: 667-673.

Khoo, C.K., Mohd-Adnan, A., Kua, B.C. & Murad, A.M.A. 2009. Fabrikasi slaid mikroatur cDNA Lates calcarifer. Sains Malaysiana 38: 609-617.

Kim, D.J., Baek, J.M., Marcel, D., Uribe, P., Kenerley, C. & Cook, D. 2002. Cloning and characterization of multiple glycosyl hydrolase genes from Trichoderma virens. Current Genetics 40: 374-384.

Kumar, M.N.V.R. 2000. A review of chitin and chitosan applications. Reactive and Functional Polymers 46: 1-27.

Kurita, K. 2006. Chitin and chitosan: Functional biopolymers from marine crustaceans. Marine Biotechnology 8: 203-226.

Li, D.C. 2006. Review of fungal chitinases. Mycopathologia 161: 345-360.

Lora, J.M., Pintor-Toro, J.A., Benítez, T. & Romero, L.C. 1995. Qid3 protein links plant bimodular proteins with fungal hydrophobins. Molecular Microbiology 18: 380-382.

Oh, S.S.L., Bakar, F.D.A., Adnan, A.M., Mahadi, N.M., Hassan, O. & Murad, A.M.A. 2009. Isolation and characterization of glyceraldehydes-3-phosphate dehydrogenase gene of Trichoderma virens UKM1. Biotechnology 8: 194-203.

Ramli, A.N.M., Mahadi, N.M., Rabu, A., Murad, A.M.A., Bakar, F.D.A. & Illias, R.M. 2011. Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarcticaPI12. Microbial Cell Factories 10: 94.

Rattanakit, N., Plikomol, A., Yano, S., Wakayama, M. & Tachiki, T. 2002. Utilization of shrimp shellfish waste as a substrate for solid-state cultivation of Aspergillus sp. S1-13: Evaluation of a culture based on chitinase formation which is necessary for chitin-assimilation. Journal of Bioscience and Bioengineering 93: 550-556.

Suraini, A.Z., Teoh, L.S., Noorjahan, A., Neelam, S. & Kamarulzaman, K. 2008. Microbial degradation of chitin materials by Trichoderma virens UKM1. Journal of Biological Science 8: 52-59.

Tanaka, T., Takahashi, F., Fukui, T., Fujiwara, S., Atomi, H. & Imanaka, T. 2005. Characterization of a novel glucosamine-6-phosphate deaminase from a hyperthermophilic archaeon. Journal of Bacteriology 187: 7038-7044.

Tharanathan, R.N. & Kittur, F.S. 2003. Chitin - the undisputed biomolecule of great potential. Critical Reviews in Food Science and Nutrition 43: 61-87.

Uria, A.R., Chasanah, E. & Fawzya, Y.N. 2005. Optimization of Bacillus sp. k29-14 chitinase production using marine crustacean waste. Journal of Coastal Development 8: 123-130.

Wang, S.L., Chen, Y.H., Wang, C.L., Yen, Y.H. & Chern, M.K. 2005. Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp and crab shell powder medium. Enzyme and Microbial Technology 36: 660-665.

*Pengarang untuk surat-menyurat; e-mail: munir@ukm.my