Sains Malaysiana 35(1): 31-35 (2006)

Isolation and Molecular Analysis of cDNA Encoding a

Partial Endoglucanase Gene From Aspergillus terreus SUK-l

(Pemencilan dan Analisis Molekul DNA kepada Sebahagian Gen Endoglukanase

daripada Aspergillus terreus SUK -1)

Nik Marzuki Sidik, Shaiful Adzni Sharifuddin, Othman Omar,

Abdul Jalil Abdul Kader & Sahidan Senafi

Pusat Pengajian Biosains dan Biotekno1ogi

Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia

43600 UKM Bangi, Se1angor D.E Malaysia

ABSTRAK

Kami melaporkan di sini pemencilan dan analisis satu fragmen cDNA untuk gen endoglukanase daripada Aspergillus terreus SUK-I. Sepasang pencetus telah direkabentuk berasaskan kepada kawasan terpelihara gen endoglukanase. Amplifikasi cDNA A. terreus SUK-I menggunakan pencetus berkenaan menghasilkan satu jalur DNA bersaiz 642 pb. Fragmen cDNA berkenaan telah ditulenkan dan diklonkan ke dalam vektor plasmid pCR®II-TOPO. cDNA selitan telah dilabelkan sebagai SA2 dan dijujukkan. Analisis terhadap jujukan SA2 menunjukkan sebahagian daripada jujukan SA2 mempunyai darjah identiti yang tinggi terhadap jujukan gen endoglukanase daripada A. aculeatus, A. kawachii dan Talaromyces emersonii. Sementara, jujukan asid amino putatif SA2 menunjukkan identiti sebanyak 74% terhadap protein endoglukanase daripada Thermoascus aurantiacus dan identiti sebanyak 69% terhadap protein endoglukanase daripada A. kawachii, A. niger, A. aculeatus dan A. nidulans. ldentiti sebanyak 66% juga diperhatikan di antara jujukan asid amino putatif SA2 dan jujukan protein endoglukanase A. oryzae. Oleh itu, kami membuat kesimpulan bahawa SA2 mengkodkan gen putative endoglukanase A. terreus SUK-I.

Kata kunci: Aspergillus terreus SUK-I, selulas, endoglukanase, CMCase, gen

ABSTRACT

We report here the isolation and sequence analysis of a cDNA fragment for an endoglucanase gene from Aspergillus terreus SUK-I. A pair of primers was designed based on conserved regions of endoglucanase gene. Amplification of A. terreus SUK-I cDNA using the primers produced a DNA band of 642 bp. The cDNA fragment was purified and cloned into pCR^®ll-TOPO plasmid vector. The cDNA insert was designated as SA2 and sequenced. Analysis of the SA2 sequence showed a high degree of identity towards endoglucanase gene sequences from A. aculeatus, A kawachii and Talaromyces emersonii. While, the putative amino acid sequence of SA2 showed 74 % identity towards endoglucanase protein from Thermoascus aurantiacus and 69 % identity towards endoglucanase protein from A. kawachii, A. niger, A. aculeatus and A. nidulans. A 66 % identity between SA2 putative amino acid sequence and A. oryzae endoglucanase protein sequence was also observed. Therefore, we concluded that SA2 codes for a putative endoglucanase gene of A. terreus SUK-I.

Keywords: Aspergillus terreus SUK-I, cellulase, endoglucanase, cMCase, gene

REFERENCES/RUJUKAN

Azevedo, M.O. & Radford, A. 1990. Sequence of the cbh-1 gene of Humicola grisea var thermoidea. Nucleic Acids Res. 18: 668.

Beguin, P. & Aubert, J.P. 1994. The biological degradation of cellulose. FEMS Microbiol. Rev. 13: 25-28.

Chikamatsu, G., Shirai, K., Kato, M., Kobayashi, T. & Tsukagoshi, N. 1999. Structure and expression properties of the endo-beta-1,4-glucanase A gene from the filamentous fungus Aspergillus nidulans. FEMS Microbiol. Let. 175: 239-­245.

Crawford, A.C., Kricker, J.A., Anderson, A.J.,Richardson, N.R. & Mather, P.B. 2004. Structure and function of a cellulose gene in redclaw crayfish, Cherax quadricarinatus. Gene 340: 267-274.

Hong, J., Tamaki, H., Yamamoto, K. & Kumagai, H. 2003.  Cloning of a gene encoding a thermo-stable endo-beta-1, 4-­glukanase from Thermoascus aurantiacus and its expression in yeast. Biotechnol. Let. 25: 657-661.

Kader, J., Zainal Abidin, Omar, O., Yusoff, W.M.W. & Daud, F. 1992. Pemencilan mikroorganisma selulolisis daripada enapcemar kelapa sawit. Sains Malaysiana 21: 75-85.

Mandel, M. & Reese, E.T. 1957. Induction of cellulose in fungi by cellobiose. J. Bacteriol. 73: 816-826.

Omar, O., Kader, J. & A1-Arikah Raja-E A1i 1984. Studies on extracellular cellu1ases during the growth of Aspergillus SUK-­1. Sains Malaysiana 13: 341-354.

Ooi, T., Sinmyo, A., Okada, H., Hara, S., Ikenaka, T., Murao, S. & Arai, M. 1990. Cloning and sequence analysis of a DNA for cellulase (F1-CMCase) from Aspergillus aculeatus. Cur. Genet. 18: 217-222.

Saha, B.C. 2003. Production, purification and properties of endog1ucanase from a newly isolated strain of Mucor circinelloides. Process Biochem. 39: 1871-1876.

Sambrook, J., Fritsch, E. & Maniatis, T. 1989. Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, New York.

Sheppard, P.O., Grant, F.J.,Oort, P.J.., Sprecher, C.A., Foster, D.C., Hagen, F.S., Upshall, A.U., McKnight, G.L. & O'Hara, P..J. 1994. The use of conserved cellulase family-specific sequences to clone cellulase homologue cDNAs from Fusarium oxysproum. Gene 150: 163-167.

Teeri, T.T., Salovuori, 1. & Knowles, J.K.C. 1983. The molecular cloning of the major cellulase gene from Trichoderma reesei. Biotechnol. 1: 696-699.

Van Peij, N.N., Gie1kens, M.M., de Vries, R.P., Visser, J. & de Graaff, L.H. 1998. The transcriptional activator XlnR regulates both xylanolytic and endog1ucanase gene expression in Aspergillus niger. Appl. Environ. Microbial. 64: 3615-3619.

Wood, T.M. 1985. Properties of cellulolytic enzyme systems. Biochem. Soc. Trans. 13: 407-410.