Sains Malaysiana
38(3): 429-434(2009)
Purification
of Enzyme and Influence of Substrate Specificity on β-1,6-Gluc
ana
se
Gene Expression
in Trichoderma harzianum
(Penulenan enzim dan pengaruh
pengkhususan substrat ke atas pemaparan
gen β-1,6-glukanase pada Trichoderma harzianum)
M.Muskhazli1*, G.L.F.Wallis2, J.F. Peberdy2, H.A.B. Salifah1, S.N.Nurul1, G. Rusea1 &A.A. Nor Azwady1
1Department of Biology, Faculty of Science, Universiti Putra Malaysia
43400 UPM Serdang, Selangor D.E. , Malaysia
2School of Biological Studies, University of Nottingham
NG7 2RD Nottingham, United Kingdom
Diserahkan: 16 Jun 2008 / Diterima: 29 Ogos 2008
ABSTRACT
ß-1,6-gluc
ana
se produced by Trichoderma harzianum has been proven as one of the prime compounds to be excreted onto the hyphae of
the pathogen causing localised cell wall lysis at the point of
interaction. This study was conducted in
the interest to investigate the regulation of ß-1,6-gluc
ana
se
gene expression in T. harzianum strain BIO10671. β-1,6-gluc
ana
se enzyme from the culture filtrate of T. harzianum was purified through
precipitation with 80% acetone followed by anion-exchange chromatography and
chromatofocusing using Neobar AQ and Mono P HR 5/20 columns, respectively. Two β-1,6-gluc
ana
se
bands at 32 kDa and 43 kDa in size has been purified. However, four restriction
endonucleases digestion revealed only a single copy of ß-1,6-gluc
ana
se gene was encoded for both ß-1,6-gluc
ana
se
iso
zymes. Fungal cell walls were able to trigger high
level expression of gene encoding ß-1,6-gluc
ana
se. The expression of ß-1,6-gluc
ana
se gene was strongly affected by substrate
specificity; where the presence of glucose or non ß-1,6-glucan linked substrate
will significantly suppress the gene transcriptions. In spite of this, 24 hours were required for
the gene transcription to achieve maximum total mRNA.
Keywords: ß-1,6-gluc
ana
se; Gene; mRNA;
purification; substrate specificity
ABSTRAK
ß-1,6-gluc
ana
se
yang dihasilkan oleh Trichoderma
harzianum telah terbukti sebagai salah satu daripada komponan utama
yang dirembeskan ke dalam hifa patogen dan mengakibatkan hakisan setempat
dinding sel di titik sentuhan. Kajian
ini dijalankan dengan tujuan menyelidik kawalaturan pemaparan gen ß-1,6-gluk
ana
se di dalam T.
harzianum strain BIO10671. Enzim β-1,6-gluk
ana
s daripada cecair kultur T. harzianum telah ditulenkan menerusi pemendapan dengan 80% asiton
diikuti oleh kromatografi penukaran ion dan kromatografi pemfokusan,
masing-masing menggunakan kolum Neobar AQ dan Mono P HR 5/20. Dua jalur β-1,6-gluk
ana
se pada saiz 32 kDa dan 43 kDa telah ditulenkan. Walau
bagaim
ana
pun pemcernaan empat
endonukleus penyekat memaparkan hanya satu salinan gen ß-1,6-gluk
ana
se yang mengkodkan kedua-dua ß-1,6-gluk
ana
se
iso
zim. Dinding sel kulat berupaya mencetuskan tahap
pemaparan gen ß-1,6-gluk
ana
se yang
tinggi. Pemaparan gen ß-1,6-gluk
ana
s
amat dipengaruhi oleh pengkhususan substrat; kehadiran glukosa atau substrat
yang tidak mempunyai jalinan ß-1,6-glukan akan menghalang secara berkesan penyalinan
gen. Namun begitu, tempoh 24 jam masih
diperlukan bagi penyalinan gen untuk mencapai jumlah maksima mRNA.
Kata kunci: ß-1,6-gluk
ana
se; Gen; Penulenan,
mRNA; Pengkhususan
substrat
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*Pengarang untuk surat-menyurat; email: muskhazli@science.upm.edu.my
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