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Purification of extrachitinase 33kDa and its gene expression in Trichoderma longibrachitum

Muskhazli Mustafa*, Nor Azwady Abd. Aziz and Salifah Hasanah Ahmad Bedawi

Plant Systematic and Microbe, Department of Biology,Faculty of Science,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

e-mail: [email protected]

ABSTRACT The chitinolytic system of Trichoderma sp. is complex with several types of chitinase working together simultaneously to fulfil the entire niche. However, this enzyme has not been studied extensively for Trichoderma longibrachiatum although it has been proven to have significant effect as biocontrol. Prior to purification, both species were subjected on chitinase activity assay using culture medium supplemented with mycelium of Ganoderma boninensis and the peak of excochitinase activity was observed at 36h. Chitinase enzyme from the culture filtrate of T. longibrachiatum was purified through precipitation with 80% acetone followed by anion-exchange chromatography using Neobar AQ. Only one chitinase band at 33kDa in size has been acquired and four restriction endonucleases digestion also revealed a single copy of chitinase gene in this species. Assessment of this chitinase gene expression showed that the expression was strongly affected by substrate specificity; where the presence of glucose or non ß-1,4- linked substrate will significantly suppressed the gene transcriptions. In spite of this, 24 hours were still required for the gene transcription to achieve maximum total mRNA, which consistent with the peak of excochitinase activity 36h. Thus, it can be concluded that characterization of exochitinases 33kDa by T. longibrachiatum in term of activities, molecular weight, number of gene, total mRNA and substrate specificity are almost identical to T. harzianum sp.

INTRODUCTION Filamentous fungi of the genus Trichoderma have long been recognized as agents for the biocontrol of plant diseases which can directly impact mycelia or survival propagules of other fungi through production of toxic secondary metabolites (Benitez et al., 2004), formation of specialized structures and secretion of cell wall degrading enzymes (Noronha and Ulhoa, 1996). The most common cell wall degrading enzymes are chitinase, ß-1,3-glucanase, ß-1,6-glucanase and proteinases (Lorito et al., 1996). Secretion of these enzymes proposed to be regulated by catabolite repression (Lorito et al., 1996) and

the presence of fungal cell walls (Djonovic et al., 2006). Chitin and ß-1,3-glucans were considered as major component in cell wall and due to this factor, chitinase and ß-1,3-glucanase have been proposed as the key enzymes in mycoparasitism against phytopathogenic fungi (Elad et al, 1982).

The existence of chitinase has been known for decades. One of the components in the chitinolytic system is the chitinase 33kDa which has a molecular weight of around 33kDa. Filamentous fungi such as T. harzianum are able to secrete high levels of protein into the culture medium, and this advantage has been used widely in biotechnology industry to produce heterologous proteins (Archer and Peberdy, 1997). Chitinase 33kDa by itself is capable of degrading fungal cell walls and its antifungal activity can be enhanced by adding chitinase 33kDa or combined with gliotoxin (Steyaert e. al., 2004). Chitinase 33kDa from different strains display different physical characteristics such as pI values and substrate specificity (Gakul et al., 2000).

Even though chitinase have been purified from Trichoderma sp. for years, their physiological function has not been conclusively. Current knowledge on chitinase is limited to studies of biochemical and lytic properties of purified enzymes.To our knowledge, no report on chitinase 33kDa purification from T. longibrachiatum has been made previously although it has been proven to have significant effect as biocontrol agent. Therefore, the aim of this study was to purify chitinase 33kDA from T. longibrachiatum T28 and also assessed gene expression of this type of chitinase.

MATERIALS AND METHODS Preparation of cultures and enzyme assays Preparation of test strain culture was carried out by subcultured T.longibrachiatum T32 from slant agar to MYG plate culture prior to the collection of culture filtrate according to the method suggested by Muskhazli et al (2009). Then, the culture filtrate was dialysed against distilled water for at least 24 hours at 4ºC before been used in chiitnase and protein assays. Chitinase activity was determined using the method described by Reissig et al. (1955). While, protein determination was performed using Bio-Rad protein assay kit based on the method by Bradford (1976).

Probe Preparations Probes (1.2kb) for CHIT33 were prepared from clones of chit33 gene. All prepared DNA fragments were labelled with radioactive ( -32P) dCTP (Amersham) using the High Prime DNA kit (Boehringer Mannheim) according to the manufacturer’s recommendations and used as probes for Southern and Northern blot analysis.

Chitinase purification Unless indicated, all steps in purification were carried out according to the method used by Muskhazli

et al. (2008) and performed at 4ºC in two steps of purification comprised of acetone precipitation and followed by anion-exchange step (Neobar AQ column). Discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was also carried out on collected fraction according to Laemmli (1970) using 10% acrylamide gel and stained with Coomasie R-250 brilliant blue (Sigma). Low molecular range standard proteins were used for molecular mass determination.

Regulation of the chitinase gene. One hundred ml of Trichoderma seed culture was grown in Trichoderma Complete Medium (TCM; pH 5.5). Seed cultures were then shaken (180rpm) at 32ºC for three days before harvested, lyophilised, homogenised and used for DNA extraction (Murray and Thomson (1980).. The determination of chitinase gene regulation was carried out via Southern blot analysis according to the method suggested by Lora et al. (1994) using four mixtures of restriction enzymes: (1) NotI+EcoRI, (2) NotI+XhoI, (3) SacI+ApaI, (4) SacII+ApaI and (5) BamHI+EcoR.

Effect of substrate and incubation period on chitinase gene expression. The seed culture of T. longbrachiatum T28 was cultured as described previously and supplemented with 1% (w/v) glucose, 1% (w/v) ball-milled chitin, 1% (w/v) P. sajor-caju and 1% (w/v) G. boninensis mycelium, respectively. All seed cultures were shaken at 180rpm at 32ºC for another 72 hours before mycelium with maximal chitinase activity was collected and mRNA activity was determined according to Muskhazli et al. (2009).

RESULTS AND DISCUSSIONS

The elution pattern of anion exchange chromatography of the crude enzyme fraction (Figure 1a) gave two peaks for chitinase activity in T. longbrachiatum T28; designated as T28 C1 for fraction at 5-9 (18.7 moles. l-1) and T28 C2 (6.29 moles. l-1) for fraction number 23-32. Analysis of the pooled fractions by SDS-PAGE (Figure 1b) showed several major proteins bands in T28 (C2) while one band was observed in T28 (C1) at approximately 33kDa which is larger than purified chitinase collected by Deane et al. from T. harzianum T198 (1998). However, these sizes are similar to the range of endochitinases that have been reported previously (de la Cruz et al., 1992; Ulhoa and Peberdy, 1993).

Figure 1: Purification of chitinase by anion exchange chromatography. (a) Elution profile of T28 on protein Neobar AQ exchanger column with fraction 5-9 (T28 C1) and faction 23-32 (C2). (b) SDS-PAGE (10%) of protein from pooled peaks and stained with Coomassie blue.

The number of hybridizing DNA band detected for each restriction enzyme was as expected for single chitinase gene (Figure 2a). Similar result also observed by Limon et al. (1995) on chit33 in T. harzianum CECT2413. Even though T. longibrachiatum T28 has been described as non-mycoparasitic and lack of chitinase activity, Southern analysis shows a clear hybridisation of one copy gene for chit33 thus indicated that the purified chitinases are not generated from a single protein by proteolysis (Garcia et al., 1994).

Figure 2: (a) Southern blot analysis of the CHIT33 using 20mg of DNA digested with NotI + EcoRI (1), NotI + XhoI (2), SacI + ApaI (3), SacII + ApaI (4) and BamHI + EcoRV (5). (b) Expression of 60mg CHIT33 mRNA at maximum chitinase production using different carbon source. (c) Expression of 60mg CHIT33 mRNA at maximum chitinase production in medium supplemented with 1% of P. sajor-caju mycelium at different period of cultivation.

Northern analysis using chit33 as probe showed that expression of mRNA for chit33 in T. longibrachiatum T28 was inducted by the carbon source with repression by glucose (Figure 2b) indicated catabolite repression of chitin33 while the difference in intensity of total mRNA transcription between G. boninensis and P. sajor-caju would probably be due to differences in the accessibility of the enzyme to the chains of ß-1,4-linked N-acetylglucosamine in the different fungal cell wall The accumulation of mRNA was maximum after 24h incubation (exponential growth phase) and almost zero towards the end of incubation (stationary phase) thus indicated that chitinase enzyme activity and mRNA accumulation were time dependent and not correlated to mycelia growth (Figure 2c). However, this maximum mRNA transcription was not followed by maximum chitinase activity probably due to the delay between period of pre- and post-translation in endoplasmic reticulum and golgi complex before enzymes were transported outside the cell (Muskhazli et al. 2009).

CONCLUSION

All results presented in this study clarified the existence of only one gene of chitinase (chit33) which control the expression of chitinase enzyme at molecular weight around 33kDa. Furthermore, the expression of chit33 gene and mRNA transcription stills much under control of substrate specificity.

ACKNOWLEDGEMENT The authors are indebted to Universiti Putra Malaysia (UPM) for their technical assistance and Ministry of Science, Technology and Innovation for the research funding under Fundmental Research Programme (05-11-02-0060F).

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