THE EVIDENCE OF NON N-GLYCAN LINKED MANNOSE IN EXOCHITINASE (42 kDa) FROM Trichoderma harzianum BIO10671 GLYCOSYLATION
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Keywords

Tricoderma harzianum
chitinase
glycosylation
N-glycan linked
mannose

Abstract

Chitinase 42 kDa produced by Trichoderma harzianum has been proven as a prime compound to be excreted onto the hyphae of the pathogen causing localised cell wall lysis at the point of interaction. This finally initiate the process of the host cell becomes empty of cytoplasm, disintegrates and shows a rapid collapse. This study investigates the existence of N-glycan linked mannose in chitinase 42 kDa produced by the Malaysian T. harzianum strain BIO10671.  The chitinase 42 kDa from T. harzianum BIO10671 was initially purified using anion exchange chromatography prior to a series of experiments such as immunoblotting against the chitinase 42 kDa antibody, lectin staining for detecting any terminal linked mannose, and galactofuranose detection to determine the presence of galatofuranose components in glycoproteins. The enzyme purification harvested about 12-fold of chitinase 42 kDa from T. harzianum BIO10671 with strong indication of the presence chitinase 42 kDa presence on SDS-Page. This was confirmed by immunoblotting with a strong response around 42 kDa after overnight incubation in chitinase 42 kDa antibody suggesting that the gene for chitinase 42 kDa was greatly expressed in this strain. There are no intervation of galatofuranose on any of the terminal mannose in chitinase 42 kDa as shown by negative results on samples treated with or without endoglycosidase-H and lectin staining. Therefore, it can be concludeed that glycosylation occurred in the chitinase 42 kDa from T. harzianum 42 kDa was not in the form of N-glycan linked mannose as expected.   

 


https://doi.org/10.29037/ajstd.259
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References

Archer, D.B. and Peberdy, J.F. (1997), The molecular biology of secreted enzyme production by fungi. Critical Reviews in Biotechnology, 17: 273-306.

Carsolio, C., Gutierrez, A., Jimenez, B., Van Montagu, M., and Herrera-Estrella, A. (1994), Characterization of ech42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism. Microbiology, 91: 10903-10907.

Correa, A. (1982), Endochitinase, a mannan-associated enzyme from Saccharomyces cerevisiae. Journal of Biological Chemistry, 257:1392-1397.

De La Cruz, J., Hidalgo-Gallego, A., Lora, J.M., Benitez, T., Pintor-Taro, J.A., and Llobell, A. (1992). Isolation and characterization of three chitinase in Trichoderma harzianum. European Journal of Biochemistry, 206: 859-867.

de las Mercedes, D.M., Limon, M.C., Mejias, R., Mach, R.L., Benitez, T., Pinto-Toro, J.A., and Kubicek, C.P. (2001), Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum. Current Genetic, 6: 335-342.

Deane, E.E., Whipps, J.M., Lynch, J.M., and Peberdy, J.F. (1998), The purification and characterization of a Trichoderma harzianum exochitinase. Biochimica et Biophysica Acta, 1383: 101-110.

Gakul, B., Lee, J.H., Song, K.B., Rhee, S.K., Kim, C.H., and Panda, T. (2000), Characterization and applications of chitinase from Trichoderma harzianum- A review. Bioprocess Engineering, 23: 691-694.

Haran, S., Schickler, H., Oppenheim, A., and Chet, I. (1995), New components of the chitinolytic system of Trichoderma harzianum. Mycological Research, 99: 441-446.

Harman, G.E., Hayes, C.K., Lorito, M., Broadway, R.M., Di-Pietro, A., Peterbauer, C. and Tronsmo, A. (1993), Chitinolytic enzymes of Trichoderma harzianum: purification of chitobiosidase and endochitinase. Phytopathology, 83: 313-318.

Kruszewska, J., Messner, R., Kubicek, C.P., and Palamarczyk, G. (1989), O-Glycosilation of protein by membrane fractions of Trichoderma reesei QM 9414. Journal of General Microbiology, 135: 301-307.

Laemmli, U.K. (1970), Clavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685.

Lima, L.H.C., Ulhoa, C.J., Fernandes, A.P., and Felix, C.R. (1997), Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rofsii and Rhzioctonia solani cell walls. Journal of Genetic Applied Microbiology, 43, pp. 31-37.

Maras, M., De Bruyn, A., Scraml, J., Herdewijn, P., Claeyssens, M., Fiers, W., and Contreras, R. (1997). Structural characterization of N-linked oligosaccharides from cellobiohydroles I secreted by filamentous fungus Trichoderma reesei RUTC 30. European Journal of Biochemistry, 245, pp. 617-625.

Matsumiya, M., Miyauchi, K., and Mochizuki, A. (2001), Characterization of 38 kDa and 42 kDa chitinase isozymes from the liver of Japanese common squid Todarodes pacificus. Fisheries Science, 68, pp. 603-609.

Matsuzawa, T., Amano, Y., Kubo, M., and Kanda, T. (1996), Mode of action of exo--1,3glucanase from Trichoderma pseudokoningii TM37 on various -D-glucans. Journal of the Society for Fermentation and Bioengineering, 74, pp. 261-267.

Neethling, D. and Nevalainen, H. (1995), Mycoparasitic species of Trichoderma harzianum produce lectins. Canada Journal of Microbiology, 42, pp. 141-146.

Nampoothiri, K.M., Baiju, T.V., Sandhya, C., Sabu, A., Szakacs, G., and Pandey, G. (2004), Process optimization for antifungal chitinase production by Trichoderma harzianum. Process Biochemistry, 39, pp. 1583-1590.

Pitson, S.M., Sevior, R.J., and Mc Dougal, B.M. (1993), Non-celluloytic fungal Bglucanase: their physiology and regulation. Enzyme Microbiology Technology, 15, pp. 178192.

Reissig, J.L., Strominger, J.L., and Lelor, L.P. (1955), A modified calorimetric method for the estimation of N-acetylamino sugar. Journal of Biological Chemistry, 217, pp. 959-966.

Steyaert, J.M., Stewart, A., Jaspers, M.V., Carpenter, M., and Ridgway, H.J. (2004), Coexpression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism by Trichoderma hamatum. Mycologia, 96, pp. 1245-1252.

Ulhoa, C.J. and Peberdy, J.F. (1993), Effect of carbon sources on chitobiase production by Trichoderma harzianum. Mycological Research, 97, pp. 45-48.

Vazquez-Garciduenas, A., Leal-Morales, C.A., and Herrera-Estrella, A. (1998), Analysis of the -1,3-glucanolytic system of the biocontrol agent Trichoderma harzianum. Applied and Environmental Microbiology, 64, pp. 1442-1446.

Viterbo, A., Haran, S., Friesem, D., Ramot, O., and Chet, I. (2001), Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM. FEMS Microbiology Letters, 200, pp. 164-174.

Wallis, G.L.F., Swift, R.J., Hemming, F.W., Trinci, A.P.J., and Peberdy, J.F. (1999), Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosyation. Biochinica et Biophysica Acta., 472, pp. 576-586.

Yan, B., Zhang, W., Ding, J., and Goa, P. (1999), Sequence pattern for the occurance of Nglycosylation in protein. Journal of Protein Chemistry, 15, pp. 511-521.

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