Isolation, purification and functional characterization of Serine protease from a biocontrol agent Trichoderma harzianum
Paper Details
Isolation, purification and functional characterization of Serine protease from a biocontrol agent Trichoderma harzianum
Abstract
Trichoderma harzianum is widely used as a biocontrol agent by virtue of its mycoparasitic abilities. An array of enzymatic proteins causing proteolysis of fungal cell wall make it competent parasite of numerous plant pathogenic fungi, protease is one such enzyme. In the present study Trichoderma harzianum producing extracellular protease was isolated from soil samples. The extracellular protease secreted by that Trichoderma isolate was purified to 41.13-fold and specific activity of the enzyme was calculated to be 145.42 U/mg. The purified enzyme was characterized for various optima. The study revealed higher stability levels of protease exhibiting upto 43% activity after an incubation of 30 min at 45°C and pH 10.0. The enzyme was found stable and highly active exclusively at strong alkaline conditions (pH=10) which showed its stability and suitability for various commercial applications. Moreover, the stability of enzyme illustrated its promising role in proficient mycoparastisic ability of Trichoderma isolate which can be employed as a biocontrol agent.
Ahmed S. 2007. Expression and characterization of Xylanases form Trichoderma harzianum and an anaerobic bacterium. Ph.D. Thesis, Dept. Chem. Bioc-hem., Univ. Agri., Faisalabad, Paksitan. http:// eprints. hec.gov.pk/2339/1/2194.htm.
Besoaín X, Maria PL, Antonio A, Llacolén L, Melna S, Jaime MR. 2007. New strains obtained after UV treatment and protoplast fusion of native Trichoderma harzianum: their biocontrol effect on Pyrenochaeta lyocpersici. Electronic Journal of Biotechnology 10, 604-617.
Bradford MM. 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72, 248-254.
Chen LL, Liu LJ, Shi M, Song XY, Zheng CY, Chen XL, Zha YZ. 2009. Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningi SMF2; FEMS Microbiology Letters 299, 135–142.
Dubey L, Adhikary S, Kuniar J, Sinha N. 2010. Isolation, Production, Purification, Assay and Charact-erization of Alkaline Protease Enzyme from Aspergillus niger and its Compatibility with Commercial Detergents. Developmental Microbiology and Molecular Biology 1(1), 75-94.
El-safy EM, Abdul-Raouf UM. 2004. Production, Purification and Characterization Of Protease Enzyme from Bacillus Subtilis. Int. Conf. Develop. Environ. in The Arab World, Assiut. Univ p. 14.
Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. (2004) Trichoderma species-opportunistic, avirulent plant symbionts. Nature Reviews Micro-biolog 2, 43-56
Harman GE. 2006. Overview of mechanisms and uses of Trichoderma spp. Phytopathology 96,190-194.
Hjeljord L, Tronsomo A. 1988. Trichoderma and Gliocladium in biological control: an overview. In: Harman, G.E. Kubicek. C.P. (Eds). Trichoderma and Gliocladium. Taylor and Frances, London pp. 13-155.
Janice LDM, Felix CR. 2002. Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches’ broom disease. B.M.C. Biochemistry 3, 3. Epub 2002 Jan 22.
Klein D, Eveleigh DE. 1998. Ecology of Trichoderma. In: Kubicek, C.P., G.E. Harman (eds). Trichoderma and Gliocladium Basic Biology, Taxonomy and Genetics. Taylor and Francis Ltd. London 57-74.
Majid M. 2012. Isolation, purification and functional characterization of Protease from a biocontrol agent Trichoderma harzianum. M.Phil. Diss., University of Agriculture Faisalabad, Pakistan.
McDonald CB, Chen LL. 1965. Lowry modification of the Folin reagent for determination of proteinase activity. Annals of Biochemistry 10, 175-186.
Migheli Q, Balmas V, Zelazowska MK, Scherm B, Fiori S, Kopchinskiy AG, Kubicek CP, Druzhinina IS. 2009. Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by PAN European, invasive species of Hypocrea/ Trichoderma. Environ-mental Microbiology 11(1), 35-46.
Negi S, Banerjee R. 2010. Optimization of culture parameters to enhance production of amylase and protease from Aspergillius awamori in a single fermentation. African Journal of Biochemistry Research 4, 73-80.
Niyonzima FN, More SS. 2015 Purification and characterization of detergent-compatible protease from Aspergillus terreus gr. 3 Biotech 5(1), 61-70.
Pérez L, Polanco R, Rios JC, Montealegre J, Valderrama L, Herrera R, Besoaín X. 2007. The increase in endochitinases and β-1,3-glucanases in the mutant Th650-NG7 of the Trichoderma harzia-num The 650, improves the biocontrol activity on Rhizoc-tonia solani infecting tomato. Biological control of fungal and bacterial plant pathogens IOBC/wprs Bulletin 30, 135-138.
Rao MB, Tanksale AM, Ghatge MS, Despande VV. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews 62, 597-635.
Sharma J, Singh A, Kumar R, Mittal A. 2006. Partial Purification of an alkaline protease from a new strain of Aspergillus oryzae AWT 20 and its enhanced stabilization in entrapped Ca-Alginate Beads. The Internet Journal of Microbiology 2(2).
Siddiquee S, Tan SG, Umi-Kalsom Y, Fatihah NHN, Hasan MM. 2012. Characterization of Malay-sian Trichoderma isolates using random amplified microsatellites (RAMS). Molecular Biology Reports 39(1), 715-722.
Siddiquee S, Tan SG, Umi-Kalsom Y. 2010. Isozyme Analysis and Relationships Among Three Species in Malaysian Trichoderma Isolates. Journal of Microbiology and Biotechnology 20, 1266-1275.
Siddiquee S, Yusuf U, Kausar H, Jahan S. 2009. In vitro studies on the potential Trichoderma harzianum for antagonistic properties against Gano-derma boninense. Journal of Food, Agriculture and Environment 7, 970-976.
Vanitha N, Rajan S, Murugesan AG. 2014. Optimization and Production of Alkaline Protease enzyme from Bacillus subtilis168 isolated from food industry waste. International Journal of Current Microbiology and Applied Sciences 6, 36-44.
Vijay KM, Jesse D, Aitken F, Carvalho A, Tyler CC, Simon M, Shanthi S, Shanthi VS, Rob K, Ruth EL, Andrew TG. 2010. Metabolic Syndrome and Altered Gut Microbiota in Mice Lacking Toll-Like Receptor. Science 5, 228-328.
Weindling R, 1932. Trichoderma lignorum as a parasite of other soil fungi. Phytopathology 22, 837-845.
Muhammad Majid, Faiz Ahmad Joyia, Ghulam Mustafa, Muhammad Anjum Zia, Ghazala, Muhammad Sarwar Khan, 2016. Isolation, purification and functional characterization of Serine protease from a biocontrol agent Trichoderma harzianum. Int. J. Biosci., 9(1), 376-383.
Copyright © 2016 by the Authors. This article is an open access article and distributed under the terms and conditions of the Creative Commons Attribution 4.0 (CC BY 4.0) license.