(10e) Selection and Characterization of Acid-Stable Cellulases Against Pretreated Oil Palm Empty Fruit Bunch From a Colombian Extreme Environment

The rapid growth of the oil palm industry around the world generates environmental concerns, with a production of more than 28.0 million tons per year of oil palm around the world, generating a great amount of by-products and wastes, such as liquid effluent, gaseous emission, and solid waste materials. Nowadays, solid waste is used commonly as manure, nevertheless the composting process is reported to be complex[1]. Therefore this agro-industry should take into consideration the use of sustainable technology. A material balance of an oil palm plant yields, in terms of mass, for every Ton of fresh fruit processed 220 kg of oil palm empty fruit bunch (OPEFB), 125-130 kg of fiber, around 50 kg of shell and 195-200 kg of palm oil [2]. Studies on the lignocellulosic content of the OPEFB have shown that display a high cellulose and hemicellulose and low lignin content [2]. This feature allows to use this waste as a substrate to obtain simple sugars, which can be used as carbon source in different fermentation processes to enable the production of alcohols that can be used as fuels. The biodegradation of cellulose is carried out by three classes of enzymes: endoglucanase (carboxymethylcellulase), exogluconase (cellobiohydrolase) and β-glucosidase, which together with a preliminary treatment of cellulose, manage to break the glycosidic bonds to produce simple sugars [3]. However, these enzymes hydrolyze in different parts of the polymer so the relation between the substrate and the enzyme should not be extrapolated due to different lignocellulosic structure [4]. Taking all this into account the objective of this study was to explore the cellulolytic potential of endophytes from a Colombian extreme environment for the saccharification of oil palm empty fruit bunch. It was determined the cellulolytic capacity for the saccharification in vitro of the pretreated oil palm empty fruit bunch of one hundred Colombian native endophytes isolated from young leaves attacked by insects (with herbivory) as well with no symptom of attack (without herbivory) from Espelieta grandiflora and E. corymbosa plants from the Páramo Crúz Verde. The microorganisms were screened to asses which of them have cellulolytic capacity on solid agar with CMC as substrate followed by Congo red staining method [5]. The microorganisms that showed cellulolytic activity were grown on cellulase inducting medium, using pretreated OPEFB as substrate, the activity of the three main components of cellulase (CMCase, β-glucosidase and Cellobiohydrolase) was measured, and total cellulolytic activity (Filter paper activity. FPA), using the procedures described by Zhang et. al (2009). Candidates obtained, (two entophytes), were partially purified through partial precipitation with ammonium sulfate (90%) [6] and chromatography using DAE-shepadex A-50 column previously equilibrated with sodium acetate buffer (pH 5, 0.05 M) and the fractions were withdrawn with a linear NaCl gradient, the fractions with cellulolytic activity were pooled and then concentrated by ultrafiltration with a centriprep (Amicon) having a 10 kDa molecular mass cut-off. The screenings allowed us to identify 28 endophytes with cellulolytic activity, and the best obtained were a Penicilium sp. and an Aspergillus sp. with maximum enzyme activities, CMCase, FPase, exogluconase and β-glucosidase, of 0.50-0.16, 0.035-0.032, 0.87-0.99 and 0.068-0.01 U/ml respectively. CMCase and the FPA activities of the partially purified cellulase was assessed at pHs ranging from 1 to 10, and temperatures ranging from 30 to 80 °C in order to find the optimum conditions for the cellulases. The cellulases from Penicillium sp. and Aspergillus sp. were stable at low pH which could be a valuable tool for hydrolysis of cellulose under acidic conditions.

References

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5.            Kasana, R., et al., A Rapid and Easy Method for the Detection of Microbial Cellulases on Agar Plates Using Gram’s Iodine. Current Microbiology, 2008. 57(5): p. 503-507.

6.            Scopes, R.K., Protein purification principles and practice Second ed. Spinger Advanced texts in chemistry, ed. C.R. Cantor. 1982: Springer-Verlag.