ABSTRACT
Abstract The cellulolytic activity of fungi growing in the subtropical rainforest of Misiones (Argentina) represents a challenge in the technological development of the production of cellulosic bioethanol in the region using native sources. These fungi are promising to obtain sustainable enzyme cocktails using their enzymes. Cellulolytic ability of 22 white-rot fungi isolated from the subtropical rainforest of Misiones-Argentina in agar medium with two types of cellulosic substrates, carboxy-methylcellulose or crystalline cellulose, were comparatively analyzed, and the activity of two cellulolytic enzymes was evaluated in liquid medium. Although all isolates were able to grow and degrade both substrates in agar medium, and to produce total cellulase Filter paper (FPase) and endo-β-1,4-glucanase (EG) activities in broth, the isolate Irpex sp. LBM 034 showed the greatest enzymatic levels (FPase, 65.45 U L-1; EG, 221.21 U L-1). Therefore, the ITS sequence of this fungus was sequenced and analyzed through a phylogenetic analysis. These results indicate that the isolate LBM 034, corresponding to Irpex lacteus, has a promising cellulolytic ability and enzymes such as EG useful in sustainable saccharification of cellulosic materials in the region. Rev. Biol. Trop. 66(3): 1034-1045. Epub 2018 September 01.
Resumen La actividad celulolítica de hongos autóctonos asociados a la selva subtropical de Misiones (Argentina) representa un desafío en el desarrollo tecnológico de la producción de bioetanol celulósico en la región, mediante el uso de recursos nativos. Los sistemas enzimáticos de estos hongos tienen potencial aplicación en la obtención de cocteles enzimáticos rentables. La habilidad celulolítica de 22 hongos causantes de pudrición blanca se analizó comparativamente, que fueron aislados de la selva subtropical de Misiones-Argentina, en cultivos agarizados con dos tipos de sustratos celulósicos, carboxi-metilcelulosa o celulosa cristalina. También se evaluó la actividad de dos enzimas celulolíticas en cultivos líquidos. Aunque todos los aislamientos fueron capaces de crecer y degradar ambos sustratos en medio agarizado y revelar actividad celulolítica total y endo-β-1,4-glucanasa en cultivo líquido, el aislamiento Irpex sp. LBM 034 mostró las mayores actividades en papel de filtro con 65.45 U L-1 y endo-β-1,4-glucanasa con 221.21 U L-1, respectivamente. Por tanto, se secuenció y analizó la secuencia ITS de este hongo a través de un análisis filogenético. Estos resultados indicaron que el aislamiento LBM 034, correspondiente a Irpex lacteus, tiene una habilidad celulolítica prometedora en la producción de enzimas con actividad endo-β-1,4-glucanasa, útil en la sacarificación sustentable de materiales celulósicos de la región.
Subject(s)
Basidiomycota , Polyporales , Fungi , Argentina , beta-Glucosidase , CellulosomesABSTRACT
ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.