AMPLIFICATION AND CLONING OF CARBOHYDRATE ACTIVE ENZYMES FROM Pycnoporus sanguineus

WIRTH SONIA; GARRIDO, MERCEDES; CAMPOS, ELEONORA

San Miguel de Tucumán

Congreso; XII CONGRESO DE MICROBIOLOGÍA GENERAL; 2017

Asociación Civil de Microbiología General SAMIGE

Lignocellulosic biomass, which is composed of cellulose, hemicellulose and lignin, is an attractive substratefor the feed, pulp and paper industries and in the production of second generation bioethanol.Cellulose is a large linear homopolymer of beta-1,4 linked D-glucose while hemicelluloses are ramifiedheteropolymers with a xylan backbone. Complete breakdown of cellulose and xylan generate glucoseand xylose that can be fermented to ethanol and hence their importance in the obtention of biofuels.The present work focuses on one of the critical steps for the utilization of biomass that is the complexityto breakdown of the lignocellulosic wall. The general aim is the development of enzymatic complexesof fungal recombinant cellulases, hemicellulases and accessory enzymes and their potential industrialapplications. Using the transcriptomic information of expressed genes from the xylophagous fungusPycnoporus sanguineus BAFC2126, we identified the coding sequences for several putative carbohydrateactive enzymes selecting three of them for further analysis. We have amplified and cloned thecoding sequences of a GH3 b-xilosidase (2270bp), a key enzyme for obtaining the fermentable pentosexylose from xylobiose, a GH43 arabinofuranosidase (940bp), which helps in the debranching of xylan,and an AA9 (former GH61) lytic polysaccharide mono-oxigenase (1040bp) which has been reported toenhace glucanase activity. In silico analysis of proteins encoded by cloned sequences showed identitiesof 85%, 88% and 80% respectively with other fungal enzymes of the corresponding families. All thethree enzymes were cloned in vectors for expression in Pichia pastoris for recombinant production assecreted proteins, purification and further characterization.