MICROBIOLOGICAL, ENZYMATIC AND GENOMIC CHARACTERIZATION OF A Paenibacillus sp. XYLANOLYTIC ISOLATE

GHIO, S; PICCINI F.E:; INSANI M; GRASSO D; CAMPOS E

Cordoba

Congreso; XI CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE 2015; 2015

Asociación Civil de Microbiología General SAMIGE

Paenibacillus sp. strains isolated from soil and lignocellulosic sources have been reported to produce extracellular enzymes useful for industrial applications. In second generation bioethanol production, enzymatic degradation of lignocellulosic biomass is considered the bottleneck, due to the recalcitrant nature of biomass, composed mainly by cellulose and hemicelluloses, mostly arabinoxylan. Therefore, improving its deconstruction is of key importance for full utilization of biomass. In this context, a cellulolytic and hemicellulolytic strain has been isolated from a previously characterized bacterial consortium obtained from a forest soil sample. By 16S rRNA sequencing and phylogenetic analysis it was classified as Paenibacillus sp, closely related to a cluster formed by P. taichungensis and P. pabuli. We named this strain Paenibacillus sp A59. It is a Gram positive, mesophilic and facultative anaerobe endospore-forming bacillus. Physiological and biochemical characterizations showed a high hydrolytic potential, indicated by good degrading activities of substrates such as xylan, cellulose, casein, citrus pectin and starch. It also showed tolerance to a wide range of pH and high salinity, being able to grow in culture from pH 5 to 10 and up to 7% NaCl. Scanning electron microscopy of bacteria growing on xylan in anaerobiosis showed high adherence to the substrate. In order to optimize culture media for enzymatic secretion, carboxymethyl cellulose, xylan from beechwood and sugarcane residue were used as sole carbon sources. The maximum xylanase activity was obtained when growing on minimal medium supplemented with xylan from beechwood, for 72 hours at 30°C. The optimal xylanase reaction condition was achieved at 50 °C, both pH 6 and pH 10, and the enzymatic extract was stable at 50 °C for 48 hours. Zymograms of cell free supernatants using xylan as substrate, revealed the presence of 30 and 70 kDa protein bands with xylanolytic activity. HPLC analysis of hydrolysis products released from xylan by cell-free supernatants showed three main peaks corresponding to xylotriose, xylobiose and xylose, indicating that this microorganism secretes the set of enzymes to obtain a complete degradation of xylan. A draft genome sequence was obtained by Illumina Miseq. In accordance to the observed enzymatic activities, genes coding for different enzymes were identified in the bacterial genome, in particular those involved in xylan hydrolysis, xylanases (of GH10 and GH11 families) and a beta xilosidase (GH43). These results showed the high xylanolytic potential of Paenibacillus sp A59 for industrial applications as well as for studying the xylanolytic bacterial system.