PROIMI   05436
PLANTA PILOTO DE PROCESOS INDUSTRIALES MICROBIOLOGICOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics analysis
Autor/es:
VILLEGAS LILIANA; CALLEGARI, EDUARDO; DI MARCO, ENZO; MARTINEZ, M ALEJANDRA
Lugar:
Tucuman
Reunión:
Congreso; XII Congreso de SAMIGE (Sociedad Argentina de Microbiologia General); 2017
Institución organizadora:
Sociedad Argentina de Microbiología General
Resumen:
Three Paenibacillus strains, identified according to their 16S rDNA gene sequence and named as AR247, AR460-1 AR489, were selected due to their ability to produce glycoside hydrolases (GH) for second generation ethanol and other biotechnological applications. The assessment of extracellular enzyme production was previously approached by utilizing a mineral-based medium, MM0.2, addedwith agricultural by-products. These substrates are low-cost and abundantly available carbon sources for biotechnological purposes. Among the tested carbon sources, an alkali pretreated sugarcane bagasse (OH-SCB) was the one that better promoted the production of extracellular xylanases for all strains. The aim of this work was to study the differential extracellular enzymes expression by these strains through gel-free proteomic analysis method. Firstly, crude extracts were obtained by centrifugation after 72 h of cultivation in MM0.2 - OH-SCB 1% medium. Then, samples were concentrated by lyophilization and digested by using trypsin for further mass spectrometry analysis. Tryptic peptides obtained were analyzed using 2D nano-Ultra Performance Liquid Chromatography, coupled to tandem mass spectrometry. Bioinformatics analysis for protein identification was performed by searching against Swiss-Prot database, using Mascot server and ProteoIQ v2.8. Peptide summary report provided by Mascot evidenced hemicellulases which are active not only over b-1,4-linkages of xylose units but also on substituents of xylan. An endo-b-1,4-xylanase with 20.1 kDamolecular weight and 9.2 isoelectric point (pI) was found exclusively in crude extract samples of strain AR247. This enzyme, identified as a GH11, was also detected as a band between 18-20 kDa in zymograms and showed a pairwise similarity of ³ 99 with an homologous from Paenibacillus sp. Y412MC10. In addition, a second b-1,4-xylanase was identified in the secretome samples, yet not detectable through zymography, from both AR247 and AR489 strains. It was of 140.9 kDa, 4.7 pI (potential GH10), and showed to be closely related to a b-1,4-xylanase from P. glucanolyticus. Moreover, additional extracellular xylanase were found, which were related to a GH30 detected in P. favisporus and to two GH25 from P. polymyxa. Other proteins related to xylan utilization identified were: S?layer protein; sugar ABC transporters and sequences from substrate-binding proteins towards beta glucans. On the other hand, strains AR460-1 and AR489, revealed sequences corresponding to a carbohydrate binding module type CBM54 and an ABC transporter protein. Finally, a chitosanase belonging to GH8 family was only found in strain AR489.In conclusion gel-free proteomics analysis proved to be a useful methodology to achieve a widespread knowledge of the enzymatic repetory contributing to better quality and quantity of results.