Extração e caracterização de xilana do milho e da cana-de-açúcar para prospecção de xilanases em isolados bacterianos

Abstract

The reuse of agro-industrial residues has been the focus of several researches, mainly in the field of biotechnology, due to the biomass of these residues being rich in polymers that have a high added value, such as xylan. Xylan is the main hemicellulose, being the second most abundant polysaccharide, it has several applications in the food, pharmaceutical and biotechnological industry, and can be used in enzymatic prospecting using microorganisms, such as bacteria. This work aimed to extract xylan from three agricultural municipalities, corn cob, sugarcane bagasse and sugarcane straw for bioprospecting of xylanases used isolated from soil bacteria from Usina Japungu Agroindustrial S/A, located in Santa Rita – Paraiba. As a result of the xylan extraction step from sugarcane residues, a yield of 4.62g for straw and 3.80g for bagasse was obtained, starting from 30g of residue. To characterize the xylans obtained, the Fourier Transform Infrared Spectroscopy (FT-IR) method was used, which showed the main characteristic bands of xylan. For xylanase prospection, 16 isolates were selected from the microorganism bank of the Laboratory of Cellular and Molecular Biotechnology, in which for enzymatic production, the isolates were added in Petri dishes containing the agar/Luria Bertani medium, added 0.4% xylan from the cob from corn, bagasse and sugarcane straw and commercial xylan (Beechwood), separately. The strains were incubated in a bacteriological oven at 37°C for 72 h, and after this period, staining with Congo red (0.1%) was used to observe the degradation halo and determine the enzymatic index (IE). Among the xylanase positive strains (11 in total), 5 strains were positive for the 4 types of xylans used and 6 strains were positive only for commercial xylan. For enzymatic production, strains JS19, which presented IE ≥ 2, in all tested media, and JS20, which presented enzyme production only in the medium containing commercial xylan, were selected. The strains were cultivated in nutrient broth added with 0.5% of industrial residue, corn cob, straw and sugarcane bagasse, and incubated in a shaker (150 rpm) at 37°C for 72 h to evaluate the enzymatic activity (U/mL), by the reducing sugar method (3,5-dinitrosalicylic acid - DNS). The results showed that strain JS19 had a higher enzyme production, ranging from 0.74 to 0.92 (U/mL), compared to strain JS20 (0.12 to 0.30 U/mL). The xylanase bands visualized in polyacrylamide gel (Zymogram) were around 60-66.2 kDa. The sequencing of isolates JS19 and JS20 was performed using the 16S rRNA, in which both were identified at the level of Genus, Bacillus and Priestia respectively. Isolate JS19 has been deposited with GenBank as Bacillus sp., JS19 under code (OM759815). These preliminary data show that the reuse of agro-industrial residues, such as corn and sugar cane, are promising for obtaining xylans, as well as for enzymatic bioprospecting using microorganisms such as bacteria isolated from the soil.