Isolation, identification and screening of hidrolytic enzymes producing phylloplane yeasts

Abstract

Background Microorganisms are common colonizers of superficial (phylloplane) and internal tissues (endophyte) of a vari-ety plant species [1]. Phylloplane yeasts are considered a promising source of new and interesting biotechnologi-cal activities, particularly hydrolytic enzymes; however, the diversity of yeasts colonizing plant phylloplane and is still poorly known for most plant species. Considering the immense diversity of Brazilian flora, which include many species with medicinal properties, this knowledge is even more limited. Given the above, this study objec-tive to isolate, identify and to evaluate the production of hidrolytic enzymes of yeasts associated with leaves, stems and flowers of alecrim-do-campo (Baccharis dra-cunculifolia DC), a plant recognized as the main source of exudates used by bees to produce green propolis and also, for its medicinal properties. Methods For isolation of yeasts, samples leaves, stems and flowers of B. dracunculifolia were colected; 10 g were macered in 90 mL of saline solution (0,85%) and dilutions of the sus-pension were spread in solid medium BDA and TSA. After purification and preservation, the isolates were char-acterized by colony morphology and grouped. Isolates representant of each group were subsequently identified by molecular techniques based on the amplification and sequence analysis of ITS1-5, 8S-ITS2 rDNA. For evalua-tion and screening of potential enzymes producers, enzy-matic activity was performed in solid media [2,3]. Proteolitic and celulolitic activities were tested in YEPG media with 2% casein 0.5% carboxymethylcellulose (CMC). Activitities of the enzymes pectinase, amylase, lipase and esterase were performed in YNB media contain-ing 1% pectin, 0.2% starch, 1% oil and 1% Tween 80, respectively. After growth for 3-5 days at 28 ° C the enzy-matic activities of the isolates were evaluated by the pro-duction of degradation halo around the colony, which is the visual indication of hydrolysis of their respective substrates. Assays were performed with three replicates and expressed by enzymatic indice (IE), which was obtained by ration between diameter of halo and diamater of colony (mm). Results and conclusions Were obtained 69 isolates, being 74% of these isolates of yeasts and yeast-like fungi. On the basis of on colony mor-phology the isolates were grouped into four morphotypes (white, brown, black or salmon colony). The majority of the isolates was originating from samples of flowers and showed black colonies on PDA media. By analysis of ITS1-5,8S-ITS2 rDNA sequences in Genbank database, the isolates were identified as members of the species Star-merella bombicola, Aureobasidium leucospermi, A. pullu-lans, Rhodotorulla mucilaginosa and Occultifur externus. The isolates were promissing for enzyme production, spe-cially for amylolytic and lipolytic activities which were pre-dominant among the isolates. The most promising isolates were belonging to the genus Aureobasidium sp., which showed activities for the five enzymes tested. The isolates identified as promising for the production of enzymes, especially those of the genus Aureobasidium sp. were selected for more specific analyzes aimed at quantification and perfecting of culture conditions that allow greater pro-duction and can contribute to to the development of alter-natives to enzymes already available in market.