Identificação molecular e produção de enzimas celulolíticas por Trichoderma spp

Abstract

Trichoderma are anamorphic fungi belonging to the class of Hifomicetos, also called imperfect fungi, or asexual conidial and whose gender Hypocrea teleomorph. They are free-living fungi, distributed throughout the world and found in different soil temperatures, especially those containing organic matter. Usually are not considered important human pathogens, but there are some reports indicating occasional pathogenicity of some species. Its easy handling and in vitro, stability and viability of colonies preserved this kind are a big target for biotechnology research. Because of these characteristics were isolated Trichoderma from plant Victoria amazonica, Rollinia sp. Murraya paniculata and Strychnos cogens; wood Scleronema micranthum, known as cardeiro; jatoba (Hymenaea courbaril), land of cubiu culture (Solanum sessiliflorum) and Indian black earth, in order to identify the molecular biology, the species level, such isolates and to assess their ability to produce cellulolytic enzymes. Of the 30 lines obtained were cultured spore, which were preserved in mineral oil, Castellani and method in 10% glycerol. From the suspension in glycerol, each sample was inoculated in 20ml of 10μL BD and cultivated 26oC, 100 rpm for 40:00 h. Next was extracted genomic DNA, performed PCR of specific regions of the ITS-1 and ITS-2 ribosomal DNA sequencing and subsequently. For the production of enzymes, the isolates were first grown in induction medium. Were inoculated 10μL of spore solution (glycerol 10%) in 50 mL of solution Manachini where the substratum was used to carboxymethylcellulose. The fungi were incubated at 27 ° C, 120 rpm for 120 hours. The dosage of CMCase was performed using the method of acid Dinitrosalicílico. For the determination of β-glucosidase was used p-nitrophenyl-β-D-glucopyranoside (PNPG) as substrate for the enzyme. The total protein concentration was determined by the Bradford method, using the reagent concentrate commercial Bio-RadTM and bovine serum albumin (ASB) as standard. The result of molecular identification of the first 13 samples revealed the species: T. harzianum, T. koningii, T. asperellum, T. viride, T. ovaslisporum, T. hamatum, T. piluliferum and T. koningiopsis, with a percentage between 96 and 99% identity and 100% reliability. The results indicated CMCase enzyme to low values, less than 0.100 U / mL with the exception of T. koningii (MPCE 10 3.2), T. harzianum (MPCE 2 2.2a), both isolates of M. paniculata, and isolate 1437 identified as Trichoderma sp., from Indian black earth, which showed slightly higher levels of 0.112 and 0.103 and 0.105 U / mL, respectively. For β-glucosidase, the results showed high activity in the vast majority of isolates with emphasis on T. harzianum MPCE 3 3.1 (10.45 U / mL) and T. piluliferum Vrc 2 3.2 (9.71 U / mL). All isolates produced protein in culture medium containing carboxymethylcellulose as substrate inducer