Produção e imobilização covalente da inulinase obtida a partir de microrganismos isolados de frutos nativos da região Amazônica (Colômbia)

Abstract

Inulinases are nonspecific β-fructosidases that hydrolyze inulin into fructooligosaccharides (FOS), fructose and a little glucose. Fructose syrups are widely used as sweeteners in the food industry. Fructose is obtained from inulin by a simple enzymatic reaction with inulinases that produce 95% fructose. The present work aimed to produce and characterize inulinases from filamentous fungi and yeasts isolated and identified from the pulp of fruits from the Amazon region, such as: Cupuaçu (Theobroma grandiflorum), Araçá-boi (Eugenia stipitata McVaugh) and Cubiu (Solanum sessiliflorum). The research was organized into three chapters. Chapter I provides an overview of all microorganisms phenotypically identified by the API method with the potential to produce inulinase by submerged fermentation using inulin (Yacon) as a carbon source and at different times. From this evaluation, six morphotypes were chosen (1, 3, 5, 10, 17 and 21) and the time 48 hours as the best condition for enzyme production. Chapter II describes the DNA and RNA extraction methods for molecular identification that were performed with the six selected morphotypes and the transcriptomics of morphotype No.3 chosen for its GRAS (safe) status characteristic. The microorganisms were identified in genus and species using the ITS and 18S region of the rDNA by the SANGER method and comparing the in silico sequences with others deposited at NBCI, using the BLAST program. The expression of inulinase genes were evaluated from RNA by next-generation RNAseq Solexa/Illumina technology and bioinformatics analyses. Chapter III describes the production of inulinases from morphotype No. 3 identified as Aspergillus niger, using culture media containing different carbon sources (M1, M2, M3 and M6), partially purified with DEAE Sephacel resin equilibrated with 0.25M NaCl, immobilized in oxidized graphene (GO), biochemically characterized and evaluated in storage at 6 ºC. In SDS-PAGE electrophoresis a protein band with molecular weight around 97 kDa was observed. The best results were obtained using the M2 culture medium containing inulin to produce the extracellular enzyme, a pH of 4.5 and a temperature of 70 ºC, Km equal to 5.2 mg.mL-1 and Vmax of 40 U.mg-1 for immobilized enzyme, with a yield above 80%, seven cycles of reuse and stability of 34% after 30 days of refrigerated storage (6 ºC).