Produção e caracterização de L-asparaginase de leveduras isoladas de pólen coletados de Melipona spp. da Região Amazônica

Abstract

Because biodiversity of the Amazon region and the enzyme potential of yeasts, this study aimed to select a producing yeast L-asparaginase, identify and assess the production parameters. In addition to analyzing the isolated influence of glucose in the biomass and L-asparaginase, biochemically characterize the enzymatic activity, perform pre-purification and to assess the degree of purity of the enzyme and its potential as an agent antiacrilamida. There were used 21 strains of yeasts and yeast with increased L-asparaginase activity was identified by DNA sequencing using NL-1 and NL-4 primers and used for evaluation of production parameters using two statistical planning, Plackett-Burman (PB) and Composite Central (CCD) . Glucose was evaluated by varying the concentration in the medium (0.5%, 1.0% and 5.0%) and through a factorial design 22 having as variables glucose (%) and aeration (%). A pre- purification was performed with ammonium sulfate evaluated on SDS-PAGE 12.5%. The test agent was aintiacrilamida as by inhibiting the formation of a polyacrylamide of 30% acrylamide solution. The selected yeast was identified as Tilletiopsis minor and the significant variables were presented by PB L- asparagine, yeast extract and MgSO4 , presenting 1975.67 U.mL-1 through L- aspraginase CCD 25 g.L-1 of L-asparagine, 40 g.L-1 yeast extract, 0.35 g.L-1 MgSO4 with medium adjusted to pH 5.0 at 28 °C for 72 hours. The results on the influence of glucose alone revealed that high levels of glucose decrease the production of L-asparaginase obtained with maximal activity at 1.0% glucose and aeration of 40%. The pH and optimum temperature of the enzyme was 7.0 and 50 °C respectively, and the Na+ ion with higher inhibitory effect on the L-asparaginase activity. The L-asparaginase produced showed a 7.8 purification factor in the fraction of 20-40% ammonium sulfate and 100% inhibited the formation of polyacrylamide . The electrophoretic profile showed a sharper band 80.5 kDa . The results show the Tilletiopsis minor studied as new producer of L-asparaginase source with potential for applications in the food industry.