Agroindustrial low-cost sources as substrate for production of L-asparaginase by filamentous fungi

Abstract

L-asparaginase (EC 3.5.1.1) is an enzyme produced by various microorganisms and responsible for the conversion of the amino acid L-asparagine to L-aspartic acid and ammonium. Its applications are wide ranging from the food industry providing the reduction of acrylamide in foods subjected to high temperatures as well as in the pharmaceutical field being applied as antitumor agent.Thus, the present work aimed to prospect for L-asparaginase from actinobacteria isolated from the Brazilian Cerrado (Minas Gerais), yeasts belonging to the Agricultural Microbiology Cultures Collection (CCMA) and filamentous fungi isolated from composting. All microorganisms were evaluated in semi-quantitative tests using L-asparagine-modified Czapeck Dox culture medium to select potential enzyme producers, followed by submerged fermentation and quantification of enzymatic activity by the Nessler method.The selected microorganisms were evaluated under the influence of different carbon sources as inducers on L-asparaginase production. Some actinobacterial and yeast species were positive for L-asparaginase production in the semi-quantitative assay, however none of these microorganisms produced the enzyme in FSbm. The seven filamentous fungi evaluated raised the pH of the medium in the semi-quantitative assay that indicates enzyme production.The filamentous fungus Penicillium solitum presented the highest enzymatic index in the semi-quantitative assay with 3.14 ± 0.24 (IE). An initial quantitative assay was performed with Aspergillus caepitosus, Aspergillus oryzae and Penicillium solitum strains in which the effect of carbon source (glucose and sucrose), pH (6.0; 6.5; 7.0) and fermentation time (24h, 48h, 72h, 96h and 120h) were evaluated. All results were subjected to analysis of variance (ANOVA) according to the data. It was found that the best treatments were 28, with volumetric activity (0.0320 U / mL) for Penicillium solitum, 23 with (0.0686 U / mL) for Aspergillus caepitosus and 7 with (0.0388 U / ml) for Aspergillus oryzae. Aspergillus caepitosus and Aspergillus oryzae strains were selected to produce L-asparaginase using Pereskiaaculeata Miller fibrous residue as substrate in FES and FSbm and whey in FSbm.In FES three humidifying agents were investigated, tap water, deionized water and Khanna salts. The values for enzyme production by Aspergillus caepitosus and Aspergillus oryzae in FES were (0.0245 U / mL) with Khana salts and (0.0185 U / mL) with deionized water, respectively. In FSbm for Aspergillus caepitosus extracellular L-asparaginase whey was the best carbon source with (0.0249 U/ mL). The production of intracellular L-asparaginase for the two filamentous fungi evaluated showed that both substrates in submerged fermentation were efficient for enzyme production. The possibility of using Pereskiaaculeata Miller extract as a low-cost substrate for L-asparaginase production is an innovative alternative. Such results are unpublished and have not been reported in the literature.