Screening And Identification Of Dextranase-producing Fungi And Research Of Enzymatic Properties

Two dextranase-producing fungi were screened from soil and were identified by morphological and molecular method. The fermentation of those two strains for producing dextranase and the enzymatic properties were studied. The contents were as follows.Screening of dextranase-producing microbes:screening medium using dextran as carbon source was adopted; the strains were anticipated to be obtained through series of screening and monospore isolation. The strains were primarily identified according to their colonial morphology, fermentation morphology and hyphae morphology which was observed after cellule cultivation. The enzymatic activity of dextranase from fermentation supernatant was tested using DNS reagent. The results were as follows:five microorganisms were obtained, and were numbered from D1 to D5. Through morphology identification, D1 and D5 were considered as Penicillium sp., D2 and D3 were considered as Fusarium sp., D4 was considered as Cephalosporium sp. D4 and D5 formed apparent blank area when they grew in blue dextran plates, and the enzymatic activity test also confirmed that they both had higher enzymatic activity, thus the two strain were selected for the next experiments.Identification of dextranase-producing species:The morphology of colony, conidiospores and conidiophores of D4 and D5 were studied. The ITS rDNA sequences of the two strains were cloned and sequenced, and then were compared with those available in the GenBank databases. The phylogenetic trees were derived with neighbor joining and analyzed with Bootstrapping. The results showed that the ITS sequence of D4 has a 99% similarity with Verticillium nigrescens, however, the result disagree with the identification based on morphological characteristics. After all D4 was identified as Cephalosporieae sp. according to its decided morphological characters. D5 was identified as Penicillium citreoviride according to its morphological characters, its ITS sequence analysis supported this conclusion. D5 strain had close consanguinities to P. daleae and P. janthinellum, which were 97% and 98%. Enzymatic properties studies:hydrolytic products of enzyme released from dextran, pullulan, starch and carboxymethyl cellulose were analyzed by TLC method. Enzymatic activity was measured by DNS reagent, in this way enzymatic properties, such as optimum temperature, temperature stability, optimum pH, pH stability and the effects of metal ion and ionic strength on dextranase were studied. The outcomes showed that the dextranase yielded by D4 and D5 was exo-Isomaltotriodextranase, which hydrolyzed serial a-1,6 glucosidic bond to release isomaltotriose from dextran while had no effects on other bonds. The enzyme has special substrate specificity and products homogeneity, therefore it is meaningful in theory and application. The enzyme yielded by D4 displayed maximum activity at 40℃; the half-life of the enzyme was 80 min at 40℃; the optimum pH value was 7, and the enzyme activity was stable from pH 5.6 to pH 7; the enzyme activity was enhanced by low concentration Mg2+ and Ba2+, and the ionic strength of NaCl at 150～300 mmol/L. The enzyme yielded by D5 displayed maximum activity at 55℃; the half-life of the enzyme was 3 h at 50℃; the optimum pH value was 6.5, and the enzyme activity was stable in the range of pH4.0 to pH6.5; the enzyme activity was enhanced by K+ and Ba2+, but was inhibited by Ca2+,Fe2+; the enzyme activity could be improved by the ionic strength of NaCl at 150 mmol/L.Fermentation for producing dextranase: The optimum conditions for Isomaltotriodextranase production were studied by flask-shaking fermentation and orthogonal experiments. The optimal condition for D4 strain were as follows:1% sucrose,1% dextran,0.2% casein, initial pH value 5.0,1.5 mL inoculating quantity, rate of shaking flask with 80 mL liquid in a 250 mL flask was 120 r/min. After fermentation for 11 days at 25℃, the enzyme activity reached 138.11 U/mL. The optimal condition for D5 strain were as follows:1% starch,1% dextran,0.1% NH4Cl,0.1% yeast extract, initial pH value 6.0, 1% inoculating quantity, rate of shaking flask with 50 mL liquid in a 250 mL flask was 120 r/min. After fermentation for 13 days at 25℃, the enzyme activity was 176.80 U/mL.Extraction and purification of dextranase from D5: The supernant of fermentation medium cultured for 13 days was collected and a series of purification processes were carried out: ultrafiltration concentration, ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and HPLC chromatography. The results of SDS-PAGE and Native-PAGE showed that two proteins including the enzyme was gained, the molecular weight of the enzyme was 66kDa.