Characterization And Application Of Extracellular Dextranase From Isolated Strains

Dextranase （α-1,6-D-glucan-6-glucanohydrolase; E.C.3.2.1.11） is aninducible enzyme that cleaves the α-1,6glycosidic linkages in the interior ofdextrans to produce linear oligosaccharides. It is a key role in the preparationof blood substitutes by the partial hydrolysis of native dextran.Fungi were isolated from natural soil samples and screened forextracellular dextranase synthesis in this study. Subsequently, the strainsF1001and F1002were identified as Penicillium aculeatum and Hypocrea lixiirespectively, using the methods of morphology, cultural characteristics andinternal transcribed spacer ribosomal DNA （ITS rDNA） analysis. F1001andF1002were selected for extracellular dextranase synthesis. The cultureconditions suitable for enzyme production were established. The strain F1001had a max dextranase activity after five days cultivation at pH5,28°C,containing Dextran T70as a carbon source and NaNO₃as a nitrogen source.Furthermore, the strain F1002also had a max dextranase activity after sixdays cultivation at pH6,28°C, containing Dextran T70as a carbon source andNaNO₃as a nitrogen source.Both of the Penicillium aculeatum dextranase and the Hypocrea lixiidextranase were purified via ammonium sulfate precipitation and Sepharose6B chromatography, the Penicillium aculeatum dextranase resulted in an7.884-fold increase in the specific activity and a13.2%recovery. This enzymeis a monomeric protein with a molecular mass of66kDa, as determined bysodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. Thepurified enzyme, which was identified as an endodextranase, had an optimumpH of5.0and an optimum temperature of35°C. The optimum substrate andsubstrate concentration were dextran T70and3%（w/v）. The dextranaseactivity was enhanced by Zn²⁺and Cu²⁺, and the low concentration of Cu²⁺could improve the dextranase activity to134.7%. However, the enzyme wasstrongly inhibited by Mn²⁺. Moreover, the Hypocrea lixii dextranase resultedin an8.3-fold increase in the specific activity and a10.73%recovery. Thisenzyme is a monomeric protein with a molecular mass of62kDa, as determined by SDS-PAGE. The purified enzyme, which was identified as anendodextranase, had an optimum pH of5.0and an optimum temperature of25°C. The optimum substrate and substrate concentration were Dextran T70and5%（w/v）. The dextranase activity was enhanced by Mg²⁺, Al3+, andespecially Zn²⁺at a low concentration, which improved its activity to124.22%.The enzyme has a very high hydrolytic affinity toward high-molecular weightdextrans.The oligodextrans were synthesized by the combined use ofdextransucrase and dextranase. The synthesized dextran with high-molecularweight were obtained by selecting the concentrations of dextransucrase （2EIU/mL） and the sucrose （12%）, as well as the reaction time （24h）.Subsequently, setting the concentration of the Penicillium aculeatumdextranase （0.23U/mL） and the reaction time （180min）, allowed thePenicillium aculeatum dextranase to hydrolyze dextrans of controlledmolecular weights （20–70kDa）. Three types of oligodextrans with differentmolecular weights （namely,69,376,38,251, and21,364Da） were obtained,with a total yield of80.32%.