Screening Of Glucomannanase Producing Strains And Purification Of The Enzyme

50 glucomannanase producing strains were preliminarily screened from soilby enriching culture, isolation and purification. 10 strains with high enzymeactivity were obtained by flat screening. Then the strain DK3 with the highestenzyme activity was obtained after the second screening by shaking culture. 16srDNA sequence blast analysis in Genbank revealed that DK3 was most relatedto the strain Pantoea agglomerans (Identities=99%).The glucomannanase production conditions for DK3 strain were studiedpreliminarily. The optimal medium composition is as follows: 0.6%glucomannan, 0.3% NaNO3, 0.5% yeast extract paste, 0.3% peptone, 0.5%MgSO₄·7H₂O, 0.5% KCl, 0.1% K₂HPO₄, 0.001% FeSO₄·7H₂O, pH6.0. Theoptimal cultivation conditions: volume of inoculum 1%, 37℃, 160rpm. Underthis optimal condition for 24h, the glucomannanase activity reach 58.54 U/mL,1.27 fold as before.The glucomannanase was then separated and purified to homogeneity by ultrafiltration, ion-exchange and gel flitration. The purified mannanase appearedas a single protein band on SDS-PAGE gel with a molecular mass of approx. 54kDa.The characteristics of glucomannanase produced by DK3 strain werestudied preliminarily. The optimal temperature and pH for the glucomannanaseactivity was 37℃and pH6.0 respectively. The glucomannanase was stablewithin pH 5.0-9.0 and up to 40℃at pH6.0. Fe²⁺ and Cu²⁺ strongly inhibit theglucomannanase; EDTA slightly inhibit the glucomannanase; NH₄⁺, Mg²⁺weekly inhibit this enzyme; the glucomannanase was gently activated by Na⁺.The conditions of immobilized cells producing konjac glucomannanasewere preliminarily studied. When 2% CaCl₂ was added to the culture mediumthe immobilized cells could be repeatly used for approx. 4 times after which theefficiency of enzyme production decreased.Enzymolysis of glucomannan by glucomannanase was preliminarilystudied. By repeatly supplying solid glucomannan, the final concentration ofglucomannan oligosaccharides in the solution reached 24%. The results ofsilica-gel G thin-layer chromatography showed that the composition of theoligosaccharides obtained by enzymolysis of glucomannan was verycomplicated.