Screening Of Strain Producing Cold Adapted Pectinase,Optimizing The Fermentation Conditions And Characteristics Of Enzyme

Screened of strain producing cold adapted pectinase and studied on the condition of producing enzyme, characterization of cold adapted pectinase, purification and kinetic constant in this paper.The strain LP272 producing cold adapted pectinase was gained by screened 118 soil samples, after indentification, the strain LP272 belong to Cryptococcus nuiguttulatus. The result of experiment also showed : the optimal temperature for growth of this strain was 25℃, the optimal temperature for producing cold adapted pectinase was 25℃, the optimal temperature and pH for the enzyme reaction was 25℃and 5.8. The enzyme was stable at pH5～6.4, after 3 hours at 25℃,the enzyme activity was 50.5%, at 35℃for 2 hours, the enzyme activity was 55.1%, at 45℃for 1.5 hours, the enzyme activity was 48.7%, at 55℃for 1 hours, the enzyme activity was 43.1%.Effects of composition of fermentation medium and fermentation conditions on the producion of cold adapted pectinase by strain LP272 were studied, through the orthogonal test ,the optimal composition of fermentation medium for production of cold adapted pectinase was citrus 2.0g, glucose 1.5g, peptone 0.5g, yeast extract 0.5g, CaCl2 0.1g, SDS 0.4g, K2HPO4 0.2g,KH2PO4 0.05g, H2O 100ml,pH5.6. the optimal fermentation conditions was culture temperature 25℃, culture time 36h, speed of rotation 200rpm,the holding amount 50ml. the metal ion Mg²⁺,Ca²⁺,Mn²⁺,Zn²⁺,Fe²⁺,Na+ enhanced the cold adapted pectinase activity ,but the metal ion Ba²⁺,Hg²⁺,Cu²⁺,Fe³⁺,Pb²⁺ inhibited the cold adapted pectinase activity strongly.The cold adapted pectinase was purified by ammonium sulfate salted out and the optimal range of ammonium sulfate saturation was 40-85%.The kinetic constant of the cold adapted pectinase was Km value 27.86mg/mL and Vmax152.13μmol/(min·ml)when pectin(sigma)as a substrate.