Study On The Pectinase Production By Penicillium Expansum YL-9 Using Apple Pomace

In this research, Penicillium expansum YL-9,screened and kept by our laboratory, was used as the starting strain to study the pectinase production. Orthogonal rotation experiments were carried out to optimize pectinase producing conditions by target strains using apple pomace; The high purity pectinase was obtained by (NH₄)₂SO₄ salting, CM anion exchange chromatography and Sephadex G-75 gel filtration; The properties of pectinase, and its specific application parameters in apple juice clarification were also studied. The main results as follows:1. The optimized solid-state fermentation conditions for enzyme production of Penicillium expansum YL-9 using apple pomase were as follows: wheat bran 8.15%, apple pomace 16.5%, (NH₄)₂SO₄ 0.86%, MnSO₄ 0.3%, KCl 0.15%, Tween 0.04%, water content 74.0%; culture temperature 28℃, the natural pH, volume of sample loading 10g/250mL, inoculum size 2×10⁶ spores/g dry basis and the cultured time 96h. Under the above conditions,the polygalacturonase activity reached 3621.04 (U/g dry Qu).2. By (NH₄)₂SO₄ salting out test, it was known that the enzyme protein was mostly precipitated when the saturation of (NH₄)₂SO₄ was 80%. Pectinase was purified by (NH₄)₂SO₄,CM anion exchange chromatography and Sephadex G-75 gel filteation column chromatography respectively,the final purification fold is15.3,the recovery rate is 17.4%. One clear and impurity-free band of the purified enzyme were obtained, showing that the the pectinase is electrophoretic pure with molecular weight about 30KDa.3. Through enzymology character study of target pectinase. Its optimal temperature range was 40-60℃, its enzyme activity was relatively stable stored at 4℃-30℃; Its optimal pH range is 3.0-5.0, which was suitable for fruit production and stable in the range of pH from 3.0 to 7.0; Metal ions including Mn²⁺, Mg²⁺ and Cu²⁺ could accelerate pectinase activity, when the Mn²⁺, Mg²⁺, Cu²⁺was respectively obtained 1.5mmol / L, 1.0mmol / L and 1.0mmol / L in enzyme reaction buffer, its activity corresponding increased 84.46%, 13.47%, 32.67%; K⁺, Na⁺, Ca²⁺, Fe²⁺ and Zn²⁺ have no significant effects on the enzyme activity; Al³⁺ was inhibitor of pectinase activity.The Km value of the pectinase was 12.048g/L .4. The result of single factor experiments for apple juice clarification showed that when used pectinase 0.3-0.5 mL/kg, temperature 30-55℃, apple juice pH 2.5-5.0, and treated more than 30min, the apple juice could be better clarified. Based on the single factor experiment, orthogonal experiment was conducted to optimize clarification parameters, the result showed the best clarification condition for apple fruit juice was: pH4.0, 0.5 mL/kg pectinase, 50℃, and treatment time is 60min.