Studies On Fermentation Production Conditions Of Pectin Lyase And Its Application On Synthesize Unsaturated Oligogalacturonide

The conditions of producing enzyme of Aspergillus niger wz003, characterization of pectic lyase, the synthetical conditions of unsaturated oligogalacturonide and its purification were studied.Physiological and biochemical parameter of Aspergillus niger wz003 for production of high active pectin lyase was investigated firstly. The experimental result indicated: the optimal fermention medium was wheat bran 40.0g/L, orange meal 20.0g/L, NaNO₃ 12.8g/L, KH₂PO₄ 1.2g/L, water concentration 60% （w/w）. After inoculation with 12% （v/w, 10⁶ entries/ml）Aspergillus niger wz003, the medium was cultivated at 40℃for 72h with original pH 6.6, the pectin lyase activity of moldy bran could reach 2607.82 U/g, increased approximately 56 times than which cultivated under basic conditions, while the synthesized of polygalacturonase was controlled effectivity, the enzyme activity was 462.12 U/g.The pectin lyase was purified by ammonium sulfate salted out and the optimal range of ammonium sulfate saturation was 40-85%. The result of experiment also showed: the optimal temperature and pH for the pectic lyase reaction was 40℃and 6.8; the pectic lyase was stable at pH 6.4-8.0 and temperature 25℃-55℃. The metal ion Ca²⁺, Zn²⁺ a certain extent enhanced the pectic lyase activity, but Mg²⁺, Na⁺, Mn²⁺, Ba²⁺ and Cu² had no obvious effect on pectic lyase activity. Through single factor test experiments, the optimum conditions for producing unsaturated oligogalacturonide were investigated. The results were as follows: initial pectin concentration 1.76%, enzyme liquid 2%（v/v）, pH 6.8, temperature 40℃and hydrolyzation time 20 hours.The pectic hydrolyze liquid separated by ultra-filtration technology. The effects of pressures、concentrations and pH vaules of pectic hydrolyze liquid on ultra-filtration permeate flux were investigated. The results showed that the optimal conditions are: pressure 0.14MPa, 1.76% pectins and pH vaule 6.4. Then we enabled the first filterable pectic liquid via two membranes that molecular cut-off weight were 5000Da and 1000Da. Finally, the three filterable pectic liquids were detected by HPLC. Though the MS analysis of hydrolyzed pectic liquidU₀, we find four kinds unsaturated oligogalacturonide whose moleculars were under 2000Da.