Study Of Isolation And Purification Of Lactase From Aspergillus Niger D2-26 And Enzyme Properties

Lactase is a kind of hydrolase that mainly applied in fields such as food, medicine, analyzing, etc at present. This research take a strain of Aspergillus niger D2-26 which preserved in the enzyme engineering laboratory as the fermentation strain to ferment lactase in 10 L fermentor, a set of methods which were easy and feasible in the purification of lactase from fermentation liquor were established and the properties of purified enzyme were studied in details. The Aspergillus niger D2-26 lactase which has a broad application prospects because of its low production costs, simple extraction and purification technology, fine stability, effective prevention on other strains in the production is a extracelluar and high temperature enzyme.By adopting filtration, ultrafiltration, ammonium sulfate fraction, sephadex G-25 gel filtration, DEAE ion-change chromatography, sephadex G-100 gel filtration, the lactase which came from Aspergillus niger D2-26 fermentation liquor was purified. The result of PAGE verified that a single protein band was obtained .The specific activity of lactase had reached 705.91 U/mg which was 43.7 folds of the primary fermentation liquor.The enzyme properties of the purified Aspergillus niger D2-26 lactase which take ONPG as substrate are as follows: the optimum temperature of the lactase is 70℃, it has a wide range of operative temperature, we can get a higher enzyme activity in the scope of 40 ℃ to 70 ℃;it has a good temperature tolerance between 30 ℃ to 60 ℃, the residual enzyme activity can reach up to 90% kept in the scope of 30 ℃ to 50℃ for 2 h, the lactase kept in 60℃ for 2 h the residual enzyme activity is 73.42%;it has a perfect stability in the condition of 4℃, the enzyme activity begin to go down a bit after the enzyme protein being kept in low temperature for 20 d;the optimum pH of the lactase is 2.5 with a wide range of operative pH, it has a higher enzyme activity in the pH value 2⁴.5;it has a good pH tolerance, the relative enzyme activity are all above 70% after being kept 2 d in the condition of pH 2⁹;Mn²⁺ have a significant activation on the lactase, the enzyme activity can be increased 44.94%, some other metal ions can also improve the enzyme activity in some extent, Hg²⁺, Pb²⁺ have a strong inhibition on the enzyme activity;The denaturation engendered by SDS on enzyme protein can severely affect the enzyme activity, galactose have a certain substrate inhibition effect on the lactase, EDTA have no influence on the enzyme activity;for ONPG the Vmax is 0.097umol/mL, Km is 8.77influence on the enzyme activity;for ONPG the Vmax is 0.097umol/mL, Km is 8.77 mmol/mL which tested by Bi-reciprocal graph, SDS-PAGE indicates that the molecular weight of the lactase is 116.978 KD, the lactase is a single subunit protein;the glycosylation of the lactase protein is 11.3% gained by sulfuric acid -phenol method.This paper has done some research on the Aspergillus niger D2-26 lactase including the isolation and purification, the enzyme properties which will be a foundation for its industrial production.