Study On The Breeding Of Pectinase Hyper-producer And Its Application

Based on the theory of metabolic control fermentation, the original strain Aspergillus niger PG-1 was treated by mutagen gradually,and a higher pectinase producer A.niger GJ-2 was obtained. Then fermentation conditions, mixed culture fermentation, purification and characterization of pectinase were all studied. Finally the application of pectinase was also studied. The main contents and results were as follows:The pectinase producer A.niger GJ-2 was derived from the original strain A.niger PG-1 whose pectinase activity was only 312.5 U/mL by UV and NTG treatment. In the original culture conditions, 441.2 U/mL pectinase was accumulated, which was 41.2% higher than the original strain.The fermentation medium and culture conditions were studied. It was found that the optimal fermentation medium components were orange peels 29.2 g/L; bran 32.8 g/L; ammonia sulfate 10 g/L. And the optimal culture conditions were fermentation period 72 h; seed age 24 h; inoculated volume 4%; fermentation temperature 36.3℃. The pectinase activity was 556.5 U/mL under the optimal conditions. The mixed culture of A.niger GJ-2 and Saccharomyces cerevisiae J-1 was studied. It was found that the pectinase was improved when S.cerevisiae J-1 was inoculated to the medium at 12h of culture with an inoculation volume of 5 mL/250 mL. And the pectinase activity was 586.9 U/mL, which was 87.8% higher than the original strain.The pectinase produced by the strain A.niger GJ-2 in the submerged fermentation was isolated and purified by a three-steep process: ammonia sulfate fractional precipitation; ion-exchange chromatography and gel filtration chromatography. After the three-steep purification, a electrophoresis pure pectiase was obtained, whose molecular weight is 35 KDa and isoelectric point is 3.84. The specific activity was 13478.0 U/mg and its purity multiple and recovery were 21.1 and 19.7%, respectively.The characterization of pectinase was studied. The optimal pH was 4.6, with relative activity of about 80% at the pH rang from 2.6 to 5.6, which indicated that it adapted to a wide range of pH. The optimal temperature was 50℃, with relative activity of about 76.8% when it was treated at 50℃for 60min, which indicated that it had a thermal stability. When it was treated by metal ions, different proformances of pectinase were observed. When the final concentration was 1 mmol/L, pectinase could be activated by K⁺, Na⁺, Fe²⁺, Cu²⁺, Mg²⁺, Zn²⁺ and Ca²⁺, and inhibited by Ba²⁺, Mn²⁺, Al³⁺ and Fe³⁺. When the final concentration was 5 mmol/L, pectinase could be activated by Na+, Fe²⁺, Cu²⁺, Mg²⁺ and Ca²⁺, and inhibited by K⁺, Zn²⁺, Ba²⁺, Mn²⁺, Al³⁺ and Fe³⁺. With pectic acid used as substrate, Km and Vmax were measured under the optimal pH and temperature, which were 28.4 mg/mL and 169.5 mg/(mL·min) respectively.In the application of pectinase, orange peels were hydrolyzed by crude enzyme. It was found that the ratio was that per gram of orange peels were treated by 550 U of crude pectinase. Enzymolysis time was 18 h. The optimal enzymolysis temperature and pH were 50℃and 4.0 respectively. The concentration of sugars in the hydrolysate can be up to 95.9 g/L and the yield of L-lactic acid was 75.2 g/L with the elimination of limonene. Rhamnose, galactose, glucose, fructose and xylose were considered as fermentable sugar, arabinose and galacturonic acid as nonfermentable sugar by Lactobacillus casei G-04.