Isolation,Purification And Identification Of Polygalacturonase From Aspergillus Niger SH312-26-19

In this experiment, the polygalacturonase from Aspergillus niger SH312-26-19 was purified and identified by ammonium sulfate precipitation, chromatography and nanoUPLC-MS/MS. The test strain is obtained by UV mutagenesis from Aspergillus niger SH312 which isolated from commercial pectinase by our group. Its'enzymatic properties also were determined. The main findings are as follow:1. Polygalacturonase was purified by ammonium sulfate precipitation between 30%?80%, Sephadex G-75 gel chromatography and DEAE-Sepharose Fast Flow ion exchange chromatography from the fermentation broth of SH312-26-19. The result of SDS-PAGE showed its'have the molecular weight with-20.1 kDa. The purified experiments achieved 9.76 of purified fold with 22.66% of yield and 2.32% of protein recovery.2. The purified PG was identified by nanoUPLC-MS/MS after being hydrolyzed by trypsin. This PG was expected with a molecular weight of 20000 Da. MS/MS results were searched at NCBInr 20120419 database of Matrix science and it didn't found some protein with significant matching. Homology analysis with a polygalacturonase named gi|145232359 showed there was only 12% of protein sequence coverage. As the result, the PG from Aspergillus nigex SH312-26-19 was a novel polygalacturonase.3. The enzymatic properties of studied PG were determined. The optimum substrate pH value was pH4.8 and the temperature was 48?. It could stay over 75% enzymatic activity after treat 1.5h at pH3.6-5.2 and over 70% activity at 43-53?. Most metal ions have no influence on PG with concentration of 2mmol/L. When the concentration of metal ions were increased to 5mmol/L, Mg2+, Cu2+, Ba2+, Fe2+, Zn2+and K+could promote PG activity significantly while Na+has slight inhibition, and Ca2+could obviously reduce PG activity at both two concentration. Notably, Al3+could inhibited PG activity at 2mmol/L and promoted little at 5mmol/L. Furthermore, low concentration of citric acid had obvious promoting effect on the enzyme activity while the high contrast.4. The methnol in the product of crude PG solutions was significantly less than commercial pectinase when pectin as substrate and they both lower than the regulated maximum limit of fruit distilled liquor. The result proved the researched protein is polygalacturonase from the side.In conclusion, the polygalacturonase obtained from Aspergillus niger SH312-26-19 was a novel polygalacturonase, and it has a good application prospect and development potential in fruit juice and fruit wine and other industries.