| Pectinase has been widely used in fruit juices and beverages, papermaking industry, scouring of cotton fiber, degumming of bast fiber plants, coffee and tea fermented, animal feed. Orange peel is an agricultural waste, which is rich in pectin, so pectinase production using orange peel as a fermentation inducer have the environmental and economic benefits. In this paper, screen and identification of pectinase-production strain using orange peel, fermentation process optimization and characterization of the pectinase were invested. Research content and results are as follows:(1) The plate separation method was used to screening pectinase-production strains, combine with the activities were determinated after the strains screened were inoculated for the liquid fermentation culture. At last the strain v25 with the pectinase activity of 15.13 U/mL was screened and isolated. Then the strain v25 was identified as Zygoascus sp. through the colony morphology, microscopic observation of morphology and gene sequencing and analysis. The growth curve of the strain v25 was determinated, so 18 h was selected as seed culture time.(2) The fermentation process of pectinase-production has been optimized through single factor experiment, Plackett-Burman design and central composite design. The optimum fermentation conditions were medium initial pH 5.5, rotation speed 160 r/min, inoculation amount 4%, liquid volume 25 mL/250 mL, fermentation temperature 31°C and fermentation time 48 h. The optimal fermentation medium components are orange peel powder 31.2 g/L, peptone 1 g/L, beef extract 3 g/L, K2HPO4.3H2 O 0.094 g/L, KH2PO4 0.5 g/L, CaCl2 0.5 g/L and NaCl 0.5 g/L. The predicted pectinase activity was 28.97 U/mL and the actual enzyme activity was 29.02 U/mL. The measured and predicted values are in good agreement. The enzyme activity is 0.918 times higher than that of before optimization.(3) The polymethylgalacturonase from Zygoascus sp. v25 submerged culture was purified and characterized exploiting ammonium sulphate precipitation, Sephadex G-25 desalting, DEAE-cellulose chromatography and Sephadex G-100 gel filtration. The polymethylgalacturonase was purified to electrophoretic homogeneity single component by SDS-PAGE electrophoresis analysis and had 16.89 purification fold with a recovery of 18.46%, the specific activity of 2469.77 U/mg and a molecular weight of 75.28 kDa.(4) The purified enzyme exhibited maximum activity at 60°C and pH 5.0, and was stable over a wide range of pH 3.0-11.0. Moreover, the enzyme activity was enhanced by Cu2+ and cysteine, and inhibited strongly by Hg2+. The profile of substrate specificity test indicated that the extent of enzymatic hydrolysis was negatively correlated with the degree of pectin esterification. The Km and Vmax values of the polymethylgalacturonase were 5.44 mg/mL and 61.73 ?mol/(min·mg), respectively. The enzyme was deemed as an exo-polymethylgalacturonase by TLC chromatography. The purified polymethylgalacturonase was applied in the clarification of the juices of four fruits, and the results showed that the percentage transmittance at 660 nm has increased 3.51%, 4.36%, 8.04% and 12.2%, respectively, indicating that the polymethylgalacturonase might possess potential to be used in fruit juice clarification. |
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