Screening Of Strain Producing Protopectinase,Optimizing The Fermentation Conditions And Characteristics Of Protopectinase

Protopectinase (PPase) can catalyze the solubilization of protopectin. Some microorganisms can produce protopectinase under certain conditions. This enzyme will be of potential use in industry, especially in pectin production. Pectin is often extracted by acid on the industrial scale. However, this process has several disadvantages that will be avoided efficiently when protopectinase is used. Up to now, there are no reports concerning protopectinase in China. In this thesis, the research work can be divided into four parts: (1) screening of strain producing protopectinase; (2) optimizing its liquid fermentation conditions; (3) purification and characteristics of protopectinase; (4) application of protopectinase. The main results were as follows:(1) A strain XZ-131 highly producing protopectinase was screened out of 137 strains. PPase and polygalacturonase (PGase) activity arrived about 126.4U/mL and 36.8 U/mL, respectively when XZ-131 was cultured in defatted soybean broth. The Q (PPase/PGase) value of such strain kept at 3.43. The production of PPase and PGase were synchronous with the growth of mycelium in liquid fermentation.(2) Effects of carbon resource, nitrogen resource, metal irons and surface detergents on the production of protopectinase by strain Aspergillus sp. XZ-131 were studied. The results showed that pectin substances were essential for the strain to produce protopectinase. The enzyme activity reached to 300 U/mL, when (NH4) 2864 and (NH4) 2HPO4 were used as nitrogen resource. Ca2+ and Tween-20 were able to enhance the production of protopectinase. The optimum composition of the medium was citrus peel powder lg,(NH4) 2SO4 2g, CaCl2 O.OlSg, Tween-20 0.2mL, KH2PO4 3.8g, K2HPO4 ?3H2O 0.2g, H2O 100mL,pH 6.0.(3) Protopectinase was purified with salt out and chromatography. The optimum reaction temperature of protopectinase was 40 C and the enzyme was unstable to heat. The activity of protopectinase lost 90% when it was kept at 60 C for an hour. The optimum reaction pH ofprotopectinase was 6.0 and the enzyme kept stable under pH 4-7. Cu2+ and Ca2+could inhibit the activity of protopectinase. The molecular weight of protopectinase was 14,600.The reaction mechanism was belonged to A-type.(4) The most suitable conditions for pectin production by protopectinase were achieved as follows. Citrus peel was suspended in pH 3.0 fermentation broth (l:2,W/V).and reaction proceeded for 5h at 25C. 35.7% of protopectin was extracted under that condition.