Isolation, Purification And Bioactivities Of Polysaccharide From Pichia Pastoris

Pichia pastoris is a new efficient eukaryotic expression system in recent years. It was reported that more than 400 genetic engineering products have been expressed in the system, such as:human interleukin-2, tumor necrosis factor, recombinant human serum albumin. With the development of technology, large-scale high-density fermentation have been achieved in more and more products. This method could improves production of the target protein, however, an amounts of waste biomasses were produced in this process. To solve the serious problem of environmental pollution caused by waste biomass of Pichia pastoris and find the way of comprehentive utilization of the waste biomass. The following studies were performed:Firstly, the contents of main component in Pichia pastoris were analysed. The results showed that contents of polysaccharide, protein, fat and nucleic acid were 36.8%,45.1%, 11.9%,7.4%, and 8.9%, respectively. HPLC-ELSD analysis also showed that the polysaccharide was composed of 15.9% glucan and 20.2% mannnan.Secondly, conditions of isolation and purification of the glucan and mannan were optimized. The procedure of isolation and purification of glucan mainly include:inducing autolysis, high-pressure hot water treatment, ultrasonic treatment, defatted by isopropanol, proteolysis, and lyophilization and so on. Through single factor experiments and response surface design, highest autolysis rate could reach 39.4% under the conditions of inducing of autolysis at pH 6.4,46.9℃for 32.9h. Best conditions of high-pressure hot water reatment were 1:30 in liquid-solid ratio, pH 7.5 for 4h. The optimal conditions for ultrasonic treatment were 1:40 in solid-liquid ratio,20 KHz vibration requency,10s work time,2s interval time at 400W ultrasonic power, repeated 60 times. Papain adding at 60U/g protein in pH 6.5 at 37℃for 4h can remove most amount of protein. Though the procedure above, glucan in the purity of 85.3% could be obtained with the yield of 11.7%. Furthermore, mannan was purified from the supernatants of autolysis, high-pressure hot water treatment and ultrasonic treatment successfully by enzymolysis and ethanol precipitation. The final purity of mannan was 94.2%, and the yield was 11.2%.Finally, glucan and mannan's biological activity was assayed and the activities of glucan in strengthening immunity of the organism, improving lipid metabolism level and enhancing the antioxidant ability of the body were proved in vivo. In the assay of immunity, when the mice were treatment by gavage with 150 mg/(kg·d) glucan, phagocytic index, spleen index and liver index increased by 17.8%,17.6% and 32.1%, respectively. In the assay of blood lipid metabolism, The content of HDL increased by 10.1%, while, the content of LDL decreased by 5.8%, when the mice were treatment by gavage with 150 mg/(kg·d) glucan, the decrease rate was 12.5% when the dose was increased to 150mg/(kg·d). In the assay of anti-antioxidation, activities of T-AOC, SOD and CAT increased by 14.2%,12.3% and 8.6%, respectively, when the mice were treatment by gavage with 150 mg/(kg·d) glucan, while, the content of MDA decreased by 6.2% at the same dose. Clearance rate of hydroxy radical and superoxide anion radical were 10.0% and 21.4%, respectively, when the mice were treatment by gavage with 150 mg/(kg·d) glucan. In the meantime, the antioxidant acivitites of mannan were also assay in vitro, the results showed that the EC50 of mannan in clearing hydroxyl radical assay was 2.5mg/mL, and mannan also showed activity in clearing superoxide anion radical. Both of the two activities were does-dependented.A new approach with the feature of simply operation, high purity and yield was studied in isolation and purification of polysaccharide in Pichia pastoris. The polysaccharide from Pichia pastoris showed good activities in immunoenhancement and antioxidation, suggesting that they could be used as health care products and medicine in treating human serious illness in the future. The studies above also lay a foundation in the high-value utilization of Pichia pastoris'effective components.