[Objective] This paper aims to study a new method of extracting pumpkin polysaccharide from pumpkin. Single factor experiments were conducted to examine the effects of extracting time,temperature,the solid-liquid rati...[Objective] This paper aims to study a new method of extracting pumpkin polysaccharide from pumpkin. Single factor experiments were conducted to examine the effects of extracting time,temperature,the solid-liquid ratio and pH value on the extraction yield of polysaccharide from pumpkin. [Method] The best enzyme ratio and extraction conditions for complex enzymes extraction were determined through orthogonal tests. Scavenging ·OH and O-2 activities of pumpkin polysaccharides were also investigated by salicylic acid and improved self-oxidation of o-pheno methods respectively. [Results] The results showed that the biggest extraction yield of polysaccharide from pumpkin can be got when adding 1% cellulose enzyme,1.5% pectinase,1.0% papain and Na2HPO4-citric acid buffer solution (pH was 4.6),and oscillating for 30 min under water at 40 ℃ with the solid-liquid ratio of 1:30. In addition,pumpkin polysaccharides had a strong activity of eliminating ·OH,but very weak activity to scavenge O-2. [Conclusion] This study provided basic data for research and application of Pumpkin polysaccharide.展开更多
[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was ...[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.展开更多
基金Supported by the Key Scientific and Technological Project of Henan Province (102102110157)the Scientific Research Found Project of Henan University of Urban Construction (2010JZD008)~~
文摘[Objective] This paper aims to study a new method of extracting pumpkin polysaccharide from pumpkin. Single factor experiments were conducted to examine the effects of extracting time,temperature,the solid-liquid ratio and pH value on the extraction yield of polysaccharide from pumpkin. [Method] The best enzyme ratio and extraction conditions for complex enzymes extraction were determined through orthogonal tests. Scavenging ·OH and O-2 activities of pumpkin polysaccharides were also investigated by salicylic acid and improved self-oxidation of o-pheno methods respectively. [Results] The results showed that the biggest extraction yield of polysaccharide from pumpkin can be got when adding 1% cellulose enzyme,1.5% pectinase,1.0% papain and Na2HPO4-citric acid buffer solution (pH was 4.6),and oscillating for 30 min under water at 40 ℃ with the solid-liquid ratio of 1:30. In addition,pumpkin polysaccharides had a strong activity of eliminating ·OH,but very weak activity to scavenge O-2. [Conclusion] This study provided basic data for research and application of Pumpkin polysaccharide.
文摘[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.
