High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastori...High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.展开更多
The liquid-phase oxidation of ethylamine with hydrogen peroxide was studied over tungsten-doped zeolites to develop a clean and simple route for producing acetaldehyde oxime. The investigations were firstly performed ...The liquid-phase oxidation of ethylamine with hydrogen peroxide was studied over tungsten-doped zeolites to develop a clean and simple route for producing acetaldehyde oxime. The investigations were firstly performed over W/MOR, where the coordinated state as well as the acidity of the W species were characterized. The reaction parameters, including H_2O_2 amount, solvent,temperature, tungsten content as well as catalyst amount, governed the activity and oxime selectivity. Under optimized reaction conditions, W/MOR showed an ethylamine conversion and corresponding oxime selectivity of 18.3% and 88.9%. W/MOR showed a superior performance in comparison to other tungsten-containing zeolites of W/Beta, W/MWW and W/Y. Although W/MOR exhibited lower amine conversion than titanosilicates of TS-1 and Ti-MWW, it gave higher selectivity to the main product of oxime. Moreover, W/MOR proved to be a robust catalyst, exhibiting a stable catalytic performance after being reused at least for 5 times.展开更多
基金Project(31870115)supported by the National Natural Science Foundation of ChinaProject(2015JJ5006)supported by the Natural Science of Hunan Province&Changde City Joint Foundation,ChinaProjects(2015zzts268,ZY2015823)supported by the Fundamental Research Funds for the Central Universities,China
文摘High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.
基金supported by the National Natural Science Foundation of China(21533002,21373089,21603075)the National Key Research and Development Program of China(2016YFA0202804)
文摘The liquid-phase oxidation of ethylamine with hydrogen peroxide was studied over tungsten-doped zeolites to develop a clean and simple route for producing acetaldehyde oxime. The investigations were firstly performed over W/MOR, where the coordinated state as well as the acidity of the W species were characterized. The reaction parameters, including H_2O_2 amount, solvent,temperature, tungsten content as well as catalyst amount, governed the activity and oxime selectivity. Under optimized reaction conditions, W/MOR showed an ethylamine conversion and corresponding oxime selectivity of 18.3% and 88.9%. W/MOR showed a superior performance in comparison to other tungsten-containing zeolites of W/Beta, W/MWW and W/Y. Although W/MOR exhibited lower amine conversion than titanosilicates of TS-1 and Ti-MWW, it gave higher selectivity to the main product of oxime. Moreover, W/MOR proved to be a robust catalyst, exhibiting a stable catalytic performance after being reused at least for 5 times.
