In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A,...In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.展开更多
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
Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is ...Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.展开更多
The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pa...The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pastoris directly. Thus, for β-mannanase production, developing the accurate, rapid and inexpensive screening method to substitute random screening is certainly required. A simple method based on the size of hydrolysis hole was described here, but this method was not very accurate that could only be used in preliminary screening. To further improve the accuracy, a micro-plate screening method is established, which appears to be more accurate and effective. The efficiency of this screening method is about 10 times higher than that of the general screening strategy of cultivation in shaking flasks. Two methods presented here can also be used for screening of recombinant Pichia strains with high-level expression of other heterologous protein after modification.展开更多
基金Project(13JJ9002)supported by Hunan Provincial Natural Science Foundation of ChinaProject(2012XK4081)supported by the Key Science Technology Plan Project of Hunan Provincial Science&Technology Department,ChinaProject(CX2012B124)supported by the Graduate Degree Thesis Innovation Program of Hunan Province,China
文摘In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.
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Project(31000350)supported by the National Natural Science Foundation of China
文摘Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.
基金Project(CX2012B124)supported by the Graduate Degree Thesis Innovation Program of Hunan ProvinceChina+3 种基金Project(13JJ9002)supported by the Natural Science Foundation of Hunan ProvinceChinaProject(2012XK4081)supported by the Key Science and Technology Plan of Hunan Provincial Science&Technology DepartmentChina
文摘The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pastoris directly. Thus, for β-mannanase production, developing the accurate, rapid and inexpensive screening method to substitute random screening is certainly required. A simple method based on the size of hydrolysis hole was described here, but this method was not very accurate that could only be used in preliminary screening. To further improve the accuracy, a micro-plate screening method is established, which appears to be more accurate and effective. The efficiency of this screening method is about 10 times higher than that of the general screening strategy of cultivation in shaking flasks. Two methods presented here can also be used for screening of recombinant Pichia strains with high-level expression of other heterologous protein after modification.
