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
金属材料挤出成型(metal material extrusion, MME)是采用金属粉末与聚合物黏结剂混合丝材为原料,通过成型、脱脂和烧结工艺(shaping-debinding-sintering, S-D-S)制造纯金属零部件的一种增材制造技术。随着MME技术的发展,亟须对其制品...金属材料挤出成型(metal material extrusion, MME)是采用金属粉末与聚合物黏结剂混合丝材为原料,通过成型、脱脂和烧结工艺(shaping-debinding-sintering, S-D-S)制造纯金属零部件的一种增材制造技术。随着MME技术的发展,亟须对其制品的抗冲击性能进行研究,然而相关信息非常匮乏。制备了不同过程参数下的MME试件,并采用夏比摆锤冲击试验研究了其抗冲击性能,探讨了成型方向、填充角度、挤出温度、床温及成型速度等过程参数对试件冲击吸收功的影响。结果表明:当填充角度为45°时,侧置方向成型试件的抗冲击性能最佳,水平方向次之,竖直方向最差;而在0°填充角度下,水平方向成型试件性能最好;进一步提高挤出温度、床温并降低成型速度,可以显著增强试件的抗冲击性能。研究结果为优化MME成型参数提供了理论依据,拓展了其在承受冲击载荷场景下的应用潜力。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.
文摘金属材料挤出成型(metal material extrusion, MME)是采用金属粉末与聚合物黏结剂混合丝材为原料,通过成型、脱脂和烧结工艺(shaping-debinding-sintering, S-D-S)制造纯金属零部件的一种增材制造技术。随着MME技术的发展,亟须对其制品的抗冲击性能进行研究,然而相关信息非常匮乏。制备了不同过程参数下的MME试件,并采用夏比摆锤冲击试验研究了其抗冲击性能,探讨了成型方向、填充角度、挤出温度、床温及成型速度等过程参数对试件冲击吸收功的影响。结果表明:当填充角度为45°时,侧置方向成型试件的抗冲击性能最佳,水平方向次之,竖直方向最差;而在0°填充角度下,水平方向成型试件性能最好;进一步提高挤出温度、床温并降低成型速度,可以显著增强试件的抗冲击性能。研究结果为优化MME成型参数提供了理论依据,拓展了其在承受冲击载荷场景下的应用潜力。
