To investigate the relationships among β-mannanase, β-mannosidase and a-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes...To investigate the relationships among β-mannanase, β-mannosidase and a-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA3 on them were surveyed. The activities of β-mannosidase and a-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA3 could promote the activities of β-mannanase and a-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase.展开更多
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
基金supported by the National Natural Science Foundation of China(Grant No.30370913)the Guangdong Provincial Natural Science Foundation(Grant No.32264).
文摘To investigate the relationships among β-mannanase, β-mannosidase and a-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA3 on them were surveyed. The activities of β-mannosidase and a-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA3 could promote the activities of β-mannanase and a-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase.
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.
