118 research outputs found

    <i>Citrobacter amalonaticus</i> Phytase on the Cell Surface of <i>Pichia pastoris</i> Exhibits High pH Stability as a Promising Potential Feed Supplement

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    <div><p>Phytase expressed and anchored on the cell surface of <i>Pichia pastoris</i> avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of <i>Citrobacter amalonaticus</i> was fused with the <i>Pichia pastoris</i> glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue <i>GCW61</i>. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our <i>in vitro</i> digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.</p></div

    Synthesis and Controlled Self-Assembly of UV-Responsive Gold Nanoparticles in Block Copolymer Templates

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    We demonstrate the facile synthesis of gold nanoparticles (GNPs) functionalized by UV-responsive block copolymer ligands, poly­(styrene)-<i>b</i>-poly­(<i>o</i>-nitrobenzene acrylate)-SH (PS-<i>b</i>-PNBA-SH), followed by their targeted distribution within a lamellae-forming poly­(styrene)-<i>b</i>-poly­(2-vinylpyridine) (PS-<i>b</i>-P2VP) block copolymer. The multilayer, micelle-like structure of the GNPs consists of a gold core, an inner PNBA layer, and an outer PS layer. The UV-sensitive PNBA segment can be deprotected into a layer containing poly­(acrylic acid) (PAA) when exposed to UV light at 365 nm, which enables the simple and precise tuning of GNP surface properties from hydrophobic to amphiphilic. The GNPs bearing ligands of different chemical compositions were successfully and selectively incorporated into the PS-<i>b</i>-P2VP block copolymer, and UV light showed a profound influence on the spatial distributions of GNPs. Prior to UV exposure, GNPs partition along the interfaces of PS and P2VP domains, while the UV-treated GNPs are incorporated into P2VP domains as a result of hydrogen bond interactions between PAA on the gold surface and P2VP domains. This provides an easy way of controlling the arrangement of nanoparticles in polymer matrices by tailoring the nanoparticle surface using UV light

    Phytase activity after induction with methanol and after treatment with laminarinase.

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    <p>A: Time dependence of the activity of cell surface phytase after induction with methanol. B: Cell surface phytase activity after laminarinase treatment. Column 1 represents cell wall fractions without treatment with laminarinase. Columns 2 and 4 represent cell wall fractions after laminarinase treatment, and column 3 and 5 represent supernatant fractions after laminarinase treatment. Columns 2 and 3 show phytase activities after treatment with 5 mU of laminarinase, while columns 4 and 5 represent the remaining activities after treatment with 50 mU of laminarinase. All activities were compared to the activity of the whole cell surface phytase, with GS115/ZαA as a background measurement.</p

    FOX proteins are predicted to bind to putative <i>let-7a3/b</i> enhancer regions.

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    <p><b>(A)</b> The predicted existence of an upstream enhancer for the <i>let-7a3/b</i> locus was based on the epigenetic state at a region 10kb upstream of the TSS, outlined in green. In addition to being marked by H3K27Ac ChIP-Seq peaks with a localized dip in signal intensity, and peaks for the enhancer-associated histone acetyltransferase protein P300, this region showed dynamic changes in DNAse sensitivity. Note that a large DNAse sensitivity peak appears only in ES-derived NPCs, suggesting a differentiation state-specific chromatin opening at this region. At bottom, relative intensity of forkhead box protein ChIP-Seq from multiple cell types are pooled, with the darkest regions indicating intense FOX protein binding. Outlined in red are similar regions that show DNAse sensitivity beginning at the fetal brain stage that also colocalize with FOX protein binding. <b>(B)</b> A zoomed in view of the green region of increased DNAse sensitivity in PSC-derived NPCs. In blue are computationally predicted transcription factor binding sites from the ORCAtk database. The degree of genomic conservation along this region from the PhastCons64 database is shown in purple. At bottom are transcription factor ChIP-seq mapped peaks from the ENCODE database. The regions in green mark forkhead box transcription factor conserved motifs. Note that the forkhead box motifs co-localize with a region of highly conserved sequence, and the redundant binding of the forkhead box motif by many family members predicts that many such proteins can bind there.</p

    <i>In vitro</i> digestibility test of cell surface and secreted phytases.

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    <p>A: Measured phosphate (Pi) released from a corn-based diet mixed with cell surface or secreted phytases. B: Simulation of the pelleting process (i.e., incubation for 3 min at 80°C or 5 min at 90°C prior to activity measurement), followed by determination of the amount of released Pi. The levels of released Pi were compared with samples without heat treatment. GS115/ZαA served as a background measurement.</p
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