Activation of immobilized lipase in non-aqueous systems by hydrophobic poly-DL-tryptophan tethers

Many industrially important reactions use immobilized enzymes in non-aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule ("tether") between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most "long" linkers (e.g., polyethylene oxide derivatives) are relatively hydrophilic, improving the solubility of the linker-enzyme conjugate in polar environments, but this provides little benefit in non-polar environments such as organic solvents. We present a novel method for the covalent immobilization of enzymes on solid surfaces using a long, hydrophobic polytryptophan tether. Candida antarctica lipase B (CALB) was covalently immobilized on non-porous, functionalized 1-microm silica microspheres, with and without an intervening hydrophobic poly-DL-tryptophan tether (n approximately 78). The polytryptophan-tethered enzyme exhibited 35 times greater esterification of n-propanol with lauric acid in the organic phase and five times the hydrolytic activity against p-nitrophenol palmitate, compared to the activity of the same enzyme immobilized without tethers. In addition, the hydrophobic tethers caused the silica microspheres to disperse more readily in the organic phase, while the surface-immobilized control treatment was less lipophilic and quickly settled out of the organic phase when the suspensions were not vigorously mixed.     

Biosilicification of dual-fusion enzyme immobilized on magnetic nanoparticle

Rapid recovery, immobilization, and silica encapsulation of a dual-fusion enzyme was achieved by using iminodiacetic acid (IDA) modified magnetic nanoparticle as a carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme in which a silica-precipitating peptide R5 and a metal ion complexing peptide (His)(6) were fused to its N- and C-terminal, respectively. After charging the magnetic particle with Cu(2+), the dual-fusion DAAO of 0.43 g could be directly recovered from the recombinant E. coli crude extract and immobilized on 1 g of the magnetic particle. Once in contact with hydrolyzed tetramethoxysilane (TMOS), the homogeneously dispersed immobilized dual-fusion DAAO was biosilicificated to form aggregates with size about 50 microm. The silica-encapsulated immobilized DAAO demonstrated a pyruvic acid production rate comparable with that of the naked immobilized DAAO in five repeated batch reactions when D-alanine was used as substrate. Furthermore, 85% of its activity remained after incubation at 60 degrees C for 1 h while the naked immobilized DAAO lost all its activity. This process provides the advantages that recombinant fusion enzyme can be directly recovered from crude extract, silica encapsulation protects the enzyme from leakage and denaturation, and the enzyme activity can be easily retrieved by applying a magnetic field.     

RNA isolation from yeast using silica matrices.

RNA isolation from yeast is complicated by the need to initially break the cell wall. While this can be accomplished by glass bead disruption or enzyme treatment, these approaches result in DNA contamination and/or the need for incubation periods. We have developed a protocol for the isolation of RNA samples from yeast that minimizes degradation by RNases and incorporates two purification steps: acid phenol extraction and binding to a silica matrix. The procedure requires no precipitation steps, facilitating automation, and can be completed in less than 90 min. The RNA quality is ideal for microarray analysis.     

Influence of various biological matrices (plasma, blood microdialysate) on chromatographic performance in the determination of β-blockers using an alkyl-diol silica precolumn for sample clean-up

A HPLC column-switching system with LiChrospher RP-8 ADS precolumn was applied for the determination of beta-blockers (atenolol, pindolol, propranolol) in human plasma. The influence of biological matrices on the changes of the chromatographic parameters such as retention time, peak symmetry, area and selectivity were investigated. After injection of 5 ml plasma a decrease of retention times of the analytes was observed of up to 25% and an increase of asymmetry factors of up to 5%. Peak areas and selectivities were not changed. The observed effect could indicate changes of chromatographic performance caused by contributions of the analytical column or the ADS precolumn. The experiments with microdialysis excluded the contribution of the analytical column. A detailed investigation of experiments have been discussed in this paper.     

Wood anatomy of Polygonaceae: analysis of a family with exceptional wood diversity

Quantitative and qualitative data are presented for woods of 30 species of woody Polygonaceae. Wood features that ally Polygonaceae with Plumbaginaceae include nonbordered perforation plates, storeying in narrow vessels and axial parenchyma, septate or nucleate fibres, vasicentric parenchyma, pith bundles that undergo secondary growth, silica bodies, and ability to form successive cambia. These features are consistent with pairing of Plumbaginaceae and Polygonaceae as sister families. Wood features that ally Polygonaceae with Rhabdodendraceae include nonbordered perforation plates, presence of vestured pits in vessels, presence of silica bodies and dark-staining compounds in ray cells, and ability to form successive cambia. Of the features listed above, nonbordered perforation plates and ability to form successive cambia may be symplesiomorphies basic to Caryophyllales sensu lato . The other features are more likely to be synapomorphies. Wood data thus support molecular cladograms that show the three families near the base of Caryophyllales s.l. Chambered crystals are common to three genera of the family and may indicate relationship. Ray histology suggests secondary woodiness in Antigonon, Atraphaxis, Bilderdykia, Dedeckera, Eriogonum, Harfordia, Muehlenbeckia, Polygonum , and Rumex . Other genera of the family show little or no evidence of secondary woodiness. Molecular data are needed to confirm this interpretation and to clarify the controversial systematic groupings within the family proposed by various authors. Vessel features of Polygonaceae (lumen diameter, element length, density, degree of grouping) show an extraordinary range from xeromorphy to mesomorphy, indicating that wood has played a key role in ecological and habital shifts within the family; the diversity in ecology and habit are correlated with quantitative wood data.  © 2003 The Linnean Society of London. Botanical Journal of the Linnean Society , 2003, 141 , 25−51.     

Enantiomerization of 3-carbethoxy-l,4-benzodiazepin-2-one: combined chiral HPLC and spectroscopic study.

Recently developed chiral HPLC columns CHIRIS AD1 and CHIRIS AD2 have been demonstrated to separate racemic, configurationally unstable ethyl-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine-3-carboxylate (1) and its 3-methyl congener 2; fast on-column enantiomerization of configurationally unstable 1 was observed, however. Addition of 0.1% of AcOH to the eluting mixture inhibits enantiomerization, whereas the same percentage of Et(3)N completely precludes enantioseparation, suggesting base-catalysis by free beta-aminoethyl groups, present in low percentage in chiral stationary phase (CSP). When both CSPs were prepared under conditions of nonexhaustive acylation by N-DNB-alpha-aminoacids, no separation of 1 was observed. The rate of enantiomerization on CHIRIS AD2 was determined at 25 degrees C, the mechanism is discussed, and experimental results correlated with calculated relative stabilities of the tautomers la-c. Absolute (3S) configuration of (+) enantiomers of 1 and 2 was determined by comparison of their eluation profile to that of (+/-)-3 and (3S)-(+)-3, taking into account relative (psia or psie) configuration of the prevailing conformer in solution.     

Activation of subtilisin Carlsberg in hexane by lyophilization in the presence of fumed silica

Subtilisin Carlsberg (SC) was lyophilized from an aqueous buffer solution containing different amounts of unmodified commercial fumed silica. The activity of the enzyme/fumed silica preparation in hexane was compared to pure freeze-dried enzyme, and to a freeze-dried preparation reported in the literature with potassium chloride as additive. A sharp increase in enzyme activity was found to correlate with an increasing amount of fumed silica added to the enzyme solution prior to freeze-drying. A weight-ratio of 98.5 wt.% fumed silica relative to the mass of the final enzyme/fumed silica preparation led to about 130-fold increased activity of SC in hexane (when compared to pure lyophilized SC in hexane). This is about twice the activation effect compared to including potassium chloride in the buffer solution before freeze-drying [1]. When freezing at −20 °C instead of in liquid nitrogen, even better activation was observed with fumed silica. We hypothesize that the activation of SC in hexane by immobilization of the enzyme on fumed silica is likely due to the distribution of the enzyme on the large surface area of fumed silica. This alleviates mass transfer limitations.     

以短肽组装体为模板仿生矿化二氧化硅纳米管

目的 二氧化硅纳米管由于具有生物相容性好、光学性质优异等特殊性能在不同领域展现出良好的应用前景，其尺寸和形貌可显著影响材料性质。为制备尺寸较大的纳米管并拓展其在不同领域的应用，本研究选择尺寸较大的多肽组装体为模板，进行二氧化硅的仿生矿化。方法 以Bola型多肽Ac-KI₃VK-CONH₂自组装形成的尺寸较大（直径约40 nm）的纳米管为模板，通过仿生矿化的方法制备二氧化硅纳米材料。首先考察了多肽纳米管的稳定性，发现在稀释、加入有机溶剂和改变溶液pH值时，纳米管的形貌均被破坏，展示出较差的稳定性。在此基础上，以该多肽纳米管为模板，选择反应速率较快的正硅酸甲酯（TMOS）为前驱体仿生矿化二氧化硅纳米材料，探索了不同矿化条件对二氧化硅纳米管尺寸和形貌的影响。结果 以反应速率较快的TMOS为硅源、体积百分比浓度为1.11%~3.33%、溶液为中性或者弱碱性时能够矿化形成形貌和尺寸较大且分布均匀的二氧化硅纳米管。结论 通过选择合适的多肽组装体模板和反应速率快的TMOS为硅源，成功制备出了尺寸较大的二氧化硅纳米管，这为拓展其在不同领域的应用奠定了基础，具有重要意义。     

A silicon budget for an Alaskan arctic lake

The cycling of dissolved reactive silicate (DRS) and sedimentary biogenic SiO₂ has been examined in ultraoligotrophic Toolik Lake, Alaska. Watershed output of DRS (∼ 7 mmol m ^{− 2} yr ⁻¹) is similar to other arctic study sites, but a short water residence time results in the poor retention of DRS (∼17%) within the lake. Biogenic SiO₂ concentrations in surficial sediments average 123 mg g⁻¹ with the highest concentrations arising from the production of benthic diatoms in the littoral zone. Lake water DRS concentrations are highest prior to spring flow and photic zone concentrations exceed 23 μmol 1⁻¹ at the time of greatest primary production. Wintertime increases in watercolumn DRS concentrations and spring and summer pore water DRS flux estimates indicate that internal cycling within the lake may supply an amount of DRS equivalent to that which biologically utilized.     

Stable Formamidinium‐Based Perovskite Solar Cells via In Situ Grain Encapsulation

Formamidinium (FA)‐based lead iodide perovskites have emerged as the most promising light‐absorber materials in the prevailing perovskite solar cells (PSCs). However, they suffer from the phase‐instability issue in the ambient atmosphere, which is holding back the realization of the full potential of FA‐based PSCs in the context of high efficiency and stability. Herein, the tetraethylorthosilicate hydrolysis process is integrated with the solution crystallization of FA‐based perovskites, forming a new film structure with individual perovskite grains encapsulated by amorphous silica layers that are in situ formed at the nanoscale. The silica not only protects perovskite grains from the degradation but also enhances the charge‐carrier dynamics of perovskite films. The underlying mechanism is discussed using a joint experiment‐theory approach. Through this in situ grain encapsulation method, PSCs show an efficiency close to 20% with an impressive 97% retention after 1000‐h storage under ambient conditions.