Biocatalytic synthesis of gold nanoparticles with cofactor regeneration in recombinant Escherichia coli cells

Here we report the enzymatic synthesis of gold nanoparticles (Au NPs) by an engineered Escherichia coli harboring an NADH cofactor regeneration system coupled with glycerol dehydrogenase, which can be triggered by the addition of exogenous glycerol.     

Asymmetric reduction of ketones with recombinant E. coli whole cells in neat substrates

The asymmetric reduction of ketones is performed by using lyophilized whole cells in neat substrates with defined water activity (a(w)). Ketones and alcohols prone to be unstable in aqueous media can now be converted via biocatalysis.     

Asymmetric hydrolysis of 2-hydroxy-carboxylic esters using recombinant Escherichia coli

Optically active 2-hydroxy-carboxylates are important compounds for their use as intermediates in the synthesis of pharmaceuticals and stereoblock polymers. Enterobacter sp. DS-S-75 and the recombinant Escherichia coli harbouring the 4-chloro-3-hydroxybutyrate (CHB) hydrolase gene from the strain DS-S-75 showed asymmetric hydrolytic activity towards 2-hydroxy-carboxylates, as well as towards CHB. It was discussed that the hydroxyl group in the substrate was particularly important for the asymmetric hydrolytic activity of the CHB hydrolase, and as such, it was re-designated to EnHCH (hydroxy-carboxylic ester hydrolase derived from Enterobacter sp.). Using the recombinant cell, both the reaction rate and the concentration of the substrates were significantly improved upon when compared to that of DS-S-75. Optically active 2-hydroxy-carboxylates could be synthesized on a practical basis for industrial production in this report.     

A highly fluorous room-temperature ionic liquid exhibiting fluorous biphasic behavior and its use in catalyst recycling

[formula: see text] A novel fluorous room-temperature ionic liquid, 1-butyl-3-methyl-imidazolium tetrakis[p-[dimethyl(1H, 1H, 2H, 2H-perfluorooctyl)silyl]phenyl]-borate (1), was used as a solvent for the homogeneous hydrosilylation of 1-octene catalyzed by a fluorous version of Wilkinson's catalyst. The catalyst was recycled by biphasic separation with an average retention of catalyst activity of 94%. As opposed to other ionic liquids, 1 exhibits high miscibility with apolar compounds such as alkenes and resembles fluorous solvents in its phase behavior with organic solvents.     

Asymmetric Baeyer-Villiger oxidations of 4-mono- and 4,4-disubstituted cyclohexanones by whole cells of engineered Escherichia coli.

Whole cells of an Escherichia coli strain that overexpresses Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase have been used for the Baeyer-Villiger oxidations of a variety of 4-mono- and 4,4-disubstituted cyclohexanones. In cases where comparisons were possible, this new biocatalytic reagent provided lactones with chemical yields and optical purities that were comparable to those obtained from the purified enzyme or a strain of bakers' yeast that expresses the same enzyme. The efficient production of cyclohexanone monooxygenase in the E. coli expression system (ca. 30% of total soluble protein) allowed these oxidations to reach completion in approximately half the time required for the engineered bakers' yeast strain. Surprisingly, 4,4-disubstituted cyclohexanones were also accepted by the enzyme, and the enantioselectivities of these oxidations could be rationalized by considering the conformational energies of bound substrates along with the enzyme's intrinsic enantioselectivity. The enzyme expressed in E. coli cells also oxidized several 4-substituted cyclohexanones bearing polar substituents, often with high enantioselectivities. In the case of 4-iodocyclohexanone, the lactone was obtained in > 98% ee and its absolute configuration was assigned by X-ray crystallography. The crystal belongs to the monoclinic P2(1) space group with a = 5.7400(10), b = 6.1650(10), c = 11.377(2) A, b = 99.98(2) degrees, and Z = 2. Taken together, these results demonstrate the utility of an engineered bacterial strain in delivering useful chiral building blocks in an experimentally simple manner.     

Comparison of uranyl extraction mechanisms in an ionic liquid by use of malonamide or malonamide-functionalized ionic liquid

The extraction of uranyl from acidic (HNO(3)) aqueous solutions toward an ionic liquid phase, C(1)-C(4)-imTf(2)N (1-methyl,3-butylimidazolium Tf(2)N), has been investigated as a function of initial acid concentration and ligand concentration for two different extracting moieties: a classical malonamide, N,N'-dimethyl-N,N'-dibutylmalonamide (DMDBMA) and a functionalized IL composed of the Tf(2)N(-) anion and an imidazolium cation on which a malonamide pattern has been grafted (FIL-MA). The extraction mechanism, as demonstrated through the influence of added C(1)-C(4)-imCl or added LiTf(2)N in the aqueous phase, is slightly different between the DMDBMA and FIL-MA extracting agents. Modeling of the extraction data evidences a double extraction mechanism, with cation exchange of UO(2)(2+)versus 2 H(+) for DMDBMA or versus C(1)-C(4) -im(+) and H(+) for FIL-MA at low acidic values, and through anion exchange of [UO(2)(NO(3))(3)](-)versus Tf(2)N(-) for both ligands at high HNO(3) concentrations. The FIL-MA molecule is more efficient than its classical DMDBMA parent.     

Asymmetric transfer hydrogenation of prochiral ketones catalyzed by aminosulfonamide-ruthenium complexes in ionic liquid

Chiral aminosulfonamides containing imidazolium group were used as ligands for the ruthenium(II)-catalyzed asymmetric transfer hydrogenation of prochiral ketones in ionic liquid, affording good to excellent conversions and enantiomeric excesses. The catalytic system could be easily recovered and reused several times.      

Tandem isomerisation-metathesis catalytic processes of linear olefins in ionic liquid biphasic system

trans-3-Hexene is converted to heavier linear olefins by a tandem Ru-catalysed biphasic isomerisation/metathesis sequence. The difference in olefin metathesis and isomerisation rates is modulated by keeping the ionophilic metathesis catalyst in an ionic phase whilst the isomerisation catalyst is in another organic non-polar phase.     

Partition of horseradish peroxidase with maintained activity in aqueous biphasic system based on ionic liquid

Enzyme activity and partition behavior in aqueous biphasic systems (ABSs) consisting of ionic liquid (IL) and salt (IL-ABSs) were investigated to increase our understanding of IL-ABSs and shed light on their application potential as enzyme extraction system. With horseradish peroxidase (HRP) as the model enzyme, several effects of alkylimidazolium chloride-K₂HPO₄ ABSs on activity and partition behavior of enzyme were studied including alkyl chain length of ILs and concentrations of each component. High lyotropic ILs (1-butyl-3-methylimidazolium and 1-ethyl-3-methylimidazolium chloride) and adequate water content (>40%) were both essential for the activity maintenance of HRP in IL-ABS. 1-Butyl-3-methylimidazolium chloride ([C₄mim]Cl) was found to be an appropriate IL for phase forming and HRP activity retaining. After optimization of phase condition, about 80% HRP amount was distributed in the IL-rich upper phase, and greater than 90% enzyme activity was obtained. Moreover, compared with the commonly used polymer-based ABSs, this [C₄mim]Cl-ABS has a much lower viscosity, which is very beneficial to the experimental operation. Therefore, the tested IL-ABS could be considered as a potential enzyme extraction system.     

One-step purification of a recombinant protein from a whole cell extract by reversed-phase high-performance liquid chromatography

We have developed a one-step facile, flexible and readily scalable purification method for a recombinant protein, TM 1-99 (113 amino acid residues; 12,837 Da) based on reversed-phase high-performance liquid chromatography (RP-HPLC) from an E. coli cell lysate. Following cell lysis, the cell contents were extracted with 0.1% aqueous trifluoroacetic acid (TFA), applied directly under conditions of high sample load to a narrow bore RP-HPLC C(8) column (150 mm x 2.1 mm I.D.) and eluted by a shallow gradient of acetonitrile (0.1%/min). Loads of 23 and 48 mg of lyophilized crude cell extract produced 2.4 and 4.2mg of purified product (>94% pure), respectively, at >94% recovery. Our results show the excellent potential of one-step RP-HPLC for purification of recombinant proteins from cell lysates, where high yields of purified product and greater purity are achieved compared to affinity chromatography. Such an approach was also successful in purifying just trace levels (<0.1% of total contents of crude sample) of TM 1-99 from a cell lysate.     

Beneficial effect of water as second solvent in ionic liquid biphasic catalytic hydrogenations

When reactions take place in ionic liquids, a solvent is normally used to extract the products after reaction. It is reported here how the presence of the solvent during the reaction already can seriously improve the catalytic performance. Above all, employing water as the added solvent enhanced the catalytic activities significantly, ascribed to the creation of a well mixed ‘emulsion-like’ system. The reductions of methyl 2-acetamidoacrylate with Rh-EtDuPHOS and of 2-cyclohexen-1-one with Wilkinson’s catalyst in bmimPF₆ were thus successfully performed in the presence of water. The complexes were easily recycled and Rh-EtDuPHOS was even no longer air sensitive.     

Asymmetric hydrogenation of methyl α-benzamido cinnamate in ionic liquid solvent

The room temperature ionic liquid EMIMOTf was employed as the sole reaction solvent for the asymmetric hydrogenation of methyl α-benzamido cinnamate. Under conditions of 60 psi hydrogen and 50 °C for 24 h, near quantitative conversions were observed using both the achiral DiPFc-Rh catalyst, and the chiral EtDuPHOS-Rh catalysts. Enantiomeric excess of 89% ee was observed for hydrogenations carried out with the chiral catalyst.     

New approach to regeneration of an ionic liquid containing solvent by molecular distillation

A promising new process for the regeneration of solvents with ionic liquid by molecular distillation has been proposed. The main advantage of this process compared with the classic stripping of acids with alkali solution is that the volatile organic acid is recovered with a yield of about 88 % in the form of free acid, not as its salt. Furthermore, improvement on the yield of the stripping can be assumed. The consumption of chemicals in stripping by molecular distillation is reduced.     

Antioxidant properties of recombinant allophycocyanin expressed in Escherichia coli

Allophycocyanin (APC) is the main core component of phycobilisome found in blue-green algae. The apo-allophycocyanin and its subunits were expressed in Escherichia coli and their antioxidant properties were evaluated using deoxyribose assay. The result showed that both recombinant allophycocyanin fused with maltose binding protein (MBP) tag and 6 x His-tag and their alpha or beta subunits can scavenge hydroxyl radicals successfully, and the separated alpha or beta subunits had a higher inhibition effect on hydroxyl radicals than that when they combined together. The scavenging effects increased with the increasing concentration. These results clearly suggested that apo-allophycocyanin is involved in the antioxidant and radical scavenging activity of phycocyanin, and the antioxidant activity may be partially responsible to the anti-tumor effect of the recombinant allophycocyanin.     

Extraction of proteins from biological fluids by use of an ionic liquid/aqueous two-phase system

An ionic liquid/aqueous two-phase system based on the hydrophilic ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl) and K(2)HPO(4) has been employed for direct extraction of proteins from human body fluids for the first time. Proteins present at low levels were quantitatively extracted into the BmimCl-rich upper phase with a distribution ratio of about 10 between the upper and lower phase and an enrichment factor of 5. Addition of an appropriate amount of K(2)HPO(4) to the separated upper phase results in a further phase separation, giving rise to an improved enrichment factor of 20. FTIR and UV spectroscopy demonstrated that no chemical (bonding) interactions between the ionic liquid and the protein functional groups were identifiable, while no alterations of the natural properties of the proteins were observed. The partitioning of proteins in the two-phase system was assumed to have been facilitated by the electrostatic potential difference between the coexisting phases, as well as by salting out effects. The system could be applied successfully for the quantification of proteins in human urine after on-line phase separation in a flow system. The use of an ionic liquid, as a green solvent, offers clear advantages over traditional liquid-liquid extractions, in which the use of toxic organic solvents is unavoidable.     

果糖一步转化制备呋喃基燃料：溶剂效应及离子液体修饰活性金属对产物选择性的决定作用

在众多生物基化合物中,2,5-二甲基呋喃(DMF)是一种有实用前景的可再生液体生物质燃料,也是一种具有重要价值的化学品,可作为生产对苯二甲酸的原料.2,5-二甲基四氢呋喃(DMTF)是DMF进一步加氢产物,该化合物比DMF更稳定,适合长期保存;由于具有更高的氢碳比,用作生物燃料燃烧时能够释放更多能量.研究生物质资源制备DMF和DMTF对可再生资源制备液体燃料和化学品具有重要意义.从生物质多糖出发制备这两类化合物,中间经历了水解、脱水、加氢、加氢脱氧等多个反应步骤,每一步反应都十分复杂,包含许多副反应途径.此外,由于每一步反应条件的不兼容性,大多数研究集中在分步反应阶段,鲜有文献能够实现从碳水化合物原料直接转化为DMF和DMTF.发展由生物质一锅法多步耦合转化技术制备化学品和燃料,不仅具有科学意义,而且可大大简化反应过程,避免中间产物分离和损失,节省资源和时间,历来受到化学家和工业界的关注.本文利用离子液体对Ru/C催化剂电子性质的修饰作用以及溶剂效应的影响,设计了离子液体/THF双相体系中果糖直接催化转化制备2,5-二甲基呋喃(DMF)和2,5-二甲基四氢呋喃(DMTF)的新路线.该转化过程耦合了果糖脱水制HMF、HMF加氢及加氢脱氧生成DMF和DMTF等多步反应.通常在HMF加氢转化过程中, Ru/C催化剂的高活性易导致HMF深度加氢生成大量开环产物及气体,我们借助离子液体与有机溶剂的不同溶解性,筛选出[BMIm]Cl/THF双相溶剂体系,使极性HMF在离子液体层反应,生成弱极性的DMF和DMTF能及时被THF萃取出来,有效稳定了目标产物.其次,果糖转化为HMF会产生少量水,通常水的存在易导致HMF发生水合等副反应,对下一步的加氢转化是不利因素;然而在本催化体系中,由于[BMIm]Cl能与水以较强的氢键结合形成水合物,对水分子起到了束缚作用,减少了HMF发生水解、水合等副反应的机会.另一方面,离子液体粘度较大,微量水的存在能降低离子液体层粘度,改善传质,从而提高反应速率.在HMF加氢处理过程中,离子液体对DMF和DMTF的生成起了决定作用.当反应体系中不添加离子液体,以THF为溶剂,反应结束后未检测到DMF生成, DMTF的收率仅为2%,但HMF已经完全转化.取气体样品进行GC分析,发现有部分气相产物生成,包括CO2、CH4和C2H6等.液体混合物进行GC-MS检测,发现产物主要包括DHMTF、5-甲基四氢糠醇(MTFA)、四氢糠醇(TFA)、1,2-戊二醇、DMTF、2-己醇和少量戊醇,产物中所有呋喃环结构的双键都发生加氢反应.以上结果表明,没有离子液体的THF中, Ru/C催化的HMF涉氢反应平衡已发生改变.当反应体系中添加0.2 g离子液体[BMIm]Cl进行HMF的加氢时,此时开始有DMF生成,随着[BMIm]Cl量依次增加, DMF以及DMTF的收率也呈上升趋势.1.0 g离子液体获得两种产物最高收率为68%.然而,如果进一步增加[BMIm]Cl的量到2.0 g,呋喃基液体燃料DMF和DMTF的收率却开始下降.综合以上实验结果,我们认为适量的[BMIm]Cl存在有可能会对催化剂物理化学性质造成影响,从而对产物的选择性起了决定性作用.通过对催化剂进行元素分析、XPS、H2-TPR表征以及一系列对比实验证明,离子液体不仅促进果糖脱水转化为HMF,同时在HMF选择性加氢反应中可修饰活性金属电子性质,改变催化路径,是多步串联反应能够耦合的关键因素.在[BMIm]Cl/THF双相溶剂体系中,离子液体的“溶剂笼效应”促进DMF和DMTF高效生成, THF的萃取功能对目标产物的稳定起了关键作用.以上对催化剂和溶剂的合理设计共同促进高产率呋喃基燃料的获得.该研究实现由六碳糖直接选择转化获取DMF和DMTF,为生物质高效催化转化制备生物基能源化学品提供了新思路.     

Reduction of silanophilic interactions in liquid chromatography with the use of ionic liquids

A suppression of silanophilic interactions by the selected ionic liquids added to the mobile phase in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) is reported. Acetonitrile was used as the eluent, alone or with various concentrations of water and phosphoric buffer pH 3. Selectivity of the normal (NP) and the reversed (RP) stationary phase material was examined using a series of proton-acceptor basic drugs analytes. The ionic liquids studied appeared to significantly affect analyte retention in NP-TLC, RP-TLC and RP-HPLC systems tested. Consequently, the increased separation selectivity was attained. Due to ionic liquid additives to eluent even analytes could be chromatographed, which were not eluted from the silica-based stationary phase materials with 100% of acetonitrile in the mobile phase. Addition of ionic liquid already in very small concentration (0.5%, v/v) could reduce the amount of acetonitrile used during the optimization of basic analytes separations in TLC and HPLC systems. Moreover, the influence of temperature on the separation of basic analytes was demonstrated and considered in practical HPLC method development.     

Aerobic oxidation with N-hydroxyphthalimide catalysts in ionic liquid

N-Hydroxyphthalimide (NHPI)-catalyzed aerobic oxidations in the ionic liquids were examined for the first time. Both NHPI and its ionic derivative, 3-pyridinylmethyl-N-hydroxyphthalimide (Py-NHPI), were found to have better performance in the ionic liquid than in the conventional organic solvents for the aerobic oxidation of N-alkylamides to imides. On the other hand, Py-NHPI was found to be a much better catalyst than NHPI for the aerobic oxidation of benzylic compounds in the ionic liquid.