Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

One-pot synthesis of imines from benzyl alcohol and amines on Au/ZrO2 catalyst

Imines were synthesized from benzyl alcohol and amines by using catalysts of gold nanoparticles supported on ZrO2 (Au/ZrO2). The effects of reaction time, temperature, gold loadings and base were investigated. High yields were achieved under moderate conditions (60 °C) in the presence of KOCH3. For instance, the yield of N-benzylidenebenzylamine produced from benzyl alcohol and benzylamine on 3 wt% Au/ZrO2 is 87 %. The synthesis of imine involves two reaction steps: selective oxidation of benzyl alcohol to benzaldehyde and the coupling reaction of amines with benzaldehyde. In the first step, the base promotes the selective oxidation. The reactions of benzyl alcohol with three different amines, aniline, n-butylamine and benzylamine, were conducted to produce corresponding imines. The results show that the amine with stronger nucleophilicity has better ability to react with benzaldehyde in the second step, resulting in higher yield of the corresponding imine. We proposed a tentative mechanism for the synthesis process.

Vertical graphene gas- and bio-sensors via catalyst-free, reactive plasma reforming of natural honey

A rapid reforming of natural honey exposed to reactive low-temperature Ar + H2 plasmas produced high-quality, ultra-thin vertical graphenes, without any metal catalyst or external heating. This transformation is only possible in the plasma and fails in similar thermal processes. The process is energy-efficient, environmentally benign, and is much cheaper than common synthesis methods based on purified hydrocarbon precursors. The graphenes retain the essential minerals of natural honey, feature reactive open edges and reliable gas- and bio-sensing performance.

Plasma-enabled graded nanotube biosensing arrays on a Si nanodevice platform : catalyst-free integration and in situ detection of nucleation events

Low-temperature plasmas in direct contact with arbitrary, written linear features on a Si wafer enable catalyst-free integration of carbon nanotubes into a Si-based nanodevice platform and in situ resolution of individual nucleation events. The graded nanotube arrays show reliable, reproducible, and competitive performance in electron field emission and biosensing nanodevices.

Plasma-enabled, catalyst-free growth of carbon nanotubes on mechanically-written Si features with arbitrary shape

Simple, rapid, catalyst-free synthesis of complex patterns of long, vertically aligned multiwalled carbon nanotubes, strictly confined within mechanically-written features on a Si(1 0 0) surface is reported. It is shown that dense arrays of the nanotubes can nucleate and fully fill the features when the low-temperature microwave plasma is in a direct contact with the surface. This eliminates additional nanofabrication steps and inevitable contact losses in applications associated with carbon nanotube patterns. Using metal catalyst has long been considered essential for the nucleation and growth of surface-supported carbon nanotubes (CNTs) [1] and [2]. Only very recently, the possibility of CNT growth using non-metallic (e.g., oxide [3] and SiC [4]) catalysts or artificially created carbon-enriched surface layers [5] has been demonstrated. However, successful integration of carbon nanostructures into Si-based nanodevice platforms requires catalyst-free growth, as the catalyst nanoparticles introduce contact losses, and their catalytic activity is very difficult to control during the growth [6]. Furthermore, in many applications in microfluidics, biological and molecular filters, electronic, sensor, and energy conversion nanodevices, the CNTs need to be arranged in specific complex patterns [7] and [8]. These patterns need to contain the basic features (e.g., lines and dots) written using simple procedures and fully filled with dense arrays of high-quality, straight, yet separated nanotubes. In this paper, we report on a completely metal or oxide catalyst-free plasma-based approach for the direct and rapid growth of dense arrays of long vertically-aligned multi-walled carbon nanotubes arranged into complex patterns made of various combinations of basic features on a Si(1 0 0) surface written using simple mechanical techniques. The process was conducted in a plasma environment [9] and [10] produced by a microwave discharge which typically generates the low-temperature plasmas at the discharge power below 1 kW [11]. Our process starts from mechanical writing (scribing) a pattern of arbitrary features on pre-treated Si(1 0 0) wafers. Before and after the mechanical feature writing, the Si(1 0 0) substrates were cleaned in an aqueous solution of hydrofluoric acid for 2 min to remove any possible contaminations (such as oil traces which could decompose to free carbon at elevated temperatures) from the substrate surface. A piece of another silicon wafer cleaned in the same way as the substrate, or a diamond scriber were used to produce the growth patterns by a simple arbitrary mechanical writing, i.e., by making linear scratches or dot punctures on the Si wafer surface. The results were the same in both cases, i.e., when scratching the surface by Si or a diamond scriber. The procedure for preparation of the substrates did not involve any possibility of external metallic contaminations on the substrate surface. After the preparation, the substrates were loaded into an ASTeX model 5200 chemical vapour deposition (CVD) reactor, which was very carefully conditioned to remove any residue contamination. The samples were heated to at least 800 °C to remove any oxide that could have formed during the sample loading [12]. After loading the substrates into the reactor chamber, N2 gas was supplied into the chamber at the pressure of 7 Torr to ignite and sustain the discharge at the total power of 200 W. Then, a mixture of CH4 and 60% of N2 gases were supplied at 20 Torr, and the discharge power was increased to 700 W (power density of approximately 1.49 W/cm3). During the process, the microwave plasma was in a direct contact with the substrate. During the plasma exposure, no external heating source was used, and the substrate temperature (∼850 °C) was maintained merely due to the plasma heating. The features were exposed to a microwave plasma for 3–5 min. A photograph of the reactor and the plasma discharge is shown in Fig. 1a and b.

Control of density of self-organized carbon nanotube arrays by catalyst pretreatment through plasma immersion ion implantation

A simple and effective method of controlling the growth of vertically aligned carbon nanotube arrays in a lowerature plasma is presented. Ni catalyst was pretreated by plasma immersion ion implantation prior to the nanotube growth by plasma-enhanced chemical vapor deposition. Both the size distribution and the areal density of the catalyst nanoparticles decrease due to the ion-surface interactions. Consequently, the resulting size distribution of the vertically aligned carbon nanotubes is reduced to 50 ∼ 100 nm and the areal density is lowered (by a factor of ten) to 10 8 cm -2, which is significantly different from the very-high-density carbon nanotube forests commonly produced by thermal chemical vapor deposition. The efficiency of this pretreatment is compared with the existing techniques such as neutral gas annealing and plasma etching. These results are highly relevant to the development of the next-generation nanoelectronic and optoelectronic devices that require effective control of the density of nanotube arrays.

3-Orders-of-magnitude density control of single-walled carbon nanotube networks by maximizing catalyst activation and dosing carbon supply

Tailoring the density of random single-walled carbon nanotube (SWCNT) networks is of paramount importance for various applications, yet it remains a major challenge due to the insufficient catalyst activation in most growth processes. Here we report on a simple and effective method to maximise the number of active catalyst nanoparticles using catalytic chemical vapor deposition (CCVD). By modulating short pulses of acetylene into a methane-based CCVD growth process, the density of SWCNTs is dramatically increased by up to three orders of magnitude without increasing the catalyst density and degrading the nanotube quality. In the framework of a vapor-liquid-solid model, we attribute the enhanced growth to the high dissociation rate of acetylene at high temperatures at the nucleation stage, which can be effective in both supersaturating the larger catalyst nanoparticles and overcoming the nanotube nucleation energy barrier of the smaller catalyst nanoparticles. These results are highly relevant to numerous applications of random SWCNT networks in next-generation energy, sensing and biomedical devices. © 2011 The Royal Society of Chemistry.

Control of dense carbon nanotube arrays via hierarchical multilayer catalyst

Effective control of dense, high-quality carbon nanotube arrays using hierarchical multilayer catalyst patterns is demonstrated. Scanning/transmission electron microscopy, atomic force microscopy, Raman spectroscopy, and numerical simulations show that by changing the secondary and tertiary layers one can control the properties of the nanotube arrays. The arrays with the highest surface density of vertically aligned nanotubes are produced using a hierarchical stack of iron nanoparticles and alumina and silica layers differing in thickness by one order of magnitude from one another. The results are explained in terms of the catalyst structure effect on carbon diffusivity.

Single-step, catalyst-free plasma-assisted synthesis and growth mechanism of single-crystalline aluminum nitride nanorods

Synthesis of one-dimensional AlN nanostructures commonly requires high process temperatures (>900 °C), metal catalyst, and hazardous gas/powder precursors. We report on a simple, single-step, catalyst-free, plasma-assisted growth of dense patterns of size-uniform single-crystalline AlN nanorods at a low substrate temperature (∼650 °C) without any catalyst or hazardous precursors. This unusual growth mechanism is based on highly effective plasma dissociation of N2 molecules, localized species precipitation on AlN islands, and reduced diffusion on the nitrogen-rich surface. This approach can also be used to produce other high-aspect-ratio oxide and nitride nanostructures for applications in energy conversion, sensing, and optoelectronics. © 2010 American Institute of Physics.

Plasma/ion-controlled metal catalyst saturation : enabling simultaneous growth of carbon nanotube/nanocone arrays

It is shown that the simultaneous saturation of Ni nanoparticles used as catalyst for vertically aligned carbon nanotube and nanocone arrays can be improved in low-temperature plasma- or ion-assisted processes compared with neutral gas-based routes. The results of hybrid multiscale numerical simulations of the catalyst nanoarrays (particle sizes of 2 and 10 nm) saturation with carbon show the possibility of reducing the difference in catalyst incubation times for smallest and largest catalyst particles by up to a factor of 2. This approach is generic and provides process conditions for simultaneous nucleation and growth of uniform arrays of vertically aligned nanostructures. © 2008 American Institute of Physics.

Self-organized carbon connections between catalyst particles on a silicon surface exposed to atmospheric-pressure Ar + CH4 microplasmas

Ag nanoparticles and Fe-coated Si micrograins were separately deposited onto Si(1 0 0) surfaces and then exposed to an Ar + CH4 microplasma at atmospheric pressure. For the Ag nanoparticles, self-organized carbon nanowires, up to 400 nm in length were produced, whereas for the Fe-coated Si micrograins carbon connections with the length up to 100 μm were synthesized on the plasma-exposed surface area of about 0.5 mm2. The experiment has revealed that long carbon connections and short nanowires demonstrate quite similar behavior and structure. While most connections/nanowires tended to link the nearest particles, some wires were found to 'dissolve' into the substrate without terminating at the second particle. Both connections and nanowires are mostly linear, but long carbon connections can form kinks which were not observed in the carbon nanowire networks. A growth scenario explaining the carbon structure nucleation and growth is proposed. Multiscale numerical simulations reveal that the electric field pattern around the growing connections/nanowires strongly affects the surface diffusion of carbon adatoms, the main driving force for the observed self-organization in the system. The results suggest that the microplasma-generated surface charges can be used as effective controls for the self-organized formation of complex carbon-based nano-networks for integrated nanodevices.

Catalyst size effects on the growth of single-walled nanotubes in neutral and plasma systems

The results of large-scale (∼109 atoms) numerical simulations of the growth of different-diameter vertically-aligned single-walled carbon nanotubes in plasma systems with different sheath widths and in neutral gases with the same operating parameters are reported. It is shown that the nanotube lengths and growth rates can be effectively controlled by varying the process conditions. The SWCNT growth rates in the plasma can be up to two orders of magnitude higher than in the equivalent neutral gas systems. Under specific process conditions, thin SWCNTs can grow much faster than their thicker counterparts despite the higher energies required for catalyst activation and nanotube nucleation. This selective growth of thin SWCNTs opens new avenues for the solution of the currently intractable problem of simultaneous control of the nanotube chirality and length during the growth stage.

Plasma-controlled metal catalyst saturation and the initial stage of carbon nanostructure array growth

The kinetics of the nucleation and growth of carbon nanotube and nanocone arrays on Ni catalyst nanoparticles on a silicon surface exposed to a low-temperature plasma are investigated numerically, using a complex model that includes surface diffusion and ion motion equations. It is found that the degree of ionization of the carbon flux strongly affects the kinetics of nanotube and nanocone nucleation on partially saturated catalyst patterns. The use of highly ionized carbon flux allows formation of a nanotube array with a very narrow height distribution of half-width 7 nm. Similar results are obtained for carbon nanocone arrays, with an even narrower height distribution, using a highly ionized carbon flux. As the deposition time increases, nanostructure arrays develop without widening the height distribution when the flux ionization degree is high, in contrast to the fairly broad nanostructure height distributions obtained when the degree of ionization is low.

Carbon saturation of arrays of Ni catalyst nanoparticles of different size and pattern uniformity on a silicon substrate

The kinetics of saturation of Ni catalyst nanoparticle patterns of the three different degrees of order, used as a model for the growth of carbon nanotips on Si, is investigated numerically using a complex model that involves surface diffusion and ion motion equations. It is revealed that Ni catalyst patterns of different degrees of order, with Ni nanoparticle sizes up to 12.5 nm, exhibit different kinetics of saturation with carbon on the Si surface. It is shown that in the cases examined (surface coverage in the range of 1-50%, highly disordered Ni patterns) the relative pattern saturation factor calculated as the ratio of average incubation times for the processes conducted in the neutral and ionized gas environments reaches 14 and 3.4 for Ni nanoparticles of 2.5 and 12.5 nm, respectively. In the highly ordered Ni patterns, the relative pattern saturation factor reaches 3 for nanoparticles of 2.5 nm and 2.1 for nanoparticles of 12.5 nm. Thus, more simultaneous saturation of Ni catalyst nanoparticles of sizes in the range up to 12.5 nm, deposited on the Si substrate, can be achieved in the low-temperature plasma environment than with the neutral gas-based process.

Growth of carbon nanocone arrays on a metal catalyst: The effect of carbon flux ionization

The growth of carbon nanocone arrays on metal catalyst particles by deposition from a low-temperature plasma is studied by multiscale Monte Carlo/surface diffusion numerical simulation. It is demonstrated that the variation in the degree of ionization of the carbon flux provides an effective control of the growth kinetics of the carbon nanocones, and leads to the formation of more uniform arrays of nanostructures. In the case of zero degree of ionization (neutral gas process), a width of the distribution of nanocone heights reaches 360 nm with the nanocone mean height of 150 nm. When the carbon flux of 75% ionization is used, the width of the distribution of nanocone heights decreases to 100 nm, i.e., by a factor of 3.6. A higher degree of ionization leads to a better uniformity of the metal catalyst saturation and the nanocone growth, thus contributing to the formation of more height-uniform arrays of carbon nanostructures.