Origin of rectification in boron nitride heterojunctions to silicon.

Cubic and hexagonal boron nitride (cBN and hBN) heterojunctions to n-type Si are fabricated under low-energy ion bombardment by inductively coupled plasma-enhanced chemical vapor deposition using the chemistry of fluorine. The sp2-bonded BN/Si heterojunction shows no rectification, while the cBN/sp2BN/Si heterojunction has rectification properties analogue to typical p-n junction diodes despite a large thickness (∼130 nm) of the sp2BN interlayer. The current-voltage characteristics at temperatures up to 573 K are governed by thermal excitation of carriers, and mostly described with the ideal diode equation and the Frenkel-Poole emission model at low and high bias voltages, respectively. The rectification in the cBN/sp2BN/Si heterojunction is caused by a bias-dependent change in the barrier height for holes arising from stronger p-type conduction in the cBN layer and enhanced with the thick sp2BN interlayer for impeding the reverse current flow at defect levels mainly associated with grain boundaries.

Direct deposition of cubic boron nitride films on tungsten carbide-cobalt.

Thick cubic boron nitride (cBN) films in micrometer-scale are deposited on tungsten carbide-cobalt (WC-Co) substrates without adhesion interlayers by inductively coupled plasma-enhanced chemical vapor deposition (ICP-CVD) using the chemistry of fluorine. The residual film stress is reduced because of very low ion-impact energies (a few eV to ∼25 eV) controlled by the plasma sheath potential. Two types of substrate pretreatment are used successively; the removal of surface Co binder using an acid solution suppresses the catalytic effect of Co and triggers cBN formation, and the surface roughening using mechanical scratching and hydrogen plasma etching increases both the in-depth cBN fraction and deposition rate. The substrate surface condition is evaluated by the wettability of the probe liquids with different polarities and quantified by the apparent surface free energy calculated from the contact angle. The surface roughening enhances the compatibility in energy between the cBN and substrate, which are bridged by the interfacial sp(2)-bonded hexagonal BN buffer layer, and then, the cBN overlayer is nucleated and evolved easier.

Construction of microbial platform for an energy-requiring bioprocess: practical 2'-deoxyribonucleoside production involving a C-C coupling reaction with high energy substrates.

BACKGROUND: Reproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway. RESULTS: Using E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2'-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2'-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2'-deoxyinosine, the deaminated product of 2'-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process. CONCLUSION: A critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary metabolites that accumulate at very low levels in the metabolic pool, will grow. The development of an efficient production method for these compounds will have a great impact in both fields of applied microbiology and industry and will also serve as a good example of a microbial platform for energy-requiring bioprocesses.

Screening and characterization of a phosphopentomutase useful for enzymatic production of 2'-deoxyribonucleoside.

Bacillus sphaericus AKU 229 was found to produce an acetaldehyde-tolerant and phosphorylated compound-tolerant phosphopentomutase useful for enzymatic 2'-deoxyribonucleoside production. The gene encoding the phosphopentomutase was cloned and expressed in Escherichia coli. The E. coli expressing B. sphaericus phosphopentomutase was an excellent catalyst as to production of 2'-deoxyribonucleoside in the presence of acetaldehyde and phosphorylated compounds such as fructose 1,6-diphosphate, and d-glyceraldehyde 3-phosphate, which are derived from glucose through glycolysis with yeast cells, and exist abundantly in the practical reaction mixture for enzymatic 2'-deoxyribonucleoside production.

One-pot microbial synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase.

A one-pot enzymatic synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker's yeast (Saccharomyces cerevisiae) generated ATP which was used to produce D: -glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The D: -glyceraldehyde 3-phosphate produced was transformed to 2'-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2'-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2'-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli.

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.

Biochemical retrosynthesis of 2'-deoxyribonucleosides from glucose, acetaldehyde, and a nucleobase.

2'-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2'-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2'-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2'-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker's yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5'-triphosphate generated from adenosine 5'-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via D-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2'-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2'-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2'-deoxyribonucleoside synthesis. 2'-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.

Construction of deoxyriboaldolase-overexpressing Escherichia coli and its application to 2-deoxyribose 5-phosphate synthesis from glucose and acetaldehyde for 2'-deoxyribonucleoside production.

The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2'-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

Microbial production of 2-deoxyribose 5-phosphate from acetaldehyde and triosephosphate for the synthesis of 2'-deoxyribonucleosides.

2-Deoxyribose 5-phosphate was produced from acetaldehyde and dihydroxyacetone phosphate via D-glyceraldehyde 3-phosphate by Klebsiella pneumoniae B-4-4 through deoxyriboaldolase- and triosephosphate isomerase-catalyzing reactions. Under the optimum conditions, 98.7 mM 2-deoxyribose 5-phosphate was produced from 200 mM acetaldehyde and 117 mM dihydroxyacetone phosphate in 2 h with a molar yield of 84%. The 2-deoxyriobse 5-phosphate produced was directly transformed to 2'-deoxyribonucleoside by phosphopentomutase- and nucleoside phosphorylase-catalyzing reactions.