Substrate Enrichment Research Articles

Encapsulating soluble active species into hollow crystalline porous capsules beyond integration of homogeneous and heterogeneous catalysis.

Homogeneous molecular catalysts and heterogeneous catalysts possess complementary strengths, and are of great importance in laboratory/commercial procedures. While various porous hosts, such as polymers, carbons, silica, metal oxides and zeolites, have been used in an attempt to heterogenize homogeneous catalysts, realizing the integration of both functions at the expense of discounting their respective advantages, it remains a significant challenge to truly combine their intrinsic strengths in a single catalyst without compromise. Here, we describe a general template-assisted approach to incorporating soluble molecular catalysts into the hollow porous capsule, which prevents their leaching due to the absence of large intergranular space. In the resultant yolk (soluble)–shell (crystalline) capsules, the soluble yolks can perform their intrinsic activity in a mimetic homogeneous environment, and the crystalline porous shells endow the former with selective permeability, substrate enrichment, size-selective and heterogeneous cascade catalysis, beyond the integration of the respective advantages of homogeneous and heterogeneous catalysts.

Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and insitu muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified >22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

The Protein Phosphorylation Landscape of Mouse Spermatids during Spermiogenesis.

The characteristic tadpole shape of sperm is formed from round spermatids via spermiogenesis, a process which results in dramatic morphological changes in the final stage of spermatogenesis in the testis. Protein phosphorylation, as one of the most important post-translational modifications, can regulate spermiogenesis; however, the phosphorylation events taking place during this process have not been systematically analyzed. In order to better understand the role of phosphorylation in spermiogenesis, large-scale phosphoproteome profiling is performed using IMAC and TiO2 enrichment. In total, 13835 phosphorylation sites, in 4196 phosphoproteins, are identified in purified mouse spermatids undergoing spermiogenesis in two biological replicates. Overall, 735 testis-specific proteins are identified to be phosphorylated, and are expressed at high levels during spermiogenesis. Gene ontology analysis shows enrichment of the identified phosphoproteins in terms of histone modification, cilium organization, centrosome and the adherens junction. Further characterization of the kinase-substrate phosphorylation network demonstrates enrichment of phosphorylation substrates related to the regulation of spermiogenesis. This global protein phosphorylation landscape of spermiogenesis shows wide phosphoregulation across a diverse range of processes during spermiogenesis and can help to further characterize the process of sperm generation. All MS data are available via ProteomeXchange with the identifier PXD011890.

A Chemical Strategy for Protease Substrate Profiling

The dipeptidyl peptidases (DPPs) regulate hormones, cytokines, and neuropeptides by cleaving dipeptides after proline from their amino termini. Due to technical challenges, many DPP substrates remain unknown. Here, we introduce a simple method, termed CHOPS (chemical enrichment of protease substrates), for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position. CHOPS can, in theory, discover substrates for any protease, but is particularly well suited to discover canonical DPP substrates, as cleaved but not intact DPP substrates can be identified by gel electrophoresis or mass spectrometry. Using CHOPS, we show that DPP8 and DPP9, enzymes that control the Nlrp1 inflammasome through an unknown mechanism, do not directly cleave Nlrp1. We further show that DPP9 robustly cleaves short peptides but not full-length proteins. More generally, this work delineates a practical technology for identifying protease substrates, which we anticipate will complement available "N-terminomic" approaches.

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

Glucose-dependent phosphorylation signaling pathways and crosstalk to mitochondrial respiration in insulin secreting cells

BackgroundGlucose is the main secretagogue of pancreatic beta-cells. Uptake and metabolism of the nutrient stimulates the beta-cell to release the blood glucose lowering hormone insulin. This metabolic activation is associated with a pronounced increase in mitochondrial respiration. Glucose stimulation also initiates a number of signal transduction pathways for the coordinated regulation of multiple biological processes required for insulin secretion.MethodsShotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways. Kinase substrate enrichment analysis (KSEA) was applied to identify key regulated kinases and phosphatases. Glucose-induced oxygen consumption was measured using a XF96 Seahorse instrument to reveal cross talk between glucose-regulated kinases and mitochondrial activation.ResultsOur kinetic analysis of substrate phosphorylation reveal the molecular mechanism leading to rapid activation of insulin biogenesis, vesicle trafficking, insulin granule exocytosis and cytoskeleton remodeling. Kinase-substrate enrichment identified upstream kinases and phosphatases and time-dependent activity changes during glucose stimulation. Activity trajectories of well-known glucose-regulated kinases and phosphatases are described. In addition, we predict activity changes in a number of kinases including NUAK1, not or only poorly studied in the context of the pancreatic beta-cell. Furthermore, we pharmacologically tested whether signaling pathways predicted by kinase-substrate enrichment analysis affected glucose-dependent acceleration of mitochondrial respiration. We find that phosphoinositide 3-kinase, Ca2+/calmodulin dependent protein kinase and protein kinase C contribute to short-term regulation of energy metabolism.ConclusionsOur results provide a global view into the regulation of kinases and phosphatases in insulin secreting cells and suggest cross talk between glucose-induced signal transduction and mitochondrial activation.

Agro-industrial byproducts for production of the immunosuppressant mycophenolic acid by Penicillium roqueforti under solid-state fermentation: Enhanced production by ultraviolet and gamma irradiation

Abstract Different agro-industrial materials were screened for their performance on production of mycophenolic acid (MPA) by two strains of Penicillium roqueforti (AG101 and LG109) under solid-state fermentation (SSF). Sugarcane bagasse followed by corn stalks and rice bran were the most favorable substrates supporting the MPA production. The maximum MPA concentration was achieved at initial moisture content 70% for the first substrate and 80% for the other substrates. Enrichment of the solid substrate with a modified Czapek's broth promoted a greater MPA concentration than that enriched with mineral salt solution. The AG101 and LG109 strains were improved to produce higher significant MPA titers on solid substrate by subjecting the spores to irradiation by UV light (254 nm) for 120 and 90 min, respectively; and gamma rays at 0.75 KGy. The highest MPA-producing ability of both irradiated strains was observed on sugarcane bagasse and it was increased by 1.57–1.72 fold when irradiated with UV rays and by 1.74–1.92 fold when irradiated with gamma rays, as compared with the non-irradiated cultures. These findings indicate the future possibility to reduce the cost of producing fermentation-based drugs.

Analysis of the Influence of Substrate Formulations on the Bioactive Chemical Profile of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes) by Conventional and Chemometrics Methods.

Ganoderma lucidum, a mushroom with medicinal properties, can grow on diverse lignocellulosic substrates. Substrate enrichment with additives has been used as a strategy to increase mushroom productivity. In this study, we evaluated the impact of substrate formulation on the bioactive chemical profile of the basidiome. The bioactive chemical profile of basidiomes cultivated on rice agro-residues (RA) or sunflower seed hulls (SSH) enriched with olive oil and/or copper was evaluated using conventional colorimetric methods and FT-MIR spectrometry coupled with chemometrics. The contents of total triterpenoids, ganoderic acids, high-molecular-weight carbohydrates, and phenolic compounds were sensitive to substrate formulation and harvest time. Moreover, cluster analysis and principal component analysis of the mid-IR spectra were able to discriminate between basidiomes cultivated on either RA or SSH substrates, and for SSH substrates between enriched and nonenriched formulas. These results indicate that the bioactive composition of G. lucidum can be influenced by the formulation of the cultivation substrate.

Genome sequencing and assessment of plant growth-promoting properties of a Serratia marcescens strain isolated from vermicompost

BackgroundPlant-bacteria associations have been extensively studied for their potential in increasing crop productivity in a sustainable manner. Serratia marcescens is a species of Enterobacteriaceae found in a wide range of environments, including soil.ResultsHere we describe the genome sequencing and assessment of plant growth-promoting abilities of S. marcescens UENF-22GI, a strain isolated from mature cattle manure vermicompost. In vitro, S. marcescens UENF-22GI is able to solubilize P and Zn, to produce indole compounds (likely IAA), to colonize hyphae and counter the growth of two phytopathogenic fungi. Inoculation of maize with this strain remarkably increased seedling growth and biomass under greenhouse conditions. The S. marcescens UENF-22GI genome has 5 Mb, assembled in 17 scaffolds comprising 4662 genes (4528 are protein-coding). No plasmids were identified. S. marcescens UENF-22GI is phylogenetically placed within a clade comprised almost exclusively of non-clinical strains. We identified genes and operons that are likely responsible for the interesting plant-growth promoting features that were experimentally described. The S. marcescens UENF-22GI genome harbors a horizontally-transferred genomic island involved in antibiotic production, antibiotic resistance, and anti-phage defense via a novel ADP-ribosyltransferase-like protein and possible modification of DNA by a deazapurine base, which likely contributes to its competitiveness against other bacteria.ConclusionsCollectively, our results suggest that S. marcescens UENF-22GI is a strong candidate to be used in the enrichment of substrates for plant growth promotion or as part of bioinoculants for agriculture.

Sulfurimonas subgroup GD17 cells accumulate polyphosphate under fluctuating redox conditions in the Baltic Sea: possible implications for their ecology.

The central Baltic Sea is characterized by a pelagic redox zone exhibiting high dark CO2 fixation rates below the chemocline. These rates are mainly driven by chemolithoautotrophic and denitrifying Sulfurimonas GD17 subgroup cells which are motile and fast-reacting r-strategists. Baltic Sea redox zones are unstable and a measurable overlap of nitrate and reduced sulfur, essential for chemosynthesis, is often only available on small scales and short times due to local mixing events. This raises the question of how GD17 cells gain access to electron donors or acceptors over longer term periods and under substrate deficiency. One possible answer is that GD17 cells store high-energy-containing polyphosphate during favorable nutrient conditions to survive periods of nutrient starvation. We used scanning electron microscopy with energy-dispersive X-ray spectroscopy to investigate potential substrate enrichments in single GD17 cells collected from Baltic Sea redox zones. More specific substrate enrichment features were identified in experiments using Sulfurimonas gotlandica GD1T, a GD17 representative. Sulfurimonas cells accumulated polyphosphate both in situ and in vitro. Combined genome and culture-dependent analyses suggest that polyphosphate serves as an energy reservoir to maintain cellular integrity at unfavorable substrate conditions. This redox-independent energy supply would be a precondition for sustaining the r-strategy lifestyle of GD17 and may represent a newly identified survival strategy for chemolithoautotrophic prokaryotes occupying eutrophic redox zones.

PO-019 Phosphoproteomics to characterise DNA damage response in mouse mammary tumours of different PARP inhibitor susceptibility

IntroductionTriple negative breast cancers show very poor prognosis, with only limited treatment options. A subgroup of these tumours is deficient in homologous recombination (HR) and therefore sensitive to DNA damaging drugs or PARP inhibitors (PARPi). Unfortunately, resistance to PARPi is often observed. Therefore, the identification of biomarkers for PARPi response or status of HR are of importance for personalised therapy. The DNA damage response is regulated via post-translational modifications such as phosphorylation. We thus study differential phosphorylation events in the context of different PARPi susceptibility.Material and methodsWe used a collection of biopsies from either untreated or irradiated (15 Gy, 2 hour) HR-deficient mouse mammary tumours and matched PARPi resistant tumours which restored HR. Changes in phosphorylation were examined via titanium dioxide based phosphopeptide enrichment or protein expression profiling using single-shot LC-MS/MS (label-free).Results and discussionsIn total, we identified 14 695 phosphopeptides and 11 138 high-confidence phosphosites (PS) from 48 individual tumours samples that were measured in duplicates. As proof of concept, we analysed changes in PS in PARPi naive tumours upon induction of the DNA damage response via ionising irradiation. We detect known events of the DNA damage response such as increased phosphorylation of proteins involved in DNA damage checkpoint or double strand break repair. Further evaluation of these PS, like sequence motif analysis, prediction of upstream kinases and kinase substrate enrichment analysis revealed activation of ATM/ATR in irradiated tumours as expected. To further proof the validity of our approach, we analysed samples with known PARPi resistance mechanism – loss of 53 BP1 expression – and observed downregulation of PS specific to 53 BP1. We are now extending our analysis to additional samples sets with known and unknown PARPi resistance mechanisms.ConclusionOur data point to the feasibility of using phosphoproteomics as a tool to study DNA damage response in BRCA1 deficient tumours with different status of HR and thus contrasting susceptibility to PARPi.

Phosphorus and Zinc Extractable and Total in Substrate Enriched Coconut Powder and Tomato Cultivation

Phosphorus (P) and Zinc (Zn) stand out for their interactions, however, little is known about the interaction of these elements in organic substrates used in the development of plants. The objective of this work was to evaluate the extractable and total P and Zn of the enriched coconut powder substrate and the development of tomato seedlings grown on the same substrate. The work consisted of 10 treatments and 4 replicates with different doses of P and Zn. The substrate used was coconut powder enriched with nutrient solutions in a ratio of 10:1 (v/m). Then, the experiment was conducted using the enriched substrate to observe the effect of P and Zn doses on the development of tomato seedlings in trays. The height, number of leaves and the dry matter of the plants were evaluated. For the water and Mehlich-1 extractors the highest values of P and Zn were for treatments with the highest doses and only the quadratic positive effect of P did not differ statistically in the enriched coconut powder substrate. The results showed that the coconut powder used as substrate is deficient in P and Zn. The highest development of the seedlings was obtained at the doses of 74 mg L-1 P and 3.25 and 4.75 mg L-1 Zn of the substrate enrichment solution.

PO-022 FISH probes for the detection of high-risk HPV nucleic acid in infected host cells

IntroductionTriple negative breast cancers show very poor prognosis, with only limited treatment options. A subgroup of these tumours is deficient in homologous recombination (HR) and therefore sensitive to DNA damaging drugs or PARP inhibitors (PARPi). Unfortunately, resistance to PARPi is often observed. Therefore, the identification of biomarkers for PARPi response or status of HR are of importance for personalised therapy. The DNA damage response is regulated via post-translational modifications such as phosphorylation. We thus study differential phosphorylation events in the context of different PARPi susceptibility.Material and methodsWe used a collection of biopsies from either untreated or irradiated (15 Gy, 2 hour) HR-deficient mouse mammary tumours and matched PARPi resistant tumours which restored HR. Changes in phosphorylation were examined via titanium dioxide based phosphopeptide enrichment or protein expression profiling using single-shot LC-MS/MS (label-free).Results and discussionsIn total, we identified 14 695 phosphopeptides and 11 138 high-confidence phosphosites (PS) from 48 individual tumours samples that were measured in duplicates. As proof of concept, we analysed changes in PS in PARPi naive tumours upon induction of the DNA damage response via ionising irradiation. We detect known events of the DNA damage response such as increased phosphorylation of proteins involved in DNA damage checkpoint or double strand break repair. Further evaluation of these PS, like sequence motif analysis, prediction of upstream kinases and kinase substrate enrichment analysis revealed activation of ATM/ATR in irradiated tumours as expected. To further proof the validity of our approach, we analysed samples with known PARPi resistance mechanism – loss of 53 BP1 expression – and observed downregulation of PS specific to 53 BP1. We are now extending our analysis to additional samples sets with known and unknown PARPi resistance mechanisms.ConclusionOur data point to the feasibility of using phosphoproteomics as a tool to study DNA damage response in BRCA1 deficient tumours with different status of HR and thus contrasting susceptibility to PARPi.

Phosphoproteomics-Based Profiling of Kinase Activities in Cancer Cells.

Cellular signaling, predominantly mediated by phosphorylation through protein kinases, is found to be deregulated in most cancers. Accordingly, protein kinases have been subject to intense investigations in cancer research, to understand their role in oncogenesis and to discover new therapeutic targets. Despite great advances, an understanding of kinase dysfunction in cancer is far from complete.A powerful tool to investigate phosphorylation is mass-spectrometry (MS)-based phosphoproteomics, which enables the identification of thousands of phosphorylated peptides in a single experiment. Since every phosphorylation event results from the activity of a protein kinase, high-coverage phosphoproteomics data should indirectly contain comprehensive information about the activity of protein kinases.In this chapter, we discuss the use of computational methods to predict kinase activity scores from MS-based phosphoproteomics data. We start with a short explanation of the fundamental features of the phosphoproteomics data acquisition process from the perspective of the computational analysis. Next, we briefly review the existing databases with experimentally verified kinase-substrate relationships and present a set of bioinformatic tools to discover novel kinase targets. We then introduce different methods to infer kinase activities from phosphoproteomics data and these kinase-substrate relationships. We illustrate their application with a detailed protocol of one of the methods, KSEA (Kinase Substrate Enrichment Analysis). This method is implemented in Python within the framework of the open-source Kinase Activity Toolbox (kinact), which is freely available at http://github.com/saezlab/kinact/.

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis.

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

ОЦЕНКА ЭФФЕКТИВНОСТИ ФЕРМЕНТАТИВНОГО ГИДРОЛИЗА ПЛОДОВЫХ ОБОЛОЧЕК ОВСА С ПОДПИТКОЙ ПРИ ВЫСОКИХ НАЧАЛЬНЫХ КОНЦЕНТРАЦИЯХ СУБСТРАТА

The aim of the work was to investigate the enzymatic hydrolysis of a chemical treatment product of oat bran at different substrate concentrations, as well as to evaluate the possibility of increasing the efficiency of hydrolysis at high initial substrate concentrations across different feeding variants. The process of enzymatic hydrolysis of a chemically treatment for oat bran products was carried out at various initial substrate concentrations, as well as with various ways of feeding: first - enzymes, second - substrate. The dependences of the current concentration and the yield of reducing substances (RS) from the initial substrate concentration in the range of 45-200 g / l are determined. The possibility of increasing the yield of reducing substances in both cases of feeding is indicated. It was found that an increase in the initial substrate concentration from 45 g / l to 200 g / l leads to an increase in the final concentration of RS by 81 g / l, while the RS yield is reduced by 33%; the enrichment of enzyme preparations or substrates in enzymatic hydrolysis with a maximum substrate concentration of 200 g / l makes it possible to increase the substrate conversion rate by 10-12%.

Depth trends of soil organic matter C:N and 15N natural abundance controlled by association with minerals

Plant residues show carbon:nitrogen (C:N) decreases, 15N isotopic enrichment and preferential loss of labile substrates during microbial decay. In soil profiles, strikingly similar patterns of decreasing C:N and 15N isotopic enrichment with increasing depth are well documented. The parallel trend in organic matter composition with soil depth and during plant residue decay has been used as evidence to suggest that organic products accumulate or develop in the subsoil due to increasing intensity of microbially-driven processing, although no studies to date have verified this. Here, by applying sequential density fractionation, specific surface area, oxalate extractable Fe and Al, C:N and δ15N measures with depth to soils with relatively uniform soil mineralogy (Oxisols), climates and vegetation we show that changes in organo-mineral associations drive subsoil C:N and δ15N and C:N depth patterns more than in situ organic matter decay. Our results provide the first direct evidence that soil depth trends could be driven by mineral association instead of in situ processing.

The Impact of Dummy Gate Processing on Si-Cap-Free SiGe Passivation: A Physical Characterization Study on Strained SiGe 25% and 45%

High mobility channel materials like SiGe, Ge and IIIV receive lots of interest in order to enable the continuation of Moore’s path during the upcoming technology nodes. Recently Si-cap-free SiGe passivation with the number of interface states (NIT) down to 2 1011 cm-2 have been demonstrated. [1-3] A clear correlation was established between Ge-content in the SiGe-oxide and NIT. Unfortunately no process information has been provided on how the Ge-oxide-free interlayers were obtained. In a separate study the impact of HF, HCl and SC1 cleaning solution on the SiGe surface and oxide composition was reported. [4] A comparison of SiGe Gate-All-Around (GAA) and FinFET devices using a Si-cap-free SiGe 25% passivation scheme in a replacement metal gate (RMG) integration indicate the device performance is improved by a TMAH (aq) treatment prior to SiGe interlayer formation and high-k deposition. [5] Here the formation of a SiGe wet chemical oxide will be presented. The impact of dummy gate processing on the interlayer composition is reported. Also the impact of a TMAH (aq) treatment prior to chemical oxide formation has been analyzed and will be reported. Blanket strained SiGe 25% and 45% were grown by epitaxy. The SiGe-substrate and -oxide composition were measured by angle-resolved XPS using the Si2p and Ge3d peaks. The Ge3d peak is fitted with three different compounds: elemental Ge, GeO2 and Ge-suboxide. The angle dependence lets us estimate the distribution of the different components with respect to the sample top surface. An concentration increase with increasing angle (relative to the surface normal) indicates the compound is located closer to the sample surface. And reversely a concentration increase with increasing angle indicates the compound is located deeper into the sample. A native oxide was grown on SiGe 25 and 45% by exposing the wafers to the cleanroom air for approx. 1 week. The native oxide was found to be stoichiometric or nearly stoichiometric with a uniform Ge content throughout the oxide for SiGe 25 and 45% respectively. A wet chemical oxide was grown using an ‘imec clean’ [6] having a final O3/HCl (aq) rinse. The wet chemical oxide is Ge-poor compared to the SiGe substrate. No or only a small variation in Ge content of the SiGe-oxide is seen with ARXPS. However the fraction Ge-suboxide decreases with increasing angle, indicating the Ge-suboxide are located deeper within the sample, so closer to the SiGe-oxide/SiGe-substrate interface. The Ge-content of the SiGe-substrate after wet chemical oxidation shows a weak incident-angle-dependence, indicative a limited Ge enrichment of the SiGe substrate towards the SiGe-oxide/-substrate interface. The low Ge content of the SiGe-oxide is attributed to the water solubility of GeO2. The increase in Ge-content towards the SiGe-oxide/-substrate can be (at least partially) attributed to the poor water solubility of Ge-suboxide. The small increase in Ge content of the SiGe substrate can be attributed to the thermodynamically preferred oxidation of Si over Ge. The combined impact of dummy gate deposition, spike anneal and dummy gate removal prior to SiGe interlayer formation was investigated by AR-XPS and SIMS. The temperature used during spike anneal was found to have a significant impact on the Ge-content of the SiGe-oxide formed during an O3-last imec clean. The results will be reported in detail at the conference. The impact of TMAH (aq) on the chemical oxide composition and surface roughness will also be reported. TMAH (aq) was found to improve SiGe device performance. [5] Also the interaction with the subsequent HfO2 deposition will be reported. In summary SiGe is found to behave as a non-linear combination of Si and Ge. In HF-free aqueous solutions, the SiGe oxide composition is a combination of (1) thermodynamically preferred oxidation of Si versus Ge; (2) poor water-solubility of SiO2 and/or its low solubilization rate; and (3) fast dissolution of GeO2 but slow dissolution of Ge-suboxides. [1] CH Lee et al. VLSI 2016. [2] S. Siddiqui et al. presented MRS Spring Meeting 2016. [3] CH Lee et al. IEDM 2016. [4] S.L. Heslop et al. ECS Trans 69 (2015) 287. [5] H. Mertens et al VLSI 2015. [6] M. Meuris et al. Solid State Phenom, July 1995, p. 109. Figure 1

Novel biocatalyst for productions of S-(-)-2-[6-benzyloxy -2,5,7,8-tetramethylchroman -2-yl] ethanol—precursor of natural α-tocols

A novel promising strain of actinobacteria Rhodococcus sp. 77-32 was identified. Its acetonetreated biomass the could be used as a biocatalyst for production of S-(-)-2-[6-benzyloxy-2,5,7,8-tetramethylchroman-2-yl] ethanol (S-BCE), a precursor of natural α-tocols. It was established that a reaction of enantioselective hydrolysis of racemic (±)-2-(2-acetoxyethyl)-6-benzyloxy-2,5,7,8-tetramethylchroman (BCEA) occurred in the phosphate buffer–acetone system, resulting in enrichment of the residual substrate by S-enantiomer (S-(+)-2-(2-acetoxyethyl)-6-benzyloxy-2,5,7,8-tetramethylchroman, S-BCEA). It was shown that the hydrolysis was accompanied by stereoinversion of the formed product, R-(+)-2-[6-benzyloxy-2,5,7,8-tetramethylchroman-2-yl] ethanol (R-BCE), into the S-BCE. The transformation conditions (acetone content, acidity, temperature, reaction duration) were optimized, providing simultaneous production of optically pure S-BCE and S-BCEA with an almost quantitative yield.

Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase.

Ferulic acid is a natural antioxidant found in various plants and serves as a precursor for various fine chemicals, including the flavouring agent vanillin. However, expensive extraction methods have limited the commercial application of ferulic acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487U/mg, Km =0.43mM, Kcat=0.48/min on methyl ferulate). The 36-kDa AtFAEA protein showed maximum ferulic acid esterase activity at 50°C and pH 6, suggesting potential application in industrial processes. A crude AtFAEA preparation extracted 26.56 and 8.86mg/g ferulic acid from maize bran and triticale bran, respectively, and also significantly increased the levels of p-coumaric and caffeic acid from triticale bran. The cost-effective production of AtFAEA could therefore allow for the enrichment of brans generally used as food and fodder, or for the production of fine chemicals (such as ferulic and p-coumaric acid) from plant substrates. The potential for larger-scale production of AtFAEA was demonstrated with the A. niger D15[AtfaeA] strain yielding a higher enzyme activity (185.14 vs. 83.48U/ml) and volumetric productivity (3.86 vs. 1.74U/ml/h) in fed-batch than batch fermentation.