High Substrate Conversion Research Articles

A novel biotransformation process of 4′-demethylepipodophyllotoxin to 4′-demethylepipodophyllic acid by Bacillus fusiformis CICC 20463

The novel biotransformation process of 4′-demethylepipodophyllotoxin to produce 4′-demethylepipodophyllic acid was developed for the first time. Based on the capability of modifying 4′-demethylepipodophyllotoxin structure, Bacillus fusiformis CICC 20463, Bacillus subtilis CCTCC AB93174, Pseudomonas aeruginosa CCTCC AB93066 and Pseudomonas oleovorans CGMCC 1.1641 were screened out from the eight tested strains. Among them, B. fusiformis was selected for the following study because of its high substrate conversion. The biotransformation product was separated from the biotransformation broth by macroporous resin D312, and identified as 4′-demethylepipodophyllic acid by the comparison analysis of 1H NMR, 13C NMR, and ESI-MS spectrums with those of 4′-demethylepipodophyllotoxin. This suggests that B. fusiformis, P. aeruginosa, B. subtilis and P. oleovorans had the ability to transform 4′-demethylepipodophyllotoxin into 4′-demethylepipodophyllic acid. The established biotransformation process will give reference to the biotransformation study of other bioactive natural leading compounds.

Grafting of vanadyl acetylacetonate onto organo-hexagonal mesoporous silica and catalytic activity in the allylic epoxidation of geraniol

Vanadyl(IV) acetylacetonate ([VO(acac) 2]) was grafted onto a hexagonal mesoporous silica (HMS) using three different methodologies: method A – direct complex immobilisation; method B – functionalisation of the HMS with 3-aminopropyltriethoxysilane (APTES) followed by the complex immobilisation; and method C – treatment of the APTES functionalised support prepared by method B with trimethylethoxysilane (TMS) to deactivate eventually unreacted surface silanol groups, followed by complex grafting. All the materials were characterised by nitrogen elemental analysis, XPS, FTIR, N 2 adsorption isotherms at −196 °C and the [VO(acac) 2] based materials were also characterised by vanadium ICP-AES analysis. The results indicated that, in method B, APTES was successfully grafted onto the HMS with 90% of efficiency and allowed the covalent attachment of [VO(acac) 2] complex mainly in the inner pores with an efficiency of 65%. In method C, a lower complex immobilisation efficiency was obtained, c.a. 25%, but the complex was covalently bonded throughout the functionalised material. In the case of method A, the parent HMS material immobilised a very low quantity of vanadium complex (2% of efficiency), mainly in the external surface through non-covalent interactions. The catalytic activity of [VO(acac) 2] based materials in the epoxidation of geraniol using tert-butyl hydroperoxide ( t-BuOOH) as oxygen source was assessed. The selectivities of the two epoxides formed in the heterogeneous phase reactions were similar to those observed in the homogeneous phase and the major reaction product was always 2,3-epoxygeraniol. The catalyst which allowed higher substrate conversion was obtained by method B but, when considering the leaching of the active phase, method C produced the most efficient catalyst.

Liquid Phase Oxidation of Cyclohexene Over Selenite Doped MCM-41

Liquid phase oxidation of cyclohexene was carried out under mild reaction conditions over selenite doped MCM-41 using aqueous hydrogen peroxide as oxidant and acetic acid as solvent. The major products of the reaction were 2-acetoxycyclohexanol and a small amount of 1,2-cyclohexanediol. The catalyst exhibited very high substrate conversion (~100%) and good product (2-acetoxycyclohexanol) selectivity (81%) and it can be reused with almost the same activity. The catalyst was characterized by a combination of various physicochemical techniques, such as N2 physisorption, TEM, X-ray diffraction and FT-IR.

Extrudate versus Powder Silica Alumina as Support for Re2O7 Catalyst in the Metathesis of Seed Oil-Derivatives – A Comparison

Self- and cross-metathesis of fatty acid methyl esters (FAMEs) was investigated using a silica alumina supported Re(2)O(7) catalyst. Although a 3 wt% Re(2)O(7)/SiO(2)-Al(2)O(3)/SnBu(4) is already active for the metathesis of unsaturated FAMEs, the results have shown that particle size of silica alumina support has a profound influence on its activity and selectivity. Consequently, high substrate conversions coupled with improved product yields (for mono- and diesters) and reaction rates were obtained upon using powder, as opposed to extrudate silica alumina as the support material. Diesters are platform compounds for the synthesis of polymers and fragrances. In this paper a comparative outline of the influence of particle size of silica alumina (extrudate versus powder) on catalytic performance of a 3 wt% Re(2)O(7)/SiO(2)-Al(2)O(3)/SnBu(4) for self- and cross-metathesis of FAMEs is made. Low surface area and diffusion constraints associated with extrudates were identified as some of the factors leading to low catalytic activity and selectivity.

Anchoring RhCl(CO)(PPh3)2 to –PrPPh2 Modified MCM-41 as Effective Catalyst for 1-Octene Hydroformylation

RhCl(CO)(PPh3)2 was anchored to diphenylphosphinopropyl-modified (–PrPPh2) mesoporous silicate MCM-41. Infrared spectroscopy was applied to monitor the multi-step assemble of the complex by assigning critical absorptions. The prepared catalyst was employed in 1-octene hydroformylation and showed high conversion of substrate as well as high selectivity of nonyl aldeydes (>97%) along with several stable catalyst cycles. Rhodium leaching in the reaction liquids was determined by mass spectroscopy, which was found extremely low. The catalyst showed higher activity and lower metal loss than the analogue by nano SiO2 as support, probably as a result of the confining of molecular sieves with ordered pores.

Transition metal doped mesoporous titania with a crystalline framework as catalysts for oxidation of p-bromotoluene to p-bromobenzaldehyde

Transition metal M (M = Mn, Fe, Co, Ni, Cu, and Zn) doped mesoporous titania with a crystalline framework was employed as catalysts for the oxidation of p-bromotoluene (PBT) to p-bromobenzaldehyde (BBD) in acetic acid using aqueous hydrogen peroxide as oxidant. It was found that mesoporous TiO2 doped with those metals (Fe, Co and Ni) whose atomic radii are relatively smaller exhibited higher conversion rate of PBT. Among these catalysts, the Co/meso-TiO2 exhibited high substrate conversion and good product (p-bromobenzaldehyde) selectivity plus it can be reused once with almost the same activity. The effect of different Ti/Co (molar) ratio on the activities of Co/meso-TiO2 was also investigated.

Liquid phase oxidation of 2-methyl pyridine to 2-pyridinecarboxylic acid over cobalt-doped SBA-3

Cobalt-doped SBA-3 was synthesized by cogellation method and was used as a catalyst for the oxidation of 2-methyl pyridine in acetic acid using aqueous hydrogen peroxide as oxidant. The catalyst exhibited very high substrate conversion (98%) and good product (2-pyridinecarboxylic acid) selectivity (93%). Fast hot catalyst filtration experiment proved that the catalyst acted as a heterogeneous one and it can be reused two times without losing its activity to a greater extent. The catalyst was characterized by X-ray diffraction, N 2 physisorption, diffuse reflectance UV–vis, and FT-IR.

Switching the mode of sucrose utilization by Saccharomyces cerevisiae

BackgroundOverflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.ResultsWe have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source.ConclusionHigher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations: An alternative to fed‐batch cultures

Overflow metabolism is an undesirable characteristic of aerobic cultures of Escherichia coli. It results from elevated glucose consumption rates that cause a high substrate conversion to acetate, severely affecting cell physiology and bioprocess performance. Such phenomenon typically occurs in batch cultures under high glucose concentration. Fed-batch culture, where glucose uptake rate is controlled by external addition of glucose, is the classical bioprocessing alternative to prevent overflow metabolism. Despite its wide-spread use, fed-batch mode presents drawbacks that could be overcome by simpler batch cultures at high initial glucose concentration, only if overflow metabolism is effectively prevented. In this study, an E. coli strain (VH32) lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) with a modified glucose transport system was cultured at glucose concentrations of up to 100 g/L in batch mode, while expressing the recombinant green fluorescence protein (GFP). At the highest glucose concentration tested, acetate accumulated to a maximum of 13.6 g/L for the parental strain (W3110), whereas a maximum concentration of only 2 g/L was observed for VH32. Consequently, high cell and GFP concentrations of 52 and 8.2 g/L, respectively, were achieved in VH32 cultures at 100 g/L of glucose. In contrast, maximum biomass and GFP in W3110 cultures only reached 65 and 48%, respectively, of the values attained by the engineered strain. A comparison of this culture strategy against traditional fed-batch culture of W3110 is presented. This study shows that high cell and recombinant protein concentrations are attainable in simple batch cultures by circumventing overflow metabolism through metabolic engineering. This represents a novel and valuable alternative to classical bioprocessing approaches.

Hydrogen Production from diluted molasses by anaerobic hydrogen producing bacteria in an anaerobic baffled reactor (ABR)

Abstract Hydrogen production from diluted molasses by anaerobic fermentation bacteria was investigated in a three-compartment anaerobic baffled reactor (ABR) with an effective volume of 27.48 L. After being inoculated with aerobic activated sludge and operated at chemical oxygen demand (COD) of 5000 mg/L and temperature of 35 ∘ C for 26 days, the ABR achieved stable ethanol-type fermentation. The liquid fermentation products, including volatile fatty acids (VFAs) and ethanol, stabilized at 1254, 2053, and 2761 mg/L in the three compartments, respectively. Effluent pH, oxidation–reduction potential (ORP), and alkalinity ranged at 4.3–4.4, - 241 to - 249 mV , and 306 – 334 mg CaCO 3 / L , respectively. The hydrogen yield of the ABR was 32.51 L/d at the stable operation status, specific hydrogen production rate of anaerobic activated sludge was 0.13 L/g MLVSS d, and the substrate conversion rate was 0.13 L/g COD. Hydrogen yields, fermentation types, and acclimatization durations varied in each compartment, with the 1st compartment having lowest hydrogen yield but longest acclimatization duration and the 2nd and 3rd compartments having higher hydrogen yields but shorter acclimatization durations. The study found that the individual compartment configuration in the ABR system provided a favorable environment for different types of anaerobic bacteria. Compared with complete stirring tank reactor (CSTR), the ABR system had a better operation stability and microbial activity, which led to higher substrate conversion rate and hydrogen production ability.

Optimal feed temperature for an immobilized enzyme packed-bed reactor.

Optimal feed temperature was determined for a nonisothermal immobilized enzymatic reaction with enzyme deactivation in a packed-bed reactor. The optimal feed temperature was obtained by maximizing the average substrate conversion over a given reaction period. Simulation showed the optimal feed temperature to be strongly dependent on the flow dispersion, the reaction activation energy, the corresponding enzyme inactivation energy and the heat of reaction. It was also observed that in a plug flow reactor the enzyme reaction generally exhibited a lower optimal feed temperature and higher substrate conversion than in a continuously stirred tank reactor.

Silicotungstic acid/zirconia immobilized on SBA-15 for esterifications

A simple wet-impregnation method and controlled calcination process was used for preparing nano-sized silicotungstic acid (STA) supported on zirconia embedded inside the various mesoporous silicas such as SBA-15, MCM-41 and MCM-48. Catalysts with different (%) STA loadings were also synthesized and characterized by XRD, N 2 adsorption–desorption, TEM, 29Si-MAS NMR, FT-Raman spectroscopic techniques. The acidic behavior of the catalysts was studied by TPD of ammonia and FT-IR pyridine adsorption technique. The effect of calcination temperature on the textural parameters of the catalysts and the integrity of STA on the surface of the catalyst were also examined. Moreover, the performance of these catalysts was investigated in the esterification of isoamyl alcohol (IAA) with acetic acid (AA). Catalytic activities were correlated with the acidity of the catalysts. Effects of reaction parameters such as mole ratio of reactants, reaction temperature, catalyst concentration and reusability of the catalyst were studied to get higher substrate conversions and product selectivities. Among the catalysts, 15 wt.% STA/22.4 wt.% ZrO 2/SBA-15 calcined at 1123 K was found to be the most active and acidic catalyst, showing higher conversion and selectivity than neat 15 wt.% STA/ZrO 2 in esterification of IAA by AA. The reaction was found to be heterogeneously catalyzed and no contribution from homogeneous (leached) STA into the medium under the reaction conditions was observed.

Measurements of Kinetic Parameters in a Microfluidic Reactor

Continuous flow microfluidic reactors that use immobilized components of enzymatic reactions present special challenges in interpretation of kinetic data. This study evaluates the difference between mass-transfer effects and reduced efficiencies of an enzyme reaction. The kinetic properties of immobilized alkaline phosphatase (AP) were measured by the dephosphorylation of 6,8-difluoro-4-methylumbelliferyl/phosphate to a fluorescent 6,8-difluoro-4-methylumbelliferone. A glass microfluidic chip with an in-channel weir was created for the capture of solid silica microbeads functionalized with enzyme. The input substrate concentrations and flow rates across the bed were varied to probe the flow-dependent transport and kinetic properties of the reaction in the microreactor bed. Unlike previous reactors, substrate was titrated directly over the fixed enzyme bed by controlling the air pressure over the chip reservoirs. The reactor explored substrate conversions from near zero to 100%. The average bed porosity, residence time, and bed resistance were measured with dye pulses. A simple criterion was derived to evaluate the importance of flow-dependent mass-transfer resistances when using microreactors for calculating kinetic rate constants. In the absence of mass-transfer resistances, the Michaelis-Menten kinetic parameters are shown to be flow independent and are appropriately predicted using low substrate conversion data. A comparison of the kinetic parameters with those obtained using solution-phase enzymatic reactions shows a significant decrease in enzyme activity in the immobilized conformation. The immobilized Km of AP is approximately 6 times greater while the kcat is reduced by approximately 28 times. Contradictions found in literature on the evaluation of Michaelis-Menten kinetic parameters for immobilized enzymes in microfluidic reactors are addressed. When product molecules occupy a significant number of enzymatic sites or modify the enzyme activity, the assumed Michaelis-Menten mechanism can no longer be valid. Under these conditions, the calculations of "apparent" kinetic rate constants, based on Michaelis-Menten kinetics, can superficially show a dependence on flow rate conditions even in the absence of mass-transfer resistances. High substrate conversions are shown to depend on flow rate. A kinetic model based on known mechanisms of the alkaline phosphatase enzyme reaction is tested to predict the measurements for high substrate conversion. The study provides a basis for appropriate use of mass-transfer and reaction arguments in successful application of enzymatic microreactors.

Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC–MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC–MS

The disparity of IC50s from CYP450 inhibition assays used to assess drug–drug interaction potential was investigated, in order to have evidence for selecting a reliable in vitro CYP450 inhibition assay to support drug discovery. Three assays were studied: individual rhCYP isozymes and corresponding coumarin derivative-probe substrates with fluorescent detection, human liver microsomes (HLM) and cocktail drug-probe substrates with LC–MS detection, and double cocktail rhCYP isozymes mix and drug-probe mix with LC–MS detection. Data comparisons showed that the rhCYP-fluorescent assay and the cocktail assay with HLM–LC–MS had weak correlation. Detection method and probe substrates were shown to not be the major cause of the disparity in IC50s. However, the enzyme source and composition (HLM versus, rhCYP) caused disparity in IC50s. Specifically, the high concentrations of CYP isozymes often used with HLM-based assays produced high probe substrate conversion and test compound metabolism, which should both contribute to artificially higher IC50s. Non-specific binding of substrate to higher concentration proteins and lipids in the HLM-based assays should also contribute to higher IC50s. The modified double cocktail assay was found to overcome limitations of the other two assays. It uses an rhCYP isozymes mix, drug-probe substrate mix, low protein concentration, and LC–MS detection. The double cocktail assay is sensitive, selective, and high throughout for use in drug discovery to provide an early alert to potential toxicity with regard to drug–drug interaction, prioritize chemical series, and guide structural modification to circumvent CYP450 inhibition.

Stability and catalytic activity of α‐amylase from barley malt at different pressure–temperature conditions

The impact of high hydrostatic pressure and temperature on the stability and catalytic activity of alpha-amylase from barley malt has been investigated. Inactivation experiments with alpha-amylase in the presence and absence of calcium ions have been carried out under combined pressure-temperature treatments in the range of 0.1-800 MPa and 30-75 degrees C. A stabilizing effect of Ca(2+) ions on the enzyme was found at all pressure-temperature combinations investigated. Kinetic analysis showed deviations of simple first-order reactions which were attributed to the presence of isoenzyme fractions. Polynomial models were used to describe the pressure-temperature dependence of the inactivation rate constants. Derived from that, pressure-temperature isokinetic diagrams were constructed, indicating synergistic and antagonistic effects of pressure and temperature on the inactivation of alpha-amylase. Pressure up to 200 MPa significantly stabilized the enzyme against temperature-induced inactivation. On the other hand, pressure also hampers the catalytic activity of alpha-amylase and a progressive deceleration of the conversion rate was detected at all temperatures investigated. However, for the overall reaction of blocked p-nitrophenyl maltoheptaoside cleavage and simultaneous occurring enzyme inactivation in ACES buffer (0.1 M, pH 5.6, 3.8 mM CaCl(2)), a maximum of substrate cleavage was identified at 152 MPa and 64 degrees C, yielding approximately 25% higher substrate conversion after 30 min, as compared to the maximum at ambient pressure and 59 degrees C.

Efficient Synthesis of 2-Hydroxy-3,4-dienoates by Oxidation of Zirconium­allenyl Enolates with Dimethyldioxirane

The α-hydroxylation of zirconiumallenyl enolates, obtained from β-allenic esters by deprotonation with LDA or LiN(SiMe3)2 and transmetalation with Cp2ZrCl2, with dimethyldioxirane (DMDO) selectively affords 2-hydroxy-3,4-dienoates. The oxidation proceeds usually with high yield and substrate conversion and tolerates even sensitive or unreactive substrates.

Transition-Metal (Ti,V,Cr,Mn,Fe,Co,Cu) Containing Ordered Nanoporous Materials:Novel Heterogeneous Catalysts for Selective Oxidation Reactions

Transition-metal (Ti,V,Cr,Mn,Fe,Co,Cu) containing periodic nanoporous catalysts, were synthesized hydrothermally and characterized using various analytical and spectroscopic techniques. The catalytic performance of the different catalysts was systematically evaluated with a detailed study on TiMCM-41. All the catalysts showed promise for the selective oxidation of cycloalkanes, viz., cyclohexane, cyclooctane, and cyclododecane, under mild reaction conditions. Furthermore, these mesoporous molecular sieves were also active for all the chosen reactions even after several recycling and/or washing treatments. Among the various materials under investigation, the catalysts TiMCM-41 and VMCM-41 showed much higher substrate conversion and excellent product selectivity in conjunction with a minimal leaching of the active species. More importantly, the influence of pore size on the catalytic activity of the bulkier substrates such as cyclooctane and cyclododecane is demonstrated.

Liquid oxidation of cyclohexane to cyclohexanol over cerium-doped MCM-41

Liquid phase oxidation of cyclohexane was carried out under mild reaction conditions over mesoporous Ce-MCM-41 catalysts using aqueous hydrogen peroxide (30%) as oxidant and acetic acid as solvent without adding any initiator. The catalysts exhibited high substrate conversion and good product (cyclohexanol) selectivity and it can be reused once with almost the same activity. The catalyst was characterized by a combination of various physicochemical techniques, such as N 2 physisorption, diffuse reflectance UV–vis, X-ray diffraction and FT-IR.

Catalytic oxidation of alcohols with polymer-supported ruthenium complex under mild conditions

Polymer-supported ruthenium-containing complex PS–Phen–Ru was synthesized (where PS = chloromethyl polystyrene resin, Phen = 1,10-phenanthroline) and was characterized by FT-IR, ICP, and XPS. The supported complex was used to catalyze the oxidation of primary aliphatic alcohols as well as aromatic alcohols in the presence of iodosylbenzene. The oxidations were carried out in acetonitrile solution, affording the corresponding aldehydes or ketones in high substrate conversion and high selectivities under mild reaction conditions. The catalyst can be easily prepared and can be recycled.

Optimal design of continuously stirred membrane reactors in series using Michaelis-Menten kinetics with competitive product inhibition: theoretical analysis

Analytical expressions were derived for the optimal design (based on the minimum of the volume of the total number of reactors) of N continuously stirred membrane reactors (CSMRs) performing the enzyme-catalyzed reaction described by Michaelis-Menten kinetics with competitive product inhibition. The influence of membrane selectivity for both substrate and product on the total dimensionless residence time of the reactors (overall volume) was determined. The optimal design of N CSMRs (variable volume reactors) was compared with equal volume membrane reactors required to achieve the same degree of substrate conversion. The effect of kinetic and operating parameters on the performance of membrane reactors was determined. Optimization results show that membrane reactors are superior to continuously stirred tank reactors (CSTRs) in series at a high substrate rejection coefficient and low product rejection coefficient, high substrate conversion and using a small number of reactors. Also a high dimensionless Michaelis-Menten constant, high dimensionless inhibition constant and low substrate concentration in the feed to the first reactor improved the performance of the membrane reactors vs. CSTRs in series. The reduction in total volume of the optimal membrane reactors compared to CSTRs in series was up to 86% for the conditions in this work. A comparison between the optimum and equal volume design of membrane reactors in series showed no major difference in total volume between the two design criteria at a practical range of operating conditions. A volume reduction up to 16% was observed for the conditions in this work.