The solubility of adenosine 3′,5′-cyclic monophosphate sodium (cAMPNa) in mixed solvents (water+ethanol, water+methanol, and water+acetone) was measured within 293.15−313.15 K under atmospheric pressure. The (CNIBS)/Redlich−Kister model and the modified Apelblat equation were respectively applied to correlate the solubility data to evaluate the effect of the compositional ratio of the organic solvent and the temperature on the solubility in binary solvents, and satisfactory simulation results were obtained. The solubility of cAMPNa was maximal in pure water and markedly diminished at all evaluated temperatures as the mole fraction of the organic solvent in the aqueous mixture increased. The thermodynamic functions for cAMPNa dissolution in the three solvent mixtures were obtained from the solubility data using the van’t Hoff and Gibbs equations, and the dissolution behavior was discussed. Dissolution of cAMPNa was endothermic and nonspontaneous in all cases, and the enthalpy was the major contributing force to the Gibbs energy.

•A new strategy to enhance biocatalyst stability was developed.•Highly crystalline mesoporous TiO2 microparticles were prepared.•The hydroxyl-coated microparticles can be modified by amino-silane.•Sequential application of APTES and GLU cross-linkers enabled effective ADA coupling.Fabrication of biocompatible micro- and nanoparticles is attractive because of their potential for application as enzyme immobilization tools. Mesoporous TiO2 microparticles with high crystallinity, high hydroxyl density, and large pore size (20 nm) were prepared by solid-state calcination and a soft chemistry method. The large pores of the microparticles were efficient in adenosine deaminase (ADA) encapsulation. The hydroxyl-coated microparticles could optimize amino-silane modification and be efficiently utilized as ADA-immobilization carriers. However, the adsorbed enzymes were easily leached when cycled. Sequential application of the coupling agent 3-aminopropyltriethoxysilane and cross-linker glutaraldehyde (GLU) enabled effective ADA coupling. After eight batch cycles, the immobilized ADA retained 80% of its initial activity, much higher than that by direct enzyme adsorption (30%). GLU prevented enzyme desorption and loss of activity. We thus improved ADA loading efficiency, recycling, and stability. TiO2 microparticles could be suitable ADA immobilization candidates for detection and industrial inosinic acid production.

N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) were immobilized for synthesis of N-acetylneuraminic acid (Neu5Ac) on three resins: Amberzyme oxirane resin (AOR), poly (styrene-co-DVB)-Br resin (PBR) and amino resin (AR). The loading capacity and immobilized enzyme activity showed that AR was the best carrier. Three methods of glutaraldehyde cross-linking were tested and simultaneous cross-linking and immobilization was demonstrated to be the best method. The functional properties of immobilized AGE and NAL were studied and compared to those of the free enzyme. The highest enzyme activities of free and immobilized AGE were obtained in 0.1 M potassium phosphate buffer at pH 7.5 and a temperature of 37 °C. Comparatively, the highest NAL activities were at pH 8.5. Meanwhile, an increase in Km (from 1.14 to 1.31 mg·mL−1 for AGE and from 1.05 to 1.25 mg·mL−1 for NAL) and a decrease in Vmax (from 177.53 to 106.37 µg·min−1 mL−1 for AGE and from 126.41 to 95.96 µg·min−1 mL−1 for NAL) were recorded after immobilization. The AR–glutaraldehyde–enzyme system exhibited better thermal stability than the free enzyme, and retained 72% of its initial activity even after eight repeated runs. The apparent activation energy (Ea) of the free and immobilized AGE (NAL) was 117.14 kJ·mol−1 (124.21 kJ·mol−1) and 78.45 kJ·mol−1 (66.64 kJ·mol−1), respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limit. Subsequently, Neu5Ac production from GlcNAc using immobilized enzymes in one reactor was carried out resulting 101.45 g·L−1 of Neu5Ac and the highest conversion ratio of 82%. This method of enzyme immobilization may have a promising future for Neu5Ac production in industry.

•An effective pretreatment of cornstalk by combining ethanol and NaOH was developed.•Low temperature and low-concentration alkali are used in the present process.•High yield of monosaccharide was obtained after ACOS pretreatment and enzymatic hydrolysis.•A slightly higher butanol concentration was achieved using ACOS pretreatment compared to the pure glucose fermentation.Biobutanol is an important alternative fuel source, but current processing methods suffer from low yields. This study aimed to develop and optimize a pretreatment protocol for biobutanol production from cornstalks. Fractionation of cornstalks into carbohydrate (cellulose and hemicellulose) and lignin was performed by alkali-catalyzed organosolv pretreatment (ACOS). After optimization of the process parameters, more than 80% of the total lignin was removed, with minimal hemicellulose degradation, at 110 °C, 4% (w/w dry cornstalk) NaOH, 90 min reaction time, and 60% (v/v) ethanol. After enzymatic hydrolysis, the maximum recovery of total monosaccharide was 83.7% (85.0% cellulose, 82.0% hemicellulose). In acetone-butanol-ethanol (ABE) fermentation, a slightly higher total ABE concentration (12.8 g/L vs. 11.9 g/L) was produced from the enzymatic hydrolysate, compared with that from a glucose control. The physical structure and chemical properties of alkali-catalyzed organosolv lignin (ACOSL) showed higher phenolic group content and antioxidant capacity compared with alkali lignin. ACOS pretreatment is an economical method for the production of fermentable monosaccharide and high-value lignin, for use in biofuel production.Download high-res image (132KB)Download full-size image

Conventional activated carbon used in decolorization of citric acid fermentation broth has the disadvantage of high citric acid loss. In this study, a novel biomass-based carbon material, namely HBCM, was prepared via H3PO4 activation of hydrothermally treated corncob. The material was fully characterized by SEM, BET, TG, FTIR, XPS and pHpzc. The results showed that the material's SBET was as high as 1720 m2 g−1 and several weak-acid functional groups existed on its surface, which contributed to efficient decolorization with low citric acid loss. By adjusting the solution pH value to around 7, nearly no citric acid was lost. Furthermore, the adsorption behavior of pigments on HBCM was systematically investigated under optimized pH. The results indicated that the adsorption was spontaneous and endothermic. Intra-particle diffusion was the rate-limiting step. By comparing FTIR data before and after adsorption, it was found that oxygen-containing functional groups on the HBCM surface participated in pigment adsorption. Overall, the tailor-made HBCM performed excellently with a 99% decolorization ratio and nearly no citric acid loss under optimum operating conditions. It could be a potential adsorbent in the removal of pigments from citric acid fermentation broth.

To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production.We developed a new one-pot PPK system by applying a three-enzyme cascade (CMK, NDK and PPK) with an in vitro polyP-based ATP regeneration system. The PPK was selected from twenty sources, and was made solvable by fusion expressing with soluble protein and constructing polycistronic plasmids, or co-expressing with molecular chaperones GroES/EL. Activities of other enzymes were optimized by employing fusion expression, tac-pBAD system, Rosetta host and codon optimization. After 24 h, the concentration of CDP and CTP reached 3.8 ± 0.2 and 6.9 ± 0.3 mM l−1 respectively with a yield of approximately 79%. The molar conversion rate of CTP was 51%, and its yield and conversion rate increased 100% from the traditional system.A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.

Available molecular and genetic tools for the genetic manipulation of Arthrobacter species are limited until now. In gene engineering, a continuous set of promoters with various strengths are of importance for fine-tuning gene expression in metabolic optimization and control analysis. Here, for the first time, we constructed a promoter trap system using green fluorescence protein (GFP) as a reporter, for screening and characterizing functional Arthrobacter promoters. Twenty-three Arthrobacter transformants of various GFP fluorescence strengths were isolated and characterized through the analysis of DNA sequences. Among the 23 putative promoters, 2 were selected for deletion analysis of promoter elements. As a result, the deletion of the upstream of the putative promoter P8 and P13 caused a 43.8% decrease and a 29.1% increase in the fluorescence signals, respectively. Finally, we obtained the strongest promoter P13-3 which was 4.4 times more potent than the promoter of 6–hydroxyl–d–nicotine oxidase gene which was previously reported in Arthrobacter nicotinovorans, and the obtained promoter was used to improve the production of cyclic adenosine monophosphate in Arthrobacter sp. CGMCC 3584. The screening strategy together with obtained promoters in this study would contribute to the future engineering of Arthrobacter species.

In this study, the heterogeneous expression of the formate dehydrogenase gene for nicotinamide adenine dinucleotide (NADH) regeneration is successfully achieved in Clostridium ljungdahlii. The specific activity of formate dehydrogenase is dependent on cell growth, reaching its highest value (3.16 U mg−1 for the engineered strain, 1.59 U mg−1 for the parent strain) when cells entered the log phase. The NADH concentration in engineered C. ljungdahlii (12.64 μm per g DCW) increased 4.3 fold compared with the parent strain (2.93 μm per g DCW). The genetically engineered bacterium C. ljungdahlii is employed in a microbial fuel cell to gauge the potential for bioelectricity generation improvement. The voltage of a sodium formate fed microbial fuel cell with C. ljungdahlii is enhanced to 290 mV ± 10, which is 3.8 times the parent strain (76 ± 8 mV). The higher NADH pool in engineered C. ljungdahlii can facilitate electron transfer in the system, which contributes to an increase in maximum power density from 15 mW m−2 to 35 mW m−2. On the basis of these results, the manipulation of intracellular cofactors is shown to be an efficient approach to improve bioelectricity generation in the microbial fuel cell, and indicates the great potential of metabolic engineering for improvements in bioelectricity.

The application of synthetic flavinium organocatalysts for the in situ regeneration of oxidized cofactors NAD(P)+ using O2 as the terminal oxidant without any special illumination or equipment is reported. With the aid of the highly active bridged flavinium catalyst, the rate of NAD(P)H oxidation is accelerated by 3 orders of magnitude. The results show that the catalytic activity of the bridged flavinium catalyst is not dependent on light but on only oxygen. Furthermore, this catalyst is compatible with various preparative enzymatic oxidation reactions. A hydride transfer mechanism is proposed for the presented system.Keywords: aerobic oxidation; cofactor regeneration; enzyme catalysis; flavin; metal-free catalysis; organocatalysis

Acetoin, a novel C4 platform molecule derived from new ABE (acetoin–butanol–ethanol) type fermentation via metabolic engineering, was used for the first time as a bio-based building block for the production of liquid hydrocarbon fuels. A series of diesel or jet fuel range C9–C14 straight, branched, or cyclic alkanes were produced in excellent yields by means of C–C coupling followed by hydrodeoxygenation reactions. Hydroxyalkylation/alkylation of acetoin with 2-methylfuran was investigated over a series of solid acid catalysts. Among the investigated candidates, zirconia supported trifluoromethanesulfonic acid showed the highest activity and stability. In the aldol condensation step, a basic ionic liquid [H3N+–CH2–CH2–OH][CH3COO−] was identified as an efficient and recyclable catalyst for the reactions of acetoin with furan based aldehydes. The scope of the process has also been studied by reacting acetoin with other aldehydes, and it was found that abnormal condensation products were formed from the reactions of acetoin with aromatic aldehydes through an aldol condensation–pinacol rearrangement route when amorphous aluminium phosphate was used as a catalyst. And the final hydrodeoxygenation step could be achieved by using a simple and handy Pd/C + H-beta zeolite system, and no or a negligible amount of oxygenates was observed after the reaction. Excellent selectivity was also observed using the present system, and the clean formation of hydrocarbons with a narrow distribution of alkanes occurred in most cases.

Here we report two new crystal forms of adenosine 3′,5′-cyclic monophosphate sodium and the discovery of the direct solid–solid transformation process from its methanol trihydrate (solvate) to its pentahydrate form (hydrate) that is only mediated by humidity. These findings provide a potential approach for the removal of residual solvents in the manufacturing of pharmaceuticals and food additives.

The mechanisms associated with how cells in biofilms exhibit enhanced tolerance to adverse environmental stress have attracted much recent attention. In this study, we investigated the tolerance mechanisms through observation of biofilm morphology combined with detection of fermentation activity, and discovered an improved way to culture biofilms for application in acetone–butanol–ethanol (ABE) fermentation. We found that a mature biofilm exhibited enhanced tolerance to acetic acid and butanol during ABE fermentation. A mature biofilm consists of a complex, heterogeneity three-dimensional structure, with a coated extracellular polymer substance (EPS). Therefore, when exposed to a harsh environment, cells in different regions of the biofilm displayed different levels of performance, resulting in cells with higher tolerance levels capable of survival, continued growth. The EPS acted as a barrier, limiting the transfer of harmful substances, and diluting their concentration in order to protect biofilm cells. During repeated-batch fermentations, the continuous fermentation formed biofilms, and the butanol concentration, productivity, and yield were 22.08%, 26.37%, and 61.08% higher, respectively, relative to suspended fermentation.

•GO is modified by amino poly(ethylene glycol) with property heterogeneity.•Nuclease P1 prefers to be physical adsorbed on the surface of pristine GO.•Nuclease P1 is stable chemical cross-linked on the edge of modified GO.•Stability of the immobilized enzyme on amino modified GO is improved.The use of graphene oxide (GO) nanosheets for functional enzyme support has attracted intensive interest owing to their unique planar structure and intriguing physical and chemical properties. However, the detailed effects of the interface properties of GO and its functionalized derivatives on active biomolecules are not well understood. We immobilize nuclease P1, a common industrial nucleic acid production enzyme, on pristine and amino poly(ethylene glycol) (PEG-NH2) modified GO nanosheets with interface property heterogeneity using two approaches, physical adsorption and chemical crosslinking. It is demonstrated that nuclease P1 could be stable immobilized on the surface of pristine GO by physical adsorption and on the edge of modified GO nanosheets by chemical crosslinking. The resultant loading capacity of nuclease P1 on pristine GO is as high as 6.45 mg/mg as a consequence of strong electrostatic and hydrophobic interactions between the enzyme and carrier. However, it is determined that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on PEG-NH2-modified GO are obviously improved compared to those of the enzyme immobilized on pristine GO. The enhanced catalytic behavior demonstrates that GO and its derivatives have great potential in efficient biocatalytic systems.

An efficient and biocompatible NADH regeneration system to promote ADH-catalysed oxidation reactions is reported. Carbon nanomaterials facilitate enhanced enzyme attachment within their hierarchical nano-structure. According to enzyme protein molecular computer simulation analysis, different electron transfer efficiencies on CNTs or GNSs enzyme-catalyzed electrodes result in different electric charge distributions around ADH, which affects its molecular spatial arrangement and three-dimensional conformation. The nanostructure enhances the enzyme–electrode interaction and electron transfer rate. 1.4- and 1.9-fold higher current density are reached on CNTs and GNSs, respectively, versus the carbon cloth control for the bio-electrochemical NADH regeneration. Maximum NADH production rates are 2.11 and 3.01 times higher than that on the unmodified carbon cloth control. The use of the efficient carbon nanomaterial electrochemical reactor leads to a highly conductive three-dimensional cathode for the improvement of bio-electrochemical NADH regeneration, making the nanomaterial an extremely efficient material from an engineering perspective as well.

•A tailor-made ion exchanger AD-1 showed a high adsorption capacity to Neu5Ac.•An ion-exchange model was successfully used to simulate the equilibrium data.•The average selectivity coefficient of Neu5Ac−/OH− was obtained.•The effect of pH on the uptake of Neu5Ac was successfully predicted by the model.N-acetyl-d-neuraminic acid (Neu5Ac) is a high value-added product widely applied in the food industry. A suitable equilibrium model is required for purification of Neu5Ac based on ion-exchange chromatography. Hence, the equilibrium uptake of Neu5Ac on a strong anion exchanger, AD-1 was investigated experimentally and theoretically. The uptake of Neu5Ac by the hydroxyl form of the resin occurred primarily by a stoichiometric exchange of Neu5Ac− and OH−. The experimental data showed that the selectivity coefficient for the exchange of Neu5Ac− with OH− was a non-constant quantity. Subsequently, the Saunders’ model, which took into account the dissociation reactions of Neu5Ac and the condition of electroneutrality, was used to correlate the Neu5Ac sorption isotherms at various solution pHs and Neu5Ac concentrations. The model provided an excellent fit to the binary exchange data for Cl−/OH− and Neu5Ac−/OH−, and an approximate prediction of equilibrium in the ternary system Cl−/Neu5Ac−/OH−. This basic information combined with the general mass transfer model could lay the foundation for the prediction of dynamic behavior of fixed bed separation process afterwards.

•Continuous production of GOS from a lactose in an UMR.•Purification of GOS with the NF membrane in a continuous diafiltration process.•Continuous production of high-purity GOS in the CPNSS.Continuous enzymatic production of galactosyl-oligosaccharides (GOS) from a lactose substrate in an ultrafiltration membrane reactor (UMR) coupled with a nanofiltration separation system (CPNSS) was investigated. The overall rate of production over 4 h of continuous production was 80–104 mg GOS formed/U with an average residence time of 66 min, an initial lactose concentration of 300 g/L, an inlet pressure of 2.0 bar inlet pressure, and an outlet pressure of 1.5 bar. In the continuous diafiltration (CD) process, the concentration of various sugars and the relationship between the yield and purity of oligosaccharides was well predicted by mathematical models, the increased rate of sugar rejections was less than 10%, and the decreased rate of concentrations in the tank was less than 15%. In the CPNSS, 33.4 wt.% GOS was obtained in the UMR, and 1.24 kg of high-purity GOS (specifically, GOS purity of 57.2% and lactose content less than 20%) was achieved. This final yield was 80.1% GOS, which meets industry standards.

Desorption is essential to design an integrated recovery process for 1-butanol, a potential biofuel. The modeling of desorption processes is a significant tool to optimize the separation process of 1-butanol. In this work, we systematically studied the desorption of 1-butanol from a porous polymeric resin KA-I by solvent desorption technique. The desorption equilibrium, desorption kinetics and dynamic desorption behaviors were studied experimentally and simulated theoretically. About 1.5 bed volumes of absolute ethanol could desorb 1-butanol completely with 73.6 g L−1 1-butanol in the effluent in the fixed bed system. A pore diffusion model was used to fit the desorption kinetics of 1-butanol satisfactorily. The concentration evolution of eluent and 1-butanol in the resin pore was simulated. During desorption, the concentration of 1-butanol in the resin pore increases gradually at first, and then decreases gradually due to the slower pore diffusion. Moreover, a general rate model was used to predict the dynamic desorption profiles of 1-butanol under different operating conditions successfully. Finally, repeated adsorption and desorption dynamic cycles were predicted by the general rate model quite well.

Metastable zone widths (MSZWs) and induction times (tind’s) were measured by turbidity techniques during the antisolvent crystallization of disodium guanosine 5′-monophosphate (5′-GMPNa2). Measured MSZWs can be affected by numerous process parameters, including temperature, the agitation rate, and the antisolvent addition rate. An exponential equation was used to correlate the supersolubility and solubility data for different conditions, and to afford predictions of the MSZW values. Values of tind at different temperatures and mole fractions of water were assessed to determine the primary nucleation and growth mechanisms of 5′-GMPNa2 crystals in the water–ethanol system. The measured tind’s were then correlated using mononuclear and polynuclear mechanistic models. The fitting results identified the primary nucleation mechanism for 5′-GMPNa2 as polynuclear, which relates tind and the supersaturation for various growth mechanisms. The growth mechanism of 5′-GMPNa2 was found to be diffusion-controlled at all experimental temperatures.

•TiO2 nanofibres heterogeneously wrapped with rGO are fabricated.•Pt nanoparticles supported on h-rGO@TiO2-rGO are uniform.•Pt/h-rGO@TiO2-rGO catalysts yields an enhanced performance in MOR.•The Pt/h-rGO@TiO2-rGO hybrid exhibits the largest If of 324 mA mg−1.The exploration of advanced catalyst supports is a promising route to obtain electrocatalysts with high activity and durability. Herein, TiO2 nanofibers heterogeneously wrapped with reduced graphene oxide (rGO) designated as h-rGO@TiO2-rGO were synthesized by a facile deposition and hydrothermal method and served as support for Pt nanoparticles (Pt NPs) used for methanol oxidation. The morphology and electrocatalytic properties of the catalysts were investigated by transmission electron microscopy, X-ray diffraction, Raman spectroscopy, cyclic voltammetry, and chronoamperometry. Pt NPs supported on h-rGO@TiO2-rGO showed uniform dispersion with a mean particle size of 3.4 nm. The electrocatalytic activity and stability of Pt/h-rGO@TiO2-rGO both improved remarkably compared with Pt/rGO and Pt/TiO2. This enhancement originated from the porous structure that promoted the homogeneous dispersion and enlarged the electrochemical active surface area (ECSA) of Pt NPs (ECSACO = 30.95 m2 g−1). Thus, the h-rGO@TiO2-rGO hybrid could be developed as a promising electrocatalyst support material in fuel cells.

The conventional ion exchange process used for recovery of uridine 5′-monophosphate (UMP) from the enzymatic hydrolysate of RNA is environmentally harmful and cost intensive. In this work, an innovative benign process, which comprises adsorption technology and use of a hyper-cross-linked resin as a stationary phase is proposed. The adsorption properties of this kind of resin in terms of adsorption equilibrium as well as kinetics were evaluated. The influences of the operating conditions, i.e., initial UMP concentration, feed flow rate, and bed height on the breakthrough curves of UMP in the fixed bed system were investigated. Subsequently, a chromatographic column model was established and validated for the prediction of the experimentally attained breakthrough curves of UMP and the main impurity component (phosphate ion) with a real enzymatic hydrolysate of RNA as a feed mixture. At the end of this paper, the crystallization of UMP was carried out. The purity of the final product (uridine 5′-monophosphate disodium, UMPNa2) of over 99.5 % was obtained.

The efficiency of current methods for industrial production of the enzyme nuclease P1 is limited. In this study, we sought to improve fermentation methods for the production of nuclease P1. An immobilized fermentation system using an activated carbon filter sponge as a carrier was used for the production of nuclease P1. In an airlift internal loop reactor (ALR), the fermentation performance of three different fermentation modes, including free-cell fermentation, repeated-batch fermentation, and semi-continuous immobilized fermentation, were compared. The fermentation kinetics in the fermentation broth of the three fermentation modes, including dissolved oxygen (DO), pH value, cell concentration, residual sugar concentration, and enzyme activity, were tested. The productivity of semi-continuous immobilized fermentation reached 8.76 U/mL/h, which was 33.3 and 80.2 % higher than that of repeated-batch fermentation and free-cell fermentation, respectively. The sugar consumption of free-cell, repeated-batch, and semi-continuous immobilized fermentations was 41.2, 30.8, and 25.9 g/L, respectively. These results showed that immobilized-cell fermentation by using Penicillium citrinum with activated carbon filter sponge in an ALR was advantageous for nuclease P1 production, especially in the semi-continuous immobilized fermentation mode. In spite of the significant improvement in nuclease P1 production in semi-continuous immobilized fermentation mode, the specific activity of nuclease P1 was almost equal among the three fermentation modes.

A Clostridium beijerinckii mutant M13 was derived from C. beijerinckii NCIMB 8052 by atmospheric pressure glow discharge. C. beijerinckii M13 generated a maximum output power density of 79.2 mW m−2 and a maximum output voltage of 230 mV in a microbial fuel cell containing 1 g glucose l−1 as carbon source and 0.15 g methyl viologen l−1 as an electron carrier.

The recovery and purification of biobutanol based on the adsorption method were performed in dynamic conditions. Computational and theoretical modeling is an important tool in the characterization, development, and validation of fixed-bed columns. Relevant breakthrough curves provide valuable information for designing fixed-bed adsorption processes for field applications. In the present study, a general rate model (GRM), implementing convection/diffusion approach theory and a competitive isotherm model, was used to predict the competitive sorption dynamics of acetone–butanol–ethanol (ABE) on a KA-I resin in a fixed-bed column under different operating conditions, i.e., inlet feed flow rate, initial adsorbate concentration, and bed height. The model simulation was quantified by the absolute average deviation (AAD). The calculated AAD values, ranging from 0.05 to 0.1, indicated that the GRM gives a general prediction for experimental data. The axial dispersion, external mass transfer, and pore diffusion coefficients were calculated by a series of empirical correlations. Biot number was used to identify the rate controlling step for the adsorption process of ABE on the resin. And the pore diffusion coefficient was found to be major governing factor for adsorption of ABE. The data and modeling presented are valuable for designing the continuous chromatographic separation process and simulation of ABE.

The first continuous meso-flow synthesis of natural and non-natural 5′-nucleotides and deoxynucleotides is described, representing a significant advance over the corresponding in-flask method. By means of this meso-flow technique, a synthesis with time consumption and high-energy consumption becomes facile to generate products with great efficiency. An abbreviated duration, satisfactory output, and mild reaction conditions are expected to be realized under the present procedure.

L-phenylalanine, one of the nine essential amino acids for the human body, is extensively used as an ingredient in food, pharmaceutical and nutrition industries. A suitable equilibrium model is required for purification of l-phenylalanine based on ion-exchange chromatography. In this work, the equilibrium uptake of l-phenylalanine on a strong acid-cation exchanger SH11 was investigated experimentally and theoretically. A modified Donnan ion-exchange (DIX) model, which takes the activity into account, was established to predict the uptake of l-phenylalanine at various solution pH values. The model parameters including selectivity and mean activity coefficient in the resin phase are presented. The modified DIX model is in good agreement with the experimental data. The optimum operating pH value of 2.0, with the highest l-phenylalanine uptake on the resin, is predicted by the model. This basic information combined with the general mass transfer model will lay the foundation for the prediction of dynamic behavior of fixed bed separation process.Download high-res image (224KB)Download full-size image

⿢Five carbonyl reductases from different Streptomyces were discovered by genome mining.⿢SgCR was characterized, expressed and compared with other SDRs.⿢Broad substrate spectrum of SgCR was shown.⿢Highly enanioselectivity and conversion of various chiral alcohols were obtained.⿢Conditions of bioproducing (S)-CHBE was optimized in a biphasic system.The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of enzymes in reaction, economic, ecological issues. Many carbonyl reductases for producing chiral alcohols have been reported but there is still a lack of good catalytic efficacies. Herein, five carbonyl reductases from different Streptomyces were discovered by the strategy of genome mining. These reductases were overexpressed, and we chose SgCR for further study as it owned better enzyme activity. This protein was purified to apparent homogeneity, and its amino acid sequence was analyzed in comparison with that of the reported SDRs. The biocatalytic properties of SgCR were investigated, and this enzyme was confirmed to have the ability to convert various prochiral ketones into highly optically active alcohols. SgCR exhibited the highest activity towards ethyl 4-chloro-3-oxobutanoate (COBE) and the corresponding product ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) was obtained with high yield and excellent e.e. value by optimizing the biphasic system. Eventually, using isopropanol as the co-substrate for NADH recycling in the substrate-coupled reaction, the yield and enantioselectivity of (S)-CHBE were obtained at the values of 90% and 99%, respectively. These results indicate that SgCR is a promising boicatalyst for the synthesis of chiral alcohols in industry.

•MIC of FA against C. beijerinckii 4693:int was 1.5 g/l.•Viability of C. beijerinckii 4693:int was 106.7% in the presence of 0.5 g/l FA.•FA altered expression of genes related antibacterial and adaptation mechanisms.•Enzyme ahpC/F was speculated to be related to the ferulic acid tolerance.Clostridium beijerinckii 4693:int with high ferulic acid (FA) tolerance was engineered and characterized in our lab. In this study, the minimum inhibition concentrations of FA against C. beijerinckii NCIMB 8052 (wild-type) and 4693:int were 1.0 and 1.5 g/l, respectively; cell viability was 18.5% and 106.7%, respectively, in the presence of 0.5 g/l FA. A comparative transcriptome analysis was carried out at two different growth stages to evaluate sensitivity to FA. Genes that were differentially expressed included those related to redox and associated cofactors, riboflavin metabolism, two-component system, glycolysis and butanoate metabolism, and DNA replication as well as those encoding ATP-binding cassette transporters. Cbei_2134 and Cbei_2135 encoding alkyl hydroperoxide reductases are thought to be involved in antibacterial and adaptation mechanisms in C. beijerinckii in the presence of FA.

•Compared to planktonic cells, C. acetobutylicum biofilm cells acquired a more efficient phenotype, e.g., improved butanol tolerance and enhanced reaction rate.•Comparative transcriptomic analysis revealed that 16.2% of the C. acetobutylicum genome genes within biofilm cells were differentially expressed, with most genes being up-regulated.•C. acetobutylicum biofilm cells increased metabolic activities probably by up-regulating iron and sulfur uptake and Fe-S cluster biosynthesis genes as well as glycolysis genes.•Genes involved in sporulation, granulose formation, extracellular polymer degradation and pentose catabolisms as well as various other processes were also apparently regulated within the biofilm cells.Biofilm-based immobilization of solventogenic Clostridia has been extensively exploited to overcome traditional bottlenecks in biobutanol production like solvent toxicity and low productivities. However, the molecular basis of solventogenic Clostridia biofilm is rarely explored. Here, for the first time, we report DNA array-based study of Clostridium acetobutylicum biofilm cells to elucidate the transcriptional modulation. Results showed that 16.2% of the C. acetobutylicum genome genes within the biofilm cells were differentially expressed, with most genes being up-regulated. The most dramatic changes occurred with amino acid biosynthesis, with sulfur uptake and cysteine biosynthesis being the most up-regulated and histidine biosynthesis being the most down-regulated in the biofilm cells. It was demonstrated that C. acetobutylicum biofilm cells increased metabolic activities probably by up-regulating iron and sulfur uptake and Fe-S cluster biosynthesis genes as well as glycolysis genes. Furthermore, genes involved in sporulation, granulose formation, extracellular polymer degradation, pentose catabolisms, and various other processes were also notably regulated, indicating that the biofilm mode of growth rendered the cells a distinct phenotype. This study provides valuable insights into the transcriptional regulation in C. acetobutylicum biofilm cells and should be highly useful for understanding and developing the biofilm-based processes.

•Mutated gene Cbei_4693 was found in mutant strain C. beijerinckii M11.•Disrupting Cbei_4693 in parental strain enhanced ferulic acid tolerance to 0.8 g/L.•Intracellular NADPH level dramatically increased in C. beijerinckii 4693::int.•This is the first time found Cbei_4693 related to ferulic acid tolerance.A mutant strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii M11, which exhibited ferulic acid tolerance up to 0.9 g/L, was generated using atmospheric pressure glow discharge and high-throughput screening. Comparative genomic analysis revealed that this strain harbored a mutation of the Cbei_4693 gene, which encodes a hypothetical protein suspected to be an NADPH-dependent FMN reductase. After disrupting the Cbei_4693 gene in C. beijerinckii NCIMB 8052 using the ClosTron group II intron-based gene inactivation system, we obtained the Cbei_4693 gene inactivated mutant strain, C. beijerinckii 4693::int. Compared with C. beijerinckii NCIMB 8052, 6.23 g/L of butanol was produced in P2 medium containing 0.5 g/L of ferulic acid by 4693::int, and the ferulic acid tolerance was also significantly increased up to 0.8 g/L. These data showed, for the first time, that the Cbei_4693 gene plays an important role in regulating ferulic acid tolerance in ABE fermentation by C. beijerinckii.

•Mutated gene Cbei_4693 was found in mutant strain C. beijerinckii M11.•Disrupting Cbei_4693 in parental strain enhanced ferulic acid tolerance to 0.8 g/L.•Intracellular NADPH level dramatically increased in C. beijerinckii 4693::int.•This is the first time found Cbei_4693 related to ferulic acid tolerance.A mutant strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii M11, which exhibited ferulic acid tolerance up to 0.9 g/L, was generated using atmospheric pressure glow discharge and high-throughput screening. Comparative genomic analysis revealed that this strain harbored a mutation of the Cbei_4693 gene, which encodes a hypothetical protein suspected to be an NADPH-dependent FMN reductase. After disrupting the Cbei_4693 gene in C. beijerinckii NCIMB 8052 using the ClosTron group II intron-based gene inactivation system, we obtained the Cbei_4693 gene inactivated mutant strain, C. beijerinckii 4693::int. Compared with C. beijerinckii NCIMB 8052, 6.23 g/L of butanol was produced in P2 medium containing 0.5 g/L of ferulic acid by 4693::int, and the ferulic acid tolerance was also significantly increased up to 0.8 g/L. These data showed, for the first time, that the Cbei_4693 gene plays an important role in regulating ferulic acid tolerance in ABE fermentation by C. beijerinckii.

An efficient and biocompatible NADH regeneration system to promote ADH-catalysed oxidation reactions is reported. Carbon nanomaterials facilitate enhanced enzyme attachment within their hierarchical nano-structure. According to enzyme protein molecular computer simulation analysis, different electron transfer efficiencies on CNTs or GNSs enzyme-catalyzed electrodes result in different electric charge distributions around ADH, which affects its molecular spatial arrangement and three-dimensional conformation. The nanostructure enhances the enzyme–electrode interaction and electron transfer rate. 1.4- and 1.9-fold higher current density are reached on CNTs and GNSs, respectively, versus the carbon cloth control for the bio-electrochemical NADH regeneration. Maximum NADH production rates are 2.11 and 3.01 times higher than that on the unmodified carbon cloth control. The use of the efficient carbon nanomaterial electrochemical reactor leads to a highly conductive three-dimensional cathode for the improvement of bio-electrochemical NADH regeneration, making the nanomaterial an extremely efficient material from an engineering perspective as well.