A novel fluorogenic substrate for dinuclear Zn(II)-containing metallo-β-lactamases

In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-β-lactamases, (6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3-carboxamido)-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phenylthio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CA) was synthesized and characterized. CA exhibited a fluorescence quantum yield (φ) of 0.0059, two fluorescence lifetimes of 3.63 × 10^?10 and 5.38 × 10^?9 s, and fluorescence intensity that is concentration-dependent. Steady-state kinetic assays revealed that CA is a substrate for metallo-β-lactamases (MβLs) L1 and CcrA, exhibiting K_m and k_cat values of 18 μM and 5 s^?1 and 11 μM and 17 s^?1, respectively.     

Effect of some redox mediators on FAD fluorescence of glucose oxidase from Penicillium adametzii LF F-2044.1

Glucose oxidase (GOx) of Penicillium adametzii LF F-2044.1 recovered by ultrafiltration, was characterized by spectrophotometric and spectrofluorometric methods. It was shown that spectra of GOx from P. adametzii are typical for flavoproteins. Optimal buffer composition was chosen. It was determined that the GOx is the most efficiently interacting with substrate (glucose) in phosphate buffer at pH 7.0 with k_kat/K_M = 15,217 ± 550 M⁻¹ s⁻¹. P. adametzii GOx fluorescence in the presence of different redox mediators (9,10-phenanthroline-5,6-dione, 9,10-phenanthrenequinone, 1,4-benzoquinone, methylene blue, ferrocene, ferrocenecarboxylic acid, α-methylferrocenemethanol, ferrocenecarboxaldehyde) was evaluated. Maximal differences in fluorescence emission intensity were observed in the presence of ferrocene and 9,10-phenanthrenequinone.     

Near-infrared triple-helical peptide with quenched fluorophores for optical imaging of MMP-2 and MMP-9 proteolytic activity in vivo

The gelatinase members of the MMP family have consistently been associated with tumor invasiveness, which make them an attractive target for molecular imaging. We report new activatable proteolytic optical imaging agents that consist of triple-helical peptide (THP) conjugates, with high specificity to the gelatinases, bearing quenched cypate dyes. With quenching efficiencies up to 51%, the amplified fluorescence signal upon cypate₃-THP hydrolysis by the gelatinases (k_cat/K_M values of 6.4 × 10³ M⁻¹ s⁻¹ to 9.1 × 10³ M⁻¹ s⁻¹ for MMP-2 and MMP-9, respectively) in mice bearing human fibrosarcoma xenografted tumors was monitored with fluorescence molecular tomography. There was significant fluorescence enhancement within the tumor and this enhancement was reduced by treatment with pan-MMP inhibitor, Ilomastat. These data, combined with the gelatinase substrate specificity observed in vitro, indicated the observed fluorescence at the site of the tumor was due to gelatinase mediated hydrolysis of cypate₃-THP.     

From zero to hero – Production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum

Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2 g L⁻¹ h⁻¹, and a final titer of 88 g L⁻¹. The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production.     

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (K_a), ΔH, ΔS and ΔG values to be 15.5×10⁴M^?1, ?116.2±18.1 Kcal mol^?1, ?366 cal mol^?1K^?1 and ?7.13 Kcal mol^?1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.     

Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM

d-Lactic acid and pyruvic acid are two important building block intermediates. Production of d-lactic acid and pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalyst prepared from 9 g dry cell wt l^?1 of Pseudomonas stutzeri SDM could catalyze 45.00 g l^?1 dl-lactic acid into 25.23 g l^?1 d-lactic acid and 19.70 g l^?1 pyruvic acid in 10 h. Using a simple ion exchange process, d-lactic acid and pyruvic acid were effectively separated from the biotransformation system. Co-production of d-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.     

The correction of reaction rates in continuous fluorometric assays of enzymes

The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D–Arg-NH₂, was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at μM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10^− 4 × s^− 1 were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH₂, were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was − 2.96 × 10^− 4 a.u μM^− 1 × s^− 1 that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate] ÷ (final − initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K_m and overestimation of V_max.     

Study on alanine aminotransferase kinetics by microchip electrophoresis

Alanine aminotransferase (ALT), which catalyzes the reversible conversion between l-glutamic acid (l-Glu) and l-alanine (l-Ala), is one of the most active aminotransferases in the clinical diagnosis of liver diseases. This work displays a microanalytical method for evaluating ALT enzyme kinetics using a microchip electrophoresis laser-induced fluorescence system. Four groups of amino acid (AA) mixtures, including the substrates of ALT (l-Glu and l-Ala), were effectively separated. Under the optimized conditions, the quantitative analysis of l-Glu and l-Ala was conducted and limits of detection (signal/noise = 3) for l-Glu and l-Ala were 4.0 × 10^?7 and 2.0 × 10^?7 M, respectively. In the reaction catalyzed by ALT, enzyme kinetic constants were determined for both the forward and reverse reactions by monitoring the concentration decrease of substrate AAs (l-Ala and l-Glu), and the K_m and V_max values were 10.12 mM and 0.48 mM/min for forward reaction and 3.22 mM and 0.22 mM/min for reverse reaction, respectively. Furthermore, the applicability of this assay was assessed by analysis of real serum samples. The results demonstrated that the proposed method could be used for kinetic study of ALT and shows great potential in the real application.     

Influence on dithiolopyrrolone antibiotic production by organic acids in Saccharothrix algeriensis NRRL B-24137

The influence of organic acids on growth and dithiolopyrrolone antibiotic production by Saccharothrix algeriensis NRRL B-24137 was studied. The production of dithiolopyrrolones depends upon the nature and concentration of the organic acids in the culture medium. Study of the nature of organic acids showed that the most effective organic acids for thiolutin specific production were maleic, 4-hydroxybenzoic, benzentetracarboxylic, pantothenic, pivalic and pyruvic acids (which yielded almost five-fold over the starting medium) and pimelic acid (more than three-fold). 4-Bromobenzoic acid showed the best production of senecioyl-pyrrothine (59 mg g⁻¹ DCW). Tiglic acid showed the best production of tigloyl-pyrrothine (22 mg g⁻¹ DCW). The highest yield of isobutyryl-pyrrothine (7.6 mg g⁻¹ DCW) was observed in the presence of crotonic acid. Sorbic acid yielded the best production of butanoyl-pyrrothine (26 mg g⁻¹ DCW). Methacrylic, butyric, pyruvic and 4-bromobenzoic acids also exhibited the best production of butanoyl-pyrrothine (27–11-fold).Study of organic acid concentration showed that among the selected organic acids, pimelic acid yielded the highest specific production of thiolutin (91 mg g⁻¹ DCW) at 7.5 mM; and senecioyl-pyrrothine (11 mg g⁻¹ DCW), tigloyl-pyrrothine (9 mg g⁻¹ DCW) and butanoyl-pyrrothine (3.5 mg g⁻¹ DCW) at 5 mM. Pyruvic acid at 1.25 mM enhanced the production of senecioyl-pyrrothine (4.3 mg g⁻¹ DCW). The maximum production of tigloyl-pyrrothine (18.6 mg g⁻¹ DCW) was observed in the presence of tiglic acid at 2.5 mM. Maximum production of isobutyryl-pyrrothine was observed in the presence of 7.5 mM tiglic acid. In addition, methacrylic acid (at 5 mM) and butyric acid (at 2.5 mM) enhanced the production of butanoyl-pyrrothine (26 and 20 times, respectively).The above results can be employed in the optimisation of the culture medium for the production of dithiolopyrrolone in higher quantities.     

Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation

Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using d-glucose and CO₂ as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g⁻¹ at a glucose consumption of 10 g L⁻¹, to 5.7 g g⁻¹ at a glucose consumption of 50 g L⁻¹. The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g⁻¹) at a glucose consumption of 10 g L⁻¹ to a value close to zero at 50 g L⁻¹. The highest SA yield on glucose and highest SA titre obtained were 0.91 g g⁻¹ and 48.5 g L⁻¹ respectively. Metabolic flux analysis based on the established C₃ and C₄ metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway.     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

Improving the lactic acid production of Actinobacillus succinogenes by using a novel fermentation and separation integration system

This work describes the development of a novel integrated system for lactic acid production by Actinobacillus succinogenes. Fermentation and separation were integrated with the use of a microfiltration (MF) membrane, and lactic acid was recovered by resin adsorption following MF. The fermentation broth containing residual sugar and nutrients was then recycled back into the fermenter after lactic acid adsorption. This novel approach overcame the problem of product inhibition and extended the cell growth period from 41 h to 120 h. Production of lactic acid was improved by 23% to 183.4 g L⁻¹. The overall yield and productivity for glucose were 0.97 g g⁻¹ and 1.53 g L⁻¹ h⁻¹, respectively. These experimental results indicate that the integrated system could benefit continuous production of lactic acid at high levels.     

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation

The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L⁻¹, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L⁻¹ h⁻¹ were obtained with lactic acid concentrations as high as 198.32 g L⁻¹ for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L⁻¹ h⁻¹ with a maximum concentration of 186.40 g L⁻¹ at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.     

Fluorescence kinetics of PSII crystals containing Ca2 + or Sr2 + in the oxygen evolving complex

Photosystem II (PSII) is the pigment–protein complex which converts sunlight energy into chemical energy by catalysing the process of light-driven oxidation of water into reducing equivalents in the form of protons and electrons. Three-dimensional structures from x-ray crystallography have been used extensively to model these processes. However, the crystal structures are not necessarily identical to those of the solubilised complexes. Here we compared picosecond fluorescence of solubilised and crystallised PSII core particles isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The fluorescence of the crystals is sensitive to the presence of artificial electron acceptors (K₃Fe(CN)₃) and electron transport inhibitors (DCMU). In PSII with reaction centres in the open state, the picosecond fluorescence of PSII crystals and solubilised PSII is indistinguishable. Additionally we compared picosecond fluorescence of native PSII with PSII in which Ca² in the oxygen evolving complex (OEC) is biosynthetically replaced by Sr^2 +. With the Sr^2 + replaced OEC the average fluorescence decay slows down slightly (81 ps to 85 ps), and reaction centres are less readily closed, indicating that both energy transfer/trapping and electron transfer are affected by the replacement.     

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 × His tag was found to be 22.6 U mg^?1 of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 °C, respectively and the inulinase was stable in the pH range of 3–8 and in the temperature range of 0–50 °C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g^?1 of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10 h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L^?1 citric acid and 5.3 g L^?1 iso-citric acid from inulin while 68.9 g L^?1 of citric acid and 4.1 g L^?1 iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.     

Action of tenuazonic acid,a natural phytotoxin,on photosystem II of spinach

Tenuazonic acid (TeA) is a putative phytotoxin obtained from Alternaria alternata, the organism that can cause brown leaf spot disease of Crofton weed (Eupatorium adenophorum). It is demonstrated here that the tenuazonic acid inhibits the activity of photosystem II (PSII); the I₅₀-value is 48 μg mL^?1. Evidences from chlorophyll fluorescence show that tenuazonic acid interrupts electron transport between Q_A and Q_B on the acceptor side of PSII. It does not have an effect on the antenna pigments, the oxygen-evolving complex (OEC) at the donor side of PSII. On the basis of the fluorescence induction kinetics and competition experiments with [¹⁴C]atrazine, it is shown that tenuazonic acid does not share the same binding environment with atrazine despite their common action target: the Q_B-site. It is concluded that tenuazonic acid is a member of a novel class of PSII inhibitors.     

Continuous production of ellagic acid in a packed-bed reactor

Ellagic acid is a high-value bioactive compound that is used in the food, cosmetic and pharmaceutical industries. The aim of this work was to develop a continuous system for ellagic acid production. Ellagitannase produced by solid-state fermentation and attached to polyurethane foam particles was used as a biocatalyst in a continuous bioreactor for the hydrolysis of ellagitannins from pomegranate by-product. A packed-bed reactor containing the biocatalyst (22.22 Units per gram of dry solid, U gds⁻¹) was fed with a pomegranate ellagitannins solution (0.1%, w/v) at a flow rate of 0.27 mL min⁻¹ at 60 °C. The bioreactor completed several biotransformations while maintaining the hydrolysis rate (60%) with a half-life of 10 continuous cycles of ellagic acid production. Volumetric productivity and ellagic acid yield were 1.09 g L⁻¹ h⁻¹ and 235.89 mg g⁻¹ of pomegranate ellagitannins during the first 70 min of hydrolysis, respectively. The developed biocatalyst showed good operational and mechanical stability and may be successfully used for ellagitannin hydrolysis in a continuous system. This is the first report of high-yield continuous production of ellagic acid using an auto-immobilized enzyme.     

Determination of hippuric acid in human urine by ion chromatography with conductivity detection

A simple, rapid, precise and eco-friendly ion chromatography (IC) method for the determination of hippuric acid (HA) in human urine was proposed in this paper. The separation was carried out an anion exchange column with 2.0 mmol L^?1 Na₂CO₃ + 2.0 mmol L^?1 NaHCO₃ as mobile phase at the flow-rate 0.7 mL min^?1. A suppressed conductivity detector was used and the detection limit was 1.0 μg L^?1(S/N = 3) for hippuric acid. The analysis time for one run was 30 min under the optimized IC condition. The recovery of hippuric acid was 93.2–98.0% while the relative standard deviation (RSD) was 1.4–2.3% by seven measurements.     

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1 mol of CO₂ was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD⁺ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD⁺ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L⁻¹ of glucose was consumed to generate 12.08 g L⁻¹ of succinic acid. Furthermore, under optimized condition of CO₂ supply, the succinic acid productivity and the CO₂ fixation rate reached 223.88 mg L⁻¹ h⁻¹ and 83.48 mg L⁻¹ h⁻¹, respectively.     

The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC₅₀ ∼ 70 μM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a K_i of ≈20 μM.