Enzymatic synthesis of phenol polymer and its functionalization

In phosphate buffer (pH = 7.0) containing sodium dodecyl sulfate (SDS), an environmentally friendly system, enzymatic polymerization of phenol catalyzed by horseradish peroxidase (HRP) was efficiently performed. The obtained phenol polymer is partly soluble in common solvents, such as acetone, THF and DMF. IR analysis shows that the polymer is composed of phenylene and oxyphenylene units. The functionalization of the phenol polymer was performed by reacting with epoxy chloropropane and triethylene-tetramine following, and then insoluble aminated phenol polymer was obtained. The aminated phenol polymer was adopted as carrier to prepare a novel supported palladium catalyst (PP-N-Pd) for Heck reaction. PP-N-Pd shows high catalytic performance for Heck reactions of aryl iodides with acrylic acid or styrene and the yields of trans-products were higher than 90%. Under the optimized conditions, aryl bromides and activated aryl chloride also reacted with alkenes to give the yields of above 80%. XPS analysis indicates that the main coordination atom in PP-N-Pd is N and the chemical valence of palladium in PP-N-Pd is Pd²⁺. The novel supported catalyst also shows good recyclability for Heck reaction.     

Reactivity improvement of cellulolytic enzyme lignin via mild hydrothermal modification

Isolated by the cellulolytic enzyme lignin (CEL) process, water-alcohol (1:1, v/v) was introduced as co-solvent in the process of the hydrothermal treatment. The modification parameters such as reaction temperature and time, solid-to-liquid ratio, and catalysts (NaOH and NaOAlO₂) have been investigated in terms of the specific lignin properties, such as the phenolic hydroxyl content (OH_phen), DPPH free radical scavenging rate, and formaldehyde value. The CELs were also characterized by GPC, FT-IR and ¹ H NMR spectroscopy, and Py-GC/MS. The key data are under optimal lignin modification conditions (solid-to-liquid ratio of 1:10 (w/v) and a temperature of 250 °C for 60 min) are: OH_phen content: 2.50 mmol/g; half maximal inhibitory concentration (IC₅₀) towards DPPH free radicals: 88.2 mg/L; formaldehyde value: 446.9 g/kg). Both base catalysts decrease the residue rate, but phenol reactivities of the products were also detracted. Py-GC/MS results revealed that modified lignin had a higher phenolic composition than the CEL did, especially the modified lignin without catalyst (ML), which represented 74.51% phenolic content.     

Functional expression of Coprinus cinereus peroxidase in Pichia pastoris

A Coprinus cinereus peroxidase (CiP) was successfully expressed by the methylotrophic yeast Pichia pastoris. The 1095-bp gene encoding peroxidase from C. cinereus was cloned with a highly inducible alcohol oxidase (AOX1) promoter and integrated into the genome of P. pastoris. The recombinant CiP (rCiP) fused with the α-mating factor pre-pro leader sequence derived from Saccharomyces cerevisiae accumulated neither inside the cell nor within the wall, and were efficiently secreted into the culture medium. SDS-PAGE and immunoblot analysis revealed that the rCiP was not hyper-glycosylated and its α-factor signal sequence was correctly processed. It was also found that the kinetic properties of rCiP were similar to those of native CiP. In order to produce large amounts of rCiP, the high cell density cultivation of recombinant P. pastoris was carried out in a fermentor with fed-batch mode. The peroxidase activity obtained in a 5 l fermentor cultivation became about 6 times (1200 U/ml) higher than that in shake-flask cultures (200 U/ml).     

Bioremediation of phenol-contaminated water and soil using magnetic polymer beads

In order to easily separate pollutant-absorbing polymer beads from contaminated soil or water, novel polymer beads containing magnetic particles were developed. The polymer beads containing 4.67% (w/w) magnetic particles exhibited an almost identical partitioning coefficient for phenol compared to that of the pure polymer. A 1.5 L phenol solution of 2000 mg/L added to a bioreactor was reduced to 481 mg/L phenol within 3 h by adding 100 g of these magnetic beads, and the phenol was completely degraded by microorganisms in 16 h. The magnetized beads were then readily removed from the bioreactor by a magnet with 10,000 G, and subsequently detached for re-use. 500 g of soil contaminated with 4 mg-phenol/g-soil was also contacted with 100 g beads, and greater than 97% removal of phenol from the soil was achieved within 1 day. The phenol-absorbing beads were easily separated from the soil by the magnet and transferred into a fermentor. The phenol was released from the beads and was degraded by the microorganism in 10 h. Modifying polymers to possess magnetic properties has greatly improved the ease of handling of these sequestering materials when decontaminating soil and water sources, in conjunction with contaminant release in partitioning bioreactors.     

Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water

A non-modified and modified with NaOH and ethylenediamine ultrafiltration membranes prepared from AN copolymer have been used as carriers for the immobilization of horseradish peroxidase (HRP) enzyme. The amount of bound protein onto the membranes and the activity of the immobilized enzyme have been investigated as well as the pH and thermal optimum, and the thermal stability of the free and immobilized HRP. The experiments have proved that the modified membrane is a better support for the immobilization of HRP enzyme. The latter has shown a greater thermal stability than the free enzyme.A possible application has been studied for reducing phenol concentration in water solutions through oxidation of phenol by hydrogen peroxide, in the presence of free and immobilized HRP enzyme on modified AN copolymer membranes. A higher degree of the phenol oxidation has been observed in the presence of the immobilized enzyme. A total removal of phenol has been achieved in the presence of immobilized HRP at concentration of the hydrogen peroxide 0.5 mmol L^?1 and concentration of the phenol in the model solutions within the interval 5–40 mg L^?1. A high degree of phenol oxidation (95.4%) has been achieved in phenol solution with 100 mg L^?1 concentration in the presence of hydrogen peroxide and immobilized HRP, which demonstrates the promising opportunity of using the enzyme for bioremediation of waste waters, containing phenol.The immobilized HRP has shown good operational stability. Deactivation of the immobilized enzyme to 50% of the initial activity has been observed after the 20th day of the enzyme operation.     

Screen printed graphite biosensors based on bacterial cells

A microbial biosensor was developed for the determination of phenolic compounds and the measurement was based on oxygen consumption in relation to analyte oxidation. Induced cells of Pseudomonas putida DSM 50026 were immobilised on the surface of SPG electrodes covered with cellulose acetate membrane by means of gelatine which was then cross linked with glutaraldehyde. The systems were calibrated for different phenolic substances. Detection ranges were 0.1–1.0 μM for phenol and 0.05–1.0 μM for l-tyrosine and l-DOPA, respectively, with a response time of 3 min. Furthermore, phenol detection was performed in the presence of synthetic wastewater samples.     

An aromatic amino acid within intracellular loop 2 of the prostaglandin EP2 receptor is a prerequisite for selective association and activation of Gαs

We previously demonstrated that the aromatic moiety of Tyr¹⁴³ within the intracellular loop 2 (ICL2) region of the prostaglandin EP2 receptor plays a crucial role in Gs coupling. Here we investigated whether the ICL2 of the EP2 receptor directly binds to Gαs and whether an aromatic moiety affects this interaction. In Chinese hamster ovary cells, mutations of Tyr¹⁴³ reduced the ability of the EP2 receptor to interact with G proteins as demonstrated by GTPγS sensitivity, as well as the ability of agonist-induced cAMP formation, with the rank order of Phe > Tyr (wild-type) = Trp > Leu > Ala (= 0). We found that the wild-type ICL2 peptide (i2Y) and its mutant with Phe at Tyr¹⁴³ (i2F) inhibited receptor-G protein complex formation of wild-type EP2 in membranes, whereas the Ala-substituted mutant (i2A) did not. Specific interactions between these peptides and the Gαs protein were detected by surface plasmon resonance, but Gαs showed different association rates, with a rank order of i2F > i2Y ≫ i2A, with similar dissociation rates. Moreover, i2F and i2Y, but not i2A activated membrane adenylyl cyclase. These results indicate that the ICL2 region of the EP2 receptor is its potential interaction site with Gαs, and that the aromatic side chain moiety at position 143 is a determinant for the accessibility of the ICL2 to the Gαs protein.     

Peroxidase-catalyzed copolymerization of syringaldehyde and bisphenol A

Syringaldehyde, one of the major derivatives of lignin, was copolymerized with bisphenol A via a CiP (Coprinus cinereus peroxidase)-catalyzed reaction. Although syringaldehyde was not polymerized to a solid polymer, the copolymer with bisphenol A was obtained as a dark brown powdery precipitate. The relatively hydrophobic solvent, 2-propanol, gave a better yield (yield = 95%) than hydrophilic solvents, such as methanol, ethanol or acetone. Characteristic signals corresponding to the aldehyde group of syringaldehyde in the copolymer were detected in the FT-IR and ¹³C NMR spectrum. The ratio of syringaldehyde incorporated into the copolymer was estimated by measuring the amount of monomers consumed (syringaldehyde and bisphenol A), which proportionally increased up to 80 mol% on increasing the initial ratio of syringaldehyde to bisphenol A. TGA (thermogravimetric analysis) showed that the thermally crosslinked copolymer (syringaldehyde:bisphenol A = 1:1, w/w) had a much higher thermal resistance to thermal degradation than poly(bisphenol A); 36% residue still remained under a nitrogen atmosphere, even over 800 °C. This implies that the copolymer of syringaldehyde and bisphenol A could be a new thermally stable material originating from renewable resources.     

Engineering a high-performance,metagenomic-derived novel xylanase with improved soluble protein yield and thermostability

The novel termite gut metagenomic-derived GH11 xylanase gene xyl7 was expressed in Escherichia coli BL21, and the purified XYL7 enzyme exhibited high specific activity (6340 U/mg) and broad pH active range of 5.5–10.0. Directed evolution was employed to enhance the thermostability of XYL7; two mutants (XYL7-TC and XYL7-TS) showed a 250-fold increase in half-life at 55 °C, with a 10 °C increase in optimal temperature compared to that of wild-type XYL7. A truncated enzyme (XYL7-Tr3) acquired by protein engineering showed similar catalytic properties as the wild-type, with a tenfold increase in soluble protein yield by the mutant. The reducing sugar produced by XYL7-TC was about fourfold greater than that produced by their parents when incubated with xylan at 60 °C for 4 h. The engineered novel xylanase exhibited superior enzymatic performance and showed promise as an excellent candidate for industrial application due to its high specific activity, stability and soluble protein yield.     

Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme

The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28 °C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05 M NaClO₄. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30 μmol g⁻¹ polymer. The adsorption capacity of lysozyme on the polymer was 3.4 mg g⁻¹polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5 M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.     

Rapid linkage of indole carboxylic acid to the plant cell wall identified as a component of basal defence in Arabidopsis against hrp mutant bacteria

Changes occurring to plant cell walls were examined following inoculation of Arabidopsis leaves with pathogenic and non-pathogenic (hrpA mutant) strains of Pseudomonas syringae pv. tomato. We have targeted low molecular weight, cross-linked phenolic and indolic compounds that were released from wall preparations by alkaline hydrolysis at 70 °C and in a microwave bomb. Significantly higher concentrations of syringaldehyde, p hydroxybenzaldehyde and indole carboxylic acid were recovered from cell walls isolated from leaves 24 h after challenge with the hrpA mutant compared with wild-type DC3000. Time course experiments showed that the accumulation of indole carboxylic acid and the other group of differentiating metabolites had occurred within 12 h of inoculation. The callose synthase deficient mutant pmr4-1 was more resistant than wild-type Columbia plants to P. syringae pv. tomato. Restricted bacterial multiplication was associated with increased accumulation of indole carboxylic acid on the plant cell wall. In the absence of callose deposition in the pmr 4-1 mutant, indolic derivatives may serve as a structural scaffold for wall modifications following bacterial challenge.     

Optimized synthesis of (Z)-3-hexen-1-yl caproate using germinated rapeseed lipase in organic solvent

(Z)-3-hexen-1-yl esters are important green top-note components of food flavors and fragrances. Effects of various process conditions on (Z)-3-hexen-1-yl caproate synthesis employing germinated rapeseed lipase acetone powder in organic solvent were investigated. Rapeseed lipase catalyzed ester formation more efficiently with non-polar compared to polar solvents despite high enzyme stability in both types of solvents. Maximum ester yield (90%) was obtained when 0.125 M (Z)-3-hexen-1-ol and caproic acid were reacted at 25 °C for 48 h in the presence of 50 g/L enzyme in heptane. Enzyme showed little sensitivity towards a_w with optimum yield at 0.45, while added water did not affect ester yield. Esterification reduced by increasing molecular sieves (>0.0125%, w/v). The highest yields of caproic acid were obtained with isoamyl alcohol (93%) followed by butanol and (Z)-3-hexen-1-o1 (88%) respectively reflecting the enzyme specificity for straight and branched chain alcohols. Secondary alcohols showed low reactivity, while tertiary alcohol had either very low reactivity or not esterified at all. A good relationship has been found between ester synthesis and the solvent polarity (log P value); while no correlation for the effect of solvents on residual enzyme activity was observed. It may be concluded that germinated rapeseed lipase is a promising biocatalyst for the synthesis of valuable green flavor note compound. The enzyme also showed a wide range of temperature stability (5–50 °C).     

Substitution of the catalytic acid–base Glu237 by Gln suppresses hydrolysis during glucosylation of phenolic acceptors catalyzed by Leuconostoc mesenteroides sucrose phosphorylase

Stereoselective glycosylation of a phenolic hydroxyl is a key transformation in the (bio)synthesis of natural products. Biocatalytic transglycosylation usually provides the desired glycosidic product in exquisite anomeric purity. However, loss of substrate and product to hydrolysis often limits application of the method. Kinetic studies and in situ proton NMR analysis of reaction time courses were used here to characterize glucosylation of substituted phenol acceptors by Leuconostoc mesenteroides sucrose phosphorylase in the presence of α-d-glucose 1-phosphate (αG1P) as donor substrate. In the wild-type enzyme, hydrolysis of the sugar 1-phosphate strongly prevailed (about 10-fold, ∼1.6 U/mg) over glucosyl transfer to the 2,6-difluorophenol acceptor (∼0.17 U/mg) used. A mutated phosphorylase in which the catalytic acid–base Glu²³⁷ had been replaced by Gln (E237Q) did not display hydrolase activity under transglucosylation conditions and therefore provided substantial (∼7-fold) enhancement of transfer yield. Utilization of the donor substrate was however slowed down (about 400-fold, ∼0.004 U/mg) in E237Q as compared to wild-type enzyme (∼1.6 U/mg). In a series of mono- and disubstituted phenols differing in hydroxyl pK_a between 7.02 and 8.71, the transferase activity of E237Q was found to be dependent on steric rather than electronic properties of the acceptor used. Both wild-type and mutated enzyme employed 4-nitrophenyl-α-d-glucopyranoside (4-NPG) as a slow artificial substrate for phosphorolysis and hydrolysis (native: ∼0.12 U/mg; E237Q: ∼0.02 U/mg).     

Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression,purification and enzymatic characterization in Pichia pastoris

Xylanase is an important industrial enzyme. In this research, to improve the thermostability and biochemical properties of a xylanase from Aspergillus niger F19, five arginine substitutions and a disulfide bond were introduced by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were expressed in Pichia pastoris. Compare to those of the wild-type enzyme, the optimal reaction temperature for the mutant enzyme increased from 45 °C to 50 °C, the half-life of the mutant enzyme extended from 10 min to 180 min, and the specific activity increased from 2127 U/mg to 3330 U/mg. However, the V_max and K_m of the mutant xylanase decreased. The enzyme activity in broth obtained from shake flask cultures could be induced to 1850 U/mL in 7 days, which is higher than results reported previously. Furthermore, the highest achievable enzyme activity was 7340 U/mL from 140 g/L of biomass in a 3 L fermentor used in our study.     

Molecular analysis of the bacterial community in digestive tract of rabbit

This work aimed to study the stability over time of the bacterial community in cæcum and fæces of the rabbit (diversity index and structure) without experimental disturbance and to evaluate its relationships with environmental parameters. Soft and hard fæces of 14 rabbits were sampled for 5 weeks while cæcal content was sampled on the 3rd week (by surgery) and the 5th week (at slaughter). Bacterial communities were assessed by studying CE-SSCP profiles of 16S rRNA genes fragments. Redox potential, pH, NH3-N concentration and volatile fatty acid concentrations were measured in the cæcum. Data showed that bacterial communities of soft and hard fæces barely differed from that of the cæcum (ANOSIM-R < 0.25; p < 0.05). Without disturbance, the bacterial communities of fæces were stable over time (ANOSIM-R < 0.25; p < 0.001). However, the bacterial communities of cæcum and fæces were affected by the surgery (ANOSIM-R = 0.22–0.33; p < 0.001). The cæcal content was an acidic (pH = 6.03 ± 0.33) and an anaerobic environment (redox potential = ?160 ± 43 mV). Only the redox potential was correlated with the diversity index of the bacterial community of the cæcum (R² = 0.35; p < 0.05) and no environmental parameters were correlated to its structure.     

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: Comparison of wild-type protein and active-site mutant D27E

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl₂ concentration-dependence of the ¹H–¹⁵N HSQC spectra of the wild-type DHFR–folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased K_m and K_d values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250 MPa together with negative activation volumes of ? 4.0 or ? 4.8 mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7 mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.     

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10⁻⁵) was roughly similar to that of Pfu DNA polymerase (4.8 × 10⁻⁵), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10⁻⁵), Neq A523R DNA polymerase (13.1 × 10⁻⁵), and Neq N540R DNA polymerase (37.7 × 10⁻⁵). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.     

The study on microwave assisted enzymatic digestion of ginkgo protein

Microwave-assisted enzymatic digestion (MAED) technique was applied for ginkgo protein digestion with both free and immobilized enzyme. Under the optimized conditions of MAED (0.01 g/mL substrate concentration of bromelain, 4500 U/g enzyme/ginkgo protein, 30 min, 300 W microwave power), a higher digestion rate (7.50%) and a significant increase in antioxidant activity (72.7 mg/g) were obtained in contrast with the conventional methods. With the optimized digestion conditions (0.625% glutaraldehyde (v/v), 0.4 mg/mL initial concentration of bromelain and 4 h of immobilization), the activity and effectiveness factor of immobilized bromelain were respectively 86 U and 81.6%. The results of ginkgo digestion by applying MAED indicated that the digestion rate of immobilized bromelain obtained by MAED method (6.41%) was comparable to that of free bromelain in the conventional digestion (8.13%). In both case with immobilized and free bromelain while applying MAED, a homogeneously abundant distribution of peptide fragments (from 7.863 Da to 5856 Da) and a few different peptide profiles were found. This report brings in conclusion that applying MAED with immobilized enzyme has the potential to obtain the highest number of antioxidant activity peptides.     

Phenolic nature,occurrence and polymerization degree as marker of environmental adaptation in the edible halophyte Mesembryanthemum edule

Mesembryanthemum edule is an edible medicinal halophyte traditionally used to treat several human diseases. In this study, particular importance was attached to the influence of environmental conditions on phenolic composition and antioxidant activities of two M. edule provenances from contrasting climatic regions (Djerba and Monastir sampled from arid and superior semi-arid bioclimatic stages, respectively). Shoot phenolic content was evaluated using colorimetric method and its composition was identified by HPLC analysis with or without thiolysis. Antioxidant activities were assessed by five in vitro antioxidant systems. Results showed that the two M. edule provenances were significantly different according to their antioxidant activity as well as their polyphenol profiles. Indeed, plants from Djerba (lack of rainfall and long light hour periods) exhibited stronger antioxidant activity together with higher phenolic content. For instance, Djerba provenance shoots showed much lower IC₅₀ (4.8 μg ml⁻¹) and EC₅₀ (80 μg ml^− 1) values for DPPH and Fe-reducing tests, respectively. In addition, the superiority of this provenance (Djerba) was more marked as compared to positive controls (BHT, BHA, and VitC). HPLC identification revealed also an important difference between the two provenances on major flavonoid components. This difference was confirmed by the mean degrees of tannin polymerization (DPn) which was higher in Djerba plants. These data suggest that M. edule adaptation to environmental stresses proceeds through induced particular phenol quality and DPn for the improvement of their antioxidant capacities to protect plant tissues against oxidative stress.     

Dephenolisation of olive mill wastewater using adapted Trametes versicolor

Olive mill wastewater (OMW) is an effluent of the olive oil extraction process. The large volumes involved, along with the high phenolic content and chemical oxygen demand, cause major environmental problems. The presence of phenolics limits the effectiveness of aerobic or anaerobic treatment of this wastewater. In most of the studies performed on OMW, the concentration of phenolics is reduced by diluting the OMW prior to biological treatment, which leads to an increase in waste volume. Therefore, the aim of this work was to investigate the possibility of reducing the phenolic content without dilution and without any addition of nutrients or pretreatment by using the white-rot fungi Trametes versicolor FPRL 28A INI. Through an adaptation process, the fungus was able to remove 78% of total phenolics in shake flask experiments and 39% in static culture using undiluted OMW medium. In continuously stirred tank reactor (CSTR) conditions, 70% of total phenolics removal was achieved. Analysis with GC–MS showed that all simple phenolics disappeared from the medium after the 8th day of cultivation at an 0.25 vvm aeration rate. The maximum activities of phenol degrading enzymes laccase and manganese peroxidase (MnP) obtained under these conditions were 762.14 ± 42.11 and 97.80 ± 8.11 U l^?1 respectively.