Novel chemically cross-linked self-molding particulate sorbents as solid-phase extraction media.

Here, we describe novel, chemically cross-linked, self-molding particulate polymer sorbents that are utilized as a molding-type solid-phase extraction medium (M-SPEM), which exhibits high permeability and rigidness. To fabricate such M-SPEM, first, polyethyleneimine (PEI)-modified reversed-phase (RP)-type particulate sorbents were synthesized, thereafter, they were chemically cross-linked by a polymer having many epoxy groups together with additional PEI. By optimizing the binding conditions of the particulate sorbents, the resultant M-SPEM has almost the same adsorption properties as the corresponding unmolded particulate sorbent for some polar (e.g., uracil and adenine) compounds. The binding technique proposed here is expected to facilitate the fabrication of molding-type sorbents and improve the performance of the SPE procedure.

Preparation and evaluation of molding-type solid-phase extraction media binding with commercially available adhesives.

A fabrication method of molding-type solid-phase extraction media (M-SPEM) bound with commercially available adhesive is presented. Six pieces of M-SPEM were prepared by heating each kneaded product of a particulate sorbent and an adhesive inserted into a six-hole cylindrical mold for hardening under an open system and normal pressure. The particulate sorbent contained in M-SPEM was divinylbenzene-based reversed-phase mode solid-phase extractants that we have reported. An examination of several adhesives showed that the moldability of M-SPEM depended on the composition and properties of the adhesive. The optimized procedure can be used to prepare an M-SPEM containing an 85 wt% particulate sorbent (particulate sorbent/adhesive, 100 mg/17 mg; particle diameter, 90-150 µm), and the M-SPEM has a specific surface area of about 500 m2/g. The established procedure in this study can bind particulate sorbents together, which showed almost no reductions in the adsorption property and liquid permeability compared with those of the particulate sorbent.

Development and Evaluation of HILIC-type Sorbents Modified with Hydrophilic Copolymers for Solid-phase Extraction.

Hydrophilic interaction chromatography (HILIC) has attractive attention for the separation of water-soluble compounds via HPLC. There are, however, few studies on the pretreatment of the HILIC-type solid-phase extraction (SPE) due to the difficulty of obtaining the HILIC-type sorbent. Therefore, the development of HILIC-type sorbents for SPE is essential. In this study, four different hydrophilic copolymers, namely diallylamine-maleic acid copolymer (DAM), diallylamine-acrylamide copolymer (DAA), allylamine-maleic acid copolymer (MAM), and partly methylcarbonylated allylamine acetate copolymer (MAC), were immobilized on glycidyl methacrylate (GMA)-base resin, and their adsorptive properties were evaluated. The results of the physical and adsorptive properties indicated that a balance between the water content of the water-enriched layer on sorbent and the amount of hydrophilic copolymer immobilized on the GMA-base resin was vital for the adsorption in HILIC-type sorbent for SPE.

Molding-type Solid-phase Extraction Media Glued with Commercially Available Adhesives.

The handling of a particulate sorbent for solid-phase extraction is often troublesome because it causes static clinging and scattering. To overcome this problem, a production method for a simple molding-type solid-phase extraction medium (M-SPEM) was developed in this study by using commercially available adhesives. The content of a particulate sorbent can increase to as much as 85 wt% in the M-SPEM. Because of the high content, the proposed M-SPEMs have a higher specific surface area than previous monolithic media.

Evaluation of the adsorption properties of nucleobase-modified sorbents for a solid-phase extraction of water-soluble compounds.

We developed hydrophilic interaction chromatography (HILIC)-type sorbents modified with nucleobases for solid phase extraction (SPE). The synthesized hydrophilic base resins were modified by each nucleobase (adenine, guanine, and cytosine). The measurement of the amount of water content indicated that each nucleobase-modified sorbent had a water layer. To evaluate the adsorption properties in the HILIC mode, we chose two nucleobases (uracil and adenine) and four nucleosides (uridine, adenosine, cytidine, guanosine) as water-soluble analytes, which were loaded into an SPE cartridge packed with the nucleobase-modified sorbent. Firstly, 95% acetonitrile (ACN) solutions were used in the process of conditioning and sample loading of the above polar analytes. High recoveries of the analytes were observed in each nucleobase-modified sorbent, and the Diol-type sorbent (no modification with any of the nucleobases) did not adsorb each water-soluble analyte. On the basis of this result, a 98% ACN solution was used during the process of conditioning and sample loading to decrease the concentration of water in the sample, which potentially inhibited the formation of hydrogen bonding between each analyte and the modified nucleobase. Considerable improvements of recoveries were observed in Adenine- and Cytosine-modified sorbents. These results were possibly attributed to the effective expression of hydrogen bonding by decreasing water concentration in the sample solution. Although a non-aqueous (100% ACN) sample solution can be expected to obtain higher recoveries compared with the 98% ACN solution, a decrease in recoveries was observed in Adenine-modified sorbent. From these results, the highest adsorption property was observed in Adenine-modified sorbent using 98% ACN as a sample condition, and the combination of this sample condition and sorbent is effective for high adsorption under HILIC condition. Moreover, we also revealed that a balance between the thickness of water layer and the modification amount of nucleobase is important for retention in the HILIC-type sorbent.

Effects of pendant-like hydrophilic monomers on the adsorption properties of reversed-phase-type sorbents for solid-phase extraction.

Solid-phase extraction (SPE) has been extensively employed as a pretreatment method. In SPE, reversed-phase-type sorbents have been widely applied for the pretreatment of environmental or biological samples. Hydrophilic-lipophilic balance (HLB)-type sorbents, constituting the copolymers used as reversed-phase-type sorbents, have been applied for various sample pretreatment methods. In HLB-type sorbents, the hydrophilic monomer contributes to the improved wettability of sorbents and increase of polar interactions. In this study, three pendant-like hydrophilic monomers, viz. N-vinylpyrrolidone (NVP), 4-acryloylmorpholine (AMO), and 4-vinyl-1,3-dioxolan-2-one (VDO), respectively, exhibiting different Log P values and possibly causing different polar interactions, were selected to improve the adsorption properties of polar compounds, and divinylbenzene (DVB)-based HLB-type sorbents containing each hydrophilic monomer were synthesized and examined. By the optimization of the molar ratio of DVB and the hydrophilic monomer (i.e. HLB), the inert diluent, and the degree of cross-linking, the developed sorbents exhibited higher recoveries for various polar compounds (viz. cytosine, uracil, cytidine, uridine, 2'-deoxycytidine, 2'-deoxyguanosine, adenine, thymidine, adenosine, and 2'-deoxyadenosine) compared to commercially available HLB-type sorbents.

Effects of hydrophilic monomers on sorptive properties of divinylbenzene-based reversed phase sorbents.

Solid phase extraction (SPE) has been extensively used as a pretreatment method. In SPE methods, commercially available reversed phase type sorbents, which consist of macroporus styrene-divinylbenzene or copolymers including divinylbenzene (DVB) and hydrophilic monomers, have been applied to a variety of samples. The later sorbents are called hydrophilic lipophilic balanced (HLB) type sorbents. Hydrophilic monomers in hydrophilic lipophilic balanced type sorbents contribute to the increase in retention of polar compounds, because hydrophilic monomers improve the wettability and increase the interaction with polar compounds as analytes. In this study, three different methacrylate monomers (ethylene glycol dimethacrylate (EGDMA), glycerol dimethacrylate (GDMA) and trimethylolpropane trimethacrylate (TMPTMA)), which are expected to improve the retention of polar compounds, were chosen, and DVB-based copolymetric sorbents including the three monomers were newly synthesized. Among them, the sorbents including GDMA or TMPTMA gave higher recoveries to polar compounds such as uridine and adenine than that including EGDMA. The optimization studies of hydrophilic lipophilic balance, inert diluent and the purity of DVB improved the sorptive abilities of the sorbents. The developed sorbents have higher recoveries for variety of polar compounds (cytosine, uracil, cytidine, uridine, 2'-deoxycytidine, 2'-deoxyguanosine, adenine, thymidine, adenosine and 2'-deoxyadenosine) than commercially available hydrophilic lipophilic balanced type sorbents, while the recoveries for theophylline were comparable between the proposed sorbents and the commercial sorbents.

Progress in a selective method for the determination of the acetaldehyde-derived DNA adducts by using HILIC-ESI-MS/MS.

Acetaldehyde (AA), which is present in tobacco smoke, automobile exhaust gases and alcohol beverage, is a mutagen and carcinogen. AA reacts with 2'-deoxyguanosine (dG) in DNA to form N2-ethyl-dG (EtdG) and cyclic, 1, N2-propano-dG (CPrdG), which are considered to have a critical role in carcinogenesis induced by AA. In this study, we have developed a highly sensitive method for the quantitation of the two AA-derived DNA adducts by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in which hydrophilic interaction chromatography (HILIC) employing mobile phases of high organic solvent concentration was selected to improve the ionization efficiency in the ESI process. Fourteen times and 11 times larger peak areas for EtdG and CPrdG, respectively, in HILIC-ESI-MS/MS were obtained compared with those in reversed phase (RP)-LC-ESI-MS/MS. Furthermore, 6.9 times (for EtdG) and 2.4 times (for CPrdG) larger peak areas were also obtained as additional enhancement by varying additive compounds in the HILIC mobile phases from ammonium acetate to ammonium bicarbonate. In total, the enhancements in detected MS signal intensities by exchanging from the RP-LC system to the HILIC system are 97 times for EtdG and 26 times for CPrdG, respectively. Three commercially available HILIC columns with different polar functional groups were examined and sufficient separation between normal 2'-deoxynucleosides and the AA-derived DNA adducts was achieved by a carbamoyl-bonded HILIC column. Finally, we applied the established method to quantify EtdG and CPrdG in the damaged calf thymus DNA.

Engineering an ATP-dependent D-Ala:D-Ala ligase for synthesizing amino acid amides from amino acids.

We successfully engineered a new enzyme that catalyzes the formation of D-Ala amide (D-AlaNH2) from D-Ala by modifying ATP-dependent D-Ala:D-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of D-Ala-D-Ala from two molecules of D-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second D-Ala of D-Ala-D-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for D-AlaNH2 production. The S293E variant, which was selected as the best enzyme for D-AlaNH2 production, exhibited an optimal activity at pH 9.0 and 40 °C for D-AlaNH2 production. The apparent K m values of this variant for D-Ala and NH3 were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of D-AlaNH2 from 10 and 50 mM D-Ala and 3 M NH4Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.

Biosynthetic pathway for the cyanide-free production of phenylacetonitrile in Escherichia coli by utilizing plant cytochrome P450 79A2 and bacterial aldoxime dehydratase.

The biosynthetic pathway for the production of phenylacetonitrile (PAN), which has a wide variety of uses in chemical and pharmaceutical industries, was constructed in Escherichia coli utilizing enzymes from the plant glucosinolate-biosynthetic and bacterial aldoxime-nitrile pathways. First, the single-step reaction to produce E,Z-phenylacetaldoxime (PAOx) from l-Phe was constructed in E. coli by introducing the genes encoding cytochrome P450 (CYP) 79A2 and CYP reductase from Arabidopsis thaliana, yielding the E,Z-PAOx-producing transformant. Second, this step was expanded to the production of PAN by further introducing the aldoxime dehydratase (Oxd) gene from Bacillus sp. strain OxB-1, yielding the PAN-producing transformant. The E,Z-PAOx-producing transformant also produced phenethyl alcohol and PAN as by-products, which were suggested to be the metabolites of E,Z-PAOx produced by E. coli enzymes, while the PAN-producing transformant accumulated only PAN in the culture broth, which suggested that the CYP79A2 reaction (the conversion of l-Phe to E,Z-PAOx) was a potential bottleneck in the PAN production pathway. Expression of active CYP79A2 and concentration of biomass were improved by the combination of the autoinduction method, coexpression of groE, encoding the heat shock protein GroEL/GroES, N-terminal truncation of CYP79A2, and optimization of the culture conditions, yielding a >60-fold concentration of E,Z-PAOx (up to 2.9 mM). The concentration of PAN was 4.9 mM under the optimized conditions. These achievements show the potential of this bioprocess to produce nitriles and nitrile derivatives in the absence of toxic chemicals.

Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism.

LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2 and VA) lacked this lag, and H2O2-LiP (H2O2-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr181, whereas H2O2-LiP contains a hydroxylated Tyr181. No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich π stacking with Tyr181 and Phe89. EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine-VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H2O2-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H2O2-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine-VA adduct strongly promotes (>100-fold) substrate oxidation by compound II, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.

Lignin-degrading peroxidases from genome of selective ligninolytic fungus Ceriporiopsis subvermispora.

The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.

Cloning, overexpression in Escherichia coli, and characterization of a thermostable fungal acetylxylan esterase from Talaromyces emersonii.

The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels.

Determination of a catalytic tyrosine in Trametes cervina lignin peroxidase with chemical modification techniques.

Trametes cervina lignin peroxidase (LiP) lacks a catalytic tryptophan strictly conserved in other LiP and versatile peroxidases. It contains tyrosine(181) at the potential catalytic site. This protein and the well-characterized Phanerochaete chrysosporium LiP with the catalytic tryptophan(171) have been chemically modified: the tryptophan-specific modification with N-bromosuccinimide sufficiently disrupted oxidation of veratryl alcohol by P. chrysosporium LiP, whereas the activity of T. cervina LiP was not affected, suggesting no catalytic tryptophan in T. cervina LiP. On the other hand, the tyrosine-specific modification with tetranitromethane did not affect the activities of P. chrysosporium LiP lacking tyrosine but inactivated T. cervina LiP due to the nitration of tyrosine(181). These results strongly suggest that tyrosine(181) is at the catalytic site in T. cervina LiP.

Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine.

Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiPs and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques. The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146, and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by the one-electron abstraction with formation of substrate cation radical, as described for the well known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical. The crystal structure reveals a partially exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: (i) by enabling substrate recognition by aromatic interactions; (ii) by acting as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and (iii) by providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine.

Molecular characterization of lignin peroxidase from the white-rot basidiomycete Trametes cervina: a novel fungal peroxidase.

The lignin peroxidase (LiP) from Trametes cervina was cloned, characterized, and identified as a novel fungal peroxidase. The sequence of T. cervina LiP encodes the essential amino acids for shaping the heme cavity and calcium-binding sites, which are conserved in plant and fungal peroxidases. However, a sequence homology analysis showed that T. cervina LiP has two unique features: it lacks the conserved tryptophan residue corresponding to the substrate-oxidation site (Trp171) of Phanerochaete chrysosporium LiP and it has a tyrosine residue (Tyr181) that has never been reported in other lignin peroxidases. A tertiary model of T. cervina LiP showed that Tyr181 sterically adjacent to the 6-propionate group of heme is surrounded by acidic amino acids and is exposed to the exterior. These attributes indicate that Tyr181 could be a T. cervina LiP substrate-oxidation site. A phylogenetic analysis showed that T. cervina LiP does not cluster with any other fungal peroxidases, suggesting that it is a unique molecule that is evolutionarily distant from other peroxidases. Thus, we concluded that T. cervina LiP could be a novel secreted peroxidase, among those produced by fungi, with a new oxidation mechanism probably involving Tyr181.

Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue.

Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation-including incorporation of heme and structural Ca2+ ions, and formation of disulfide bridges-were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H2O2 and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (k(cat)) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis-Menten constant (K(m)) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure-function relationships, and autocatalytic activation of the unique T. cervina LiP.

Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases.

Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn(2+), the manganese peroxidase (MnP) substrate (Mn(3+) being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these 'hybrid' properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn(2+) oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not beta-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.