Main Hydrolysis Products Research Articles

Molecular cloning, expression, and functional characterization of the \u03b2-agarase AgaB-4 from Paenibacillus agarexedens

In this study, a β-agarase gene, agaB-4, was isolated for the first time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883-amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purified from the soluble fraction of E. coli cell lysate through immobilized metal ion affinity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specificity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confirmed to be neoagarotetraose. Purified rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degradation for the production of neoagarotetraose.

Biochemical Characterization of a Novel GH86 β-Agarase Producing Neoagarohexaose from Gayadomonas joobiniege G7.

A novel β-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be 30°C and 4.5, respectively. AgaJ5 was an acidic β-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at 10°C. AgaJ5 required monovalent ions such as Na+ and K+ for its maximum activity, but its activity was severely inhibited by several metal ions. The Km and Vmax of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type β-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing β-agarase having specific biochemical features that may be useful for industrial applications.

Purification and Characterization of a Biofilm-Degradable Dextranase from a Marine Bacterium.

This study evaluated the ability of a dextranase from a marine bacterium Catenovulum sp. (Cadex) to impede formation of Streptococcus mutans biofilms, a primary pathogen of dental caries, one of the most common human infectious diseases. Cadex was purified 29.6-fold and had a specific activity of 2309 U/mg protein and molecular weight of 75 kDa. Cadex showed maximum activity at pH 8.0 and 40 °C and was stable at temperatures under 30 °C and at pH ranging from 5.0 to 11.0. A metal ion and chemical dependency study showed that Mn2+ and Sr2+ exerted positive effects on Cadex, whereas Cu2+, Fe3+, Zn2+, Cd2+, Ni2+, and Co2+ functioned as inhibitors. Several teeth rinsing product reagents, including carboxybenzene, ethanol, sodium fluoride, and xylitol were found to have no effects on Cadex activity. A substrate specificity study showed that Cadex specifically cleaved the α-1,6 glycosidic bond. Thin layer chromatogram and high-performance liquid chromatography indicated that the main hydrolysis products were isomaltoogligosaccharides. Crystal violet staining and scanning electron microscopy showed that Cadex impeded the formation of S. mutans biofilm to some extent. In conclusion, Cadex from a marine bacterium was shown to be an alkaline and cold-adapted endo-type dextranase suitable for development of a novel marine agent for the treatment of dental caries.

Voltammetric detection of carbofuran determination using screen-printed carbon electrodes modified with gold nanoparticles and graphene oxide

Carbofuran is a highly toxic pesticide that is heavily used in agriculture due to its high effectiveness and low cost. Improved methods that are simpler and lower cost are needed for carbofuran detection in food and agricultural samples. Herein, we describe the development of a unique electrochemical method for carbofuran-phenol, which is the main hydrolysis product of carbofuran. We have successfully developed a highly accurate and precise method in a portable size using a screen-printed carbon electrode (SPCE) that is modified with graphene oxide (GO) and gold nanoparticles (AuNPs). Consequently, the developed electrode is highly sensitive to and selective for carbofuran. Using the central composite design (CCD) approach, we optimized the method for analysis parameters including the electrode surface loadings of GO and AuNPs as well as the working solution pH. The method exhibited a wide linear range of 1–250µM for analyte detection using differential pulse voltammetry (DPV) on AuNPs/GO-SPCE under the optimized conditions. The limits of detection and quantitation were 0.22 and 0.72µM, respectively. In addition, we also report the application of the method for carbofuran determination in real cucumber and rice samples. This sensitive and selective carbofuran detection method is very promising for simple and low cost analysis in real agricultural fields.

Sugars and char formation on subcritical water hydrolysis of sugarcane straw

Subcritical water has potential as an environmentally friendly solvent for applications including hydrolysis, liquefaction, extraction, and carbonization. Here, we report hydrolysis of sugarcane straw, an abundant byproduct of sugar production, in a semi-continuous reactor at reaction temperatures ranging from 190 to 260°C and at operating pressures of 9 and 16MPa. The target hydrolysis products were total reducing sugars. The main products of sugarcane straw hydrolysis were glucose, xylose, arabinose, and galactose in addition to 5- hydroxymethylfurfural and furfural as minor byproducts. Fourier transform infrared spectroscopy and thermogravimetric analysis provided additional information on the surface and bulk composition of the residual biomass. Char was present on samples treated at temperatures equal to and greater than 190°C. Samples treated at 260°C contained approximately 20wt% char, yet retained substantial hemicellulose and cellulose content. Hydrolysis temperature of 200°C provided the greatest TRS yield while minimizing char formation.

Secretory expression of β-mannanase from Bacillus circulans NT 6.7 in Lactobacillus plantarum

The β-mannanase gene of Bacillus circulans NT 6.7 was successfully cloned in Lactobacillus plantarum WCFS1 using the pSIP403 expression vector and secreted to the supernatant rather than accumulated in the cells. The highest activity was achieved by controlling the pH at 6 during cultivation. Maximum mannanase activities detected in the supernatant and cell-free extract of 200 ml MRS broth were 8.2 and 0.86 U/ml, respectively. Enzyme activity in the supernatant increased to 27 U/ml by fermentation in a 5-L bioreactor with automatic pH control. The optimum temperature of recombinant β-mannanase was 50 °C and stable between 30 and 50 °C. The optimum pH was 6 with stability in the range 5–7. Enzyme activity slightly increased with Co2+ but was strongly inhibited by EDTA. The enzyme exhibited high specificity to galactomannan substrates. The main products of copra meal and locust bean gum hydrolysis were manno-oligosaccharides. Therefore, recombinant β-mannanase produced from a food grade host, L. plantarum WCFS1, showed potential for use in manno-oligosaccharides production and other food-related applications.

Isothiocyanates, Nitriles, and Epithionitriles from Glucosinolates Are Affected by Genotype and Developmental Stage in Brassica oleracea Varieties.

Vegetables of the Brassica oleracea group, such as broccoli, cauliflower, and cabbage, play an important role for glucosinolate consumption in the human diet. Upon maceration of the vegetable tissue, glucosinolates are degraded enzymatically to form volatile isothiocyanates, nitriles, and epithionitriles. However, only the uptake of isothiocyanates is linked to the cancer-preventive effects. Thus, it is of great interest to evaluate especially the isothiocyanate formation. Here, we studied the formation of glucosinolates and their respective hydrolysis products in sprouts and fully developed vegetable heads of different genotypes of the five B. oleracea varieties: broccoli, cauliflower as well as white, red, and savoy cabbages. Further, the effect of ontogeny (developmental stages) during the head development on the formation of glucosinolates and their respective hydrolysis products was evaluated at three different developmental stages (mini, fully developed, and over-mature head). Broccoli and red cabbage were mainly rich in 4-(methylsulfinyl)butyl glucosinolate (glucoraphanin), whereas cauliflower, savoy cabbage and white cabbage contained mainly 2-propenyl (sinigrin) and 3-(methylsulfinyl)propyl glucosinolate (glucoiberin). Upon hydrolysis, epithionitriles or nitriles were often observed to be the main hydrolysis products, with 1-cyano-2,3-epithiopropane being most abundant with up to 5.7 μmol/g fresh weight in white cabbage sprouts. Notably, sprouts often contained more than 10 times more glucosinolates or their hydrolysis products compared to fully developed vegetables. Moreover, during head development, both glucosinolate concentrations as well as hydrolysis product concentrations changed and mini heads contained the highest isothiocyanate concentrations. Thus, from a cancer-preventive point of view, consumption of mini heads of the B. oleracea varieties is recommended.

Microwave-enhanced sub-critical hydrolysis of rice straw to produce reducing sugar catalyzed by ionic liquid

The grain industry produces a great amount of rice straw, one of agricultural wastes. Because cellulose and hemicelluloses are rich in straw, it is also a main renewable biomass resource which can be converted to ethanol fuel. An effective and profitable process at relatively mild temperature under microwave heating with green solvent of ionic liquid ([Amim]Cl) as catalyst to produce reducing sugars from the hydrolysis of straw has both economical and environmental benefit. When the straw was hydrolyzed at relatively mild temperatures ranging from 140 to 220 °C and autogenous pressure by the intensification of microwave heating, the main hydrolysis products were determined to be glucose and xylose. With weight ratio of water/straw 40, weight content of ionic liquid (IL) in IL–water mixture 10%, temperature 180 °C and reaction time 60 min, the yield of glucose and xylose can attain 29.1 and 24.3%, respectively. The new method can not only obtain high yield of reducing sugar owing to their low degradation, but also remain high efficiency with microwave heating intensification and IL [Amin]Cl as catalyst.

Kinetics and Mechanism of Degradation of Aqueous Promecarb Insecticide Studied by UV Spectrophotometry and HPLC

The present paper deals with the degradation mechanism of the insecticide promecarb, or 3-isopropyl-5-methylphenyl- N-methylcarbamate, in aqueous media. The reaction kinetics have been investigated using UV spectrophotometry and reversed phase HPLC. The determination of 3-isopropyl-5-methylphenol as the main product of promecarb hydrolysis gives evidence for the significant reactivity of this insecticide in alkaline solution. The rate constants were determined following a proposed first-order kinetic model. The positive activation entropy obtained Δ S≠= +66.82 J mol−1K−1and the absence of general basic catalysis indicates an E1cB mechanism involving unimolecular collapse of the promecarb via a methylisocyanate intermediate. These results were confirmed by the fact that promecarb fits well into Brønsted and Hammett plots obtained for a series of substituted N-methylcarbamates for which the decomposition in aqueous media was established to follow an E1cB mechanism.

Metabolic profiling of glucosinolates and their hydrolysis products in a germplasm collection of Brassica rapa turnips

About 10% of the world's vegetable production is generated from Brassicaceae, wherein Brassica rapa is a dominating species. There is growing evidence that glucosinolates (GLSs), main plant secondary metabolites in Brassicales, play an important role in promoting human health. Natural genetic diversity of B. rapa can be explored for vegetable improvement. We analyzed leaves and tubers of 16 B. rapa turnips for their GLS composition by UHPLC-DAD and the corresponding hydrolysis products by GC–MS. Thirteen GLSs were identified, 8 aliphatic, 4 indolic and one aromatic. 3-Butenyl GLS was prevailing in both plant organs while in tubers 2-hydroxy-3-butenyl GLS and 2-phenylethyl GLS occurred in high amounts. A total of 24 GLS breakdown products were detected in tubers and 16 in leaves. Epithionitriles were the main hydrolysis products in both plant organs with 4,5-epithiopentanenitrile and 3-hydroxy-4,5-epithiopentanenitrile being the main compounds. When comparing leaves and tubers, an accumulation of GLSs and their breakdown products was observed in tubers compared to leaves. Our analysis achieved the comprehensive profiling of all GLS metabolites in a collection of B. rapa turnips, underlining the natural variation not only of intact GLS, but also of their breakdown products.

Purification and characterization of alkaline chitinase from Paenibacillus pasadenensis CS0611

An extracellular chitinase produced by Paenibacillus pasadenensis CS0611 was purified by ammonium sulfate precipitation, HiTrap DEAE FF and HiLoad 26/600 Superdex 200pg column chromatography. The apparent molecular mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 69 kDa. The optimum pH and optimum temperature of the chitinase were 5.0 and 50 °C, respectively. The enzyme showed high stability at alkaline pH values and temperatures below 40 °C. Additionally, the metal ions Mn2+, Mg2+, and Co2+ inhibited activity of the chitinase. The chitinase was active on colloidal chitin with an apparent Km of 4.41 mg/mL and Vmax of 1.08 mg/min. Substrate spectrum analysis indicated that the chitinase reacted preferentially with the glucosidic bond between GlcNAc-GlcNAc. The enzymatic hydrolysate was analyzed by high-performance liquid chromatography and thin layer chromatography, and clearly showed that a subunit of (GlcNAc)2 was the main hydrolysis product.

Graphene oxide/MnO2 nanocomposite as destructive adsorbent of nerve-agent simulants in aqueous media

Graphene oxide/MnO2 nanocomposite was prepared by thermal hydrolysis of potassium permanganate (KMnO4) and 2-chloroacetamide aqueous solutions with graphene oxide (GO) suspension. The synthesized samples were characterized by specific surface area (BET) and porosity determination (BJH), X-ray Diffraction (XRD) and high-resolution electron microscopes (HRSEM, HRTEM). These nanocomposites were used in an experimental evaluation of their adsorption activity with nerve agent simulants dimethyl methyl phosphonate (DMMP) and triethyl phosphate (TEP) in aqueous media. The nanocomposites exhibited enhanced adsorptive degradation ability compared to pure manganese oxide (MnO2) and GO. The GO amount in the nanocomposites affected their degradation activity substantially. The best adsorption efficiency was observed for samples with moderate GO amount. Three methods were used to observe the mechanism of the nerve-agent simulants deactivation: Gas chromatography with mass spectrometry (GC–MS), High-Performance Liquid Chromatography (HPLC) and in situ Infrared spectroscopy (FTIR). It was shown that the hydrolysis on the surface of prepared nanocomposites yields volatile primary alcohols (methanol and ethanol) as the main hydrolysis products.

Hydrolysis of Microcrystalline Cellulose to Produce Fermentable Monosaccharides by Plasma Acid

ABSTRACTPlasma acid was obtained by treating distilled water with a dielectric barrier discharge (DBD) at atmospheric pressure. The tentative relationship between discharge conditions and pH value of the plasma acid was investigated. In order to optimize the hydrolysis conditions of microcrystalline cellulose with the plasma acid, an orthogonal experiment was carried out and the colorimetric determination of 3,5-dinitro-salicylic acid was applied to measure the concentration of total reduced sugar (TRS). The results showed that the pH value of the plasma acid was related to the discharge time. The acidity of the plasma acid was maintained for several hours and then faded gradually. The microcrystalline cellulose was hydrolyzed effectively by the plasma acid and the optimal hydrolysis conditions were as follows: pH 1.42 of the plasma acid, hydrolysis temperature of 80°C and hydrolysis time of 60 min. Under these conditions, the microcrystalline cellulose with polymerization degree of 200–300 was hydrolyzed completely to produce monosaccharides, including xylose and glucose with the mole ratio of 1:35, as shown by high-performance liquid chromatography (HPLC) analysis. Moreover, the hydrolysis of luffa cellulose with polymerization degree of 500–600 was also carried out. The luffa cellulose was hydrolyzed completely to produce monosaccharides including xylose, mannose and glucose with mole ratio of 6.7:1:218. Therefore, it could be concluded that the main hydrolysis product of both types of cellulose was glucose. The glucose yield of microcrystalline cellulose was 46%, whereas for luffa cellulose it was 41%. This method was an environmentally friendly and effective method to hydrolyze cellulose.

Functional expression of a novel \u03b1-amylase from Antarctic psychrotolerant fungus for baking industry and its magnetic immobilization

Backgroundα-Amylase plays a pivotal role in a broad range of industrial processes. To meet increasing demands of biocatalytic tasks, considerable efforts have been made to isolate enzymes produced by extremophiles. However, the relevant data of α-amylases from cold-adapted fungi are still insufficient. In addition, bread quality presents a particular interest due to its high consummation. Thus developing amylases to improve textural properties could combine health benefits with good sensory properties. Furthermore, iron oxide nanoparticles provide an economical and convenient method for separation of biomacromolecules. In order to maximize the catalytic efficiency of α-amylase and support further applications, a comprehensive characterization of magnetic immobilization of α-amylase is crucial and needed.ResultsA novel α-amylase (AmyA1) containing an open reading frame of 1482 bp was cloned from Antarctic psychrotolerant fungus G. pannorum and then expressed in the newly constructed Aspergillus oryzae system. The purified recombinant AmyA1 was approximate 52 kDa. AmyA1 was optimally active at pH 5.0 and 40 °C, and retained over 20% of maximal activity at 0–20 °C. The Km and Vmax values toward soluble starch were 2.51 mg/mL and 8.24 × 10−2 mg/(mL min) respectively, with specific activity of 12.8 × 103 U/mg. AmyA1 presented broad substrate specificity, and the main hydrolysis products were glucose, maltose, and maltotetraose. The influence of AmyA1 on the quality of bread was further investigated. The application study shows a 26% increase in specific volume, 14.5% increase in cohesiveness and 14.1% decrease in gumminess in comparison with the control. AmyA1 was immobilized on magnetic nanoparticles and characterized. The immobilized enzyme showed improved thermostability and enhanced pH tolerance under neutral conditions. Also, magnetically immobilized AmyA1 can be easily recovered and reused for maximum utilization.ConclusionsA novel α-amylase (AmyA1) from Antarctic psychrotolerant fungus was cloned, heterologous expression in Aspergillus oryzae, and characterized. The detailed report of the enzymatic properties of AmyA1 gives new insights into fungal cold-adapted amylase. Application study showed potential value of AmyA1 in the food and starch fields. In addition, AmyA1 was immobilized on magnetic nanoparticles and characterized. The improved stability and longer service life of AmyA1 could potentially benefit industrial applications.

Purification and Biochemical and Kinetic Properties of an Endo-Polygalacturonase from the Industrial Fungus Aspergillus sojae

An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with K_M and V_MAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO₂, ethanol, and various cations assayed except Hg²⁺.

A novel thermostable and halotolerant xylanase from Colletotrichum graminicola

A novel endoxylanase from Colletotrichum graminicola (Excg1) was purified. Similar apparent molecular masses were estimated by gel filtration (17.3±1.9kDa) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (20.0±2.4kDa), suggesting that Excg1 is monomeric. The enzyme showed good halotolerance, retaining about 85% and 50% of the control activity in the presence of 0.5molL−1 and 3.0molL−1 NaCl, respectively. The optimum temperature of Excg1 (65°C) was not affected by NaCl, but the optimum pH rose from 5.5 in the absence and presence of 0.5molL−1 NaCl to 6.0, in 2.5molL−1 NaCl. Excg1 was highly thermostable at 50°C, with half-lives around 48h in either water or 0.5molL−1 NaCl and a residual activity of 75% at 2.5molL−1 NaCl. Excg1 was fully stable at pH 3.0–10.0 in the absence of salt, and from pH 4.0–10.0 in the presence of 0.5molL−1 and 2.5molL−1 NaCl. The enzyme hydrolyzed beechwood xylan with maximal velocity and apparent affinity constant of 481.3±34.0Umg−1 and 3.7±0.3mgmL−1, respectively. Similar kinetic parameters were obtained in the presence of 0.5molL−1 NaCl, but a maximum velocity about 34% lower was determined in 2.5molL−1 NaCl. Excg1 was tolerant to various organic solvents at a concentration of 5% (w/v) and also to sodium acetate up to 200mmolL−1. Xylobiose and xylotriose with a 4-O-methylglucuronic acid branching were the main products of beechwood xylan hydrolysis, and the time course of hydrolysis was not affected by NaCl 0.5molL−1 or sea water. To the best of our knowledge, this is the first report of a halotolerant endoxylanase from fungal origin. The properties of Excg1 suggested good potential for use in lignocellulose saccharification processes particularly using sea water or under high salt conditions, or in the presence of residues and/or byproducts of pretreatment steps, contributing to improve the economic viability of 2G ethanol production.

Characterization of a Salt-Tolerant and Cold-Adapted Xylanase from Bacillus cellulosilyticus

A xylanase (Xyn10A) gene from the saline-alkali-tolerant microorganism Bacillus cellulosilyticus DSM 2522 was cloned and expressed in Escherichia coli BL21 (DE3). The open reading frame was composed of 1008 base pairs, and it encoded 335 amino acid residues belonging to glycosyl hydrolase family 10. The optimal temperature and pH of the purified Xyn10A were 40 °C and 8.0, respectively. The Xyn10A was sensitive to heat and showed obvious cold-adapted activity, retaining 38.3%, 55.7%, and 82.9% of the optimal activity at 4, 20, and 30 °C, respectively. Xyn10A also showed a high level of NaCl tolerance. The highest activity was observed with 1.5 M NaCl. The specific enzyme activity of Xyn10A was as much as 163.8 U/mg. Kinetic assays showed that Km, Vmax, and Kcat were 2.56 mg/mL, 202.5 μM/min/mg, and 132.6 /s, respectively. Additionally, the main hydrolysis products using birchwood xylan as substrate were xylobiose, xylotriose, and xylotetraose, as determined by thin layer chromatography analysis. As a cold-adapted and salt-tolerant enzyme, Xyn10A is an ideal candidate for further research and biotechnological applications.

Novel hybrid process for the conversion of microcrystalline cellulose to value-added chemicals: part 1: process optimization

In this paper, a novel hybrid process for the treatment of microcrystalline cellulose (MCC) under hot-compressed water was investigated by applying constant direct current on the reaction medium. Constant current range from 1A to 2A was applied through a cylindrical anode made of titanium to the reactor wall. Reactions were conducted using a specially designed batch reactor (450 mL) made of SUS 316 stainless steel for 30–120 min of reaction time at temperature range of 170–230 °C. As a proton donor H2SO4 was used at concentrations of 1–50 mM. Main hydrolysis products of MCC degradation in HCW were detected as glucose, fructose, levulinic acid, 5-HMF, and furfural. For the quantification of these products, High Performance Liquid Chromatography (HPLC) and Gas Chromatography with Mass Spectroscopy (GC–MS) were used. A ½ fractional factorial design with 2-level of four factors; reaction time, temperature, H2SO4 concentration and applied current with 3 center points were built and responses were statistically analyzed. Response surface methodology was used for process optimization and it was found that introduction of 1A current at 200 °C to the reaction medium increased Total Organic Carbon (TOC) and cellulose conversions to 62 and 81 %, respectively. Moreover, application of current diminished the necessary reaction temperature and time to obtain high TOC and cellulose conversion values and hence decreased the energy required for cellulose hydrolysis to value added chemicals. Applied current had diverse effect on levulinic acid concentration (29.9 %) in the liquid product (230 °C, 120 min., 2 A, 50 mM H2SO4).

Electrochemical sensing of thiodiglycol, the main hydrolysis product of sulfur mustard, by using an electrode composed of an ionic liquid, graphene nanosheets and silver nanoparticles

The authors describe a method for amperometric determination of thiodiglycol (TDG), the main hydrolysis product of sulfur mustard. The electrode consists of a mixture of graphene nanosheets, silver nanoparticles and the ionic liquid octylpyridinium hexafluorophosphate. Electrochemical oxidation of TDG was performed by cyclic voltammetry at pH 4 and revealed a pair of well-defined redox peaks at potentials of 0.43 and 0.19 V (vs. Ag/AgCl). Amperometric detection was accomplished over a dynamic range that is linear in the 10–3700 μM concentration range. The detection limit (at an S/N of 3) is 6 μM. The electrode was applied to the determination of TDG in (spiked) waste water and gave recoveries that ranged from 98.2 to 103.3 %.

Characterization and biotechnological application of recombinant xylanases from Aspergillus nidulans

Two xylanases from Aspergillus nidulans, XlnB and XlnC, were expressed in Pichia pastoris, purified and characterized. XlnB and XlnC achieved maximal activities at 60°C and pH 7.5 and at 50°C and pH 6.0, respectively. XlnB showed to be very thermostable by maintaining 50% of its original activity after 49h incubated at 50°C. XlnB had its highest activity against wheat arabinoxylan while XlnC had the best activity against beechwood xylan. Both enzymes were completely inhibited by SDS and HgCl2. Xylotriose at 1mg/ml also totally inibited XlnB activity. TLC analysis showed that the main product of beechwood xylan hydrolysis by XlnB and XlnC was xylotetraose. An additive effect was shown between XlnB and XlnC and the xylanases of two tested commercial cocktails. Sugarcane bagasse saccharification results showed that these two commercial enzymatic cocktails were able to release more glucose and xylose after supplementation with XlnB and XlnC.