The influence of ferrous sulfate utilization on the sugar yields from dilute-acid pretreatment of softwood for bioethanol production

By employing metal salts in dilute-acid pretreatment the severity can be reduced due to reduced activation energy. This study reports on a dilute-acid steam pretreatment of spruce chips by addition of a small amount of ferrous sulfate to the acid catalyst, i.e., either SO₂, H₂SO₃ or H₂SO₄. The utilization of ferrous sulfate resulted in a slightly increased overall glucose yield (from 74% to 78% of the theoretical value) in pretreatment with SO₂ and H₂SO₃. Impregnation with ferrous sulfate and sulfuric acid did not give any improvement compared with pretreatment based solely on H₂SO₄.     

Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa

Xylooligosaccharides (XOS) with DP 2-4 are important synbiotics used as food ingredients based on its prebiotic characteristics. In this work, the production of XOS from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylan was prepared from triploid Populas tomentosa, and bioconverted into XOS by crude xylanase solution obtained from Pichia stipitis. The effects of reaction time, temperature, enzyme dosage, and pH value on the production of XOS were fully evaluated. Under the optimal condition (25 U g⁻¹ substrate, pH 5.4 and 50 °C), 36.8% of the xylan preparation was converted to XOS, equivalent to 3.95 mg/mL of the hydrolyzate. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzymatic hydrolyzate, which together accounted for over 95% of the released oligosaccharides. Meanwhile, the effect of sonication pretreatment on the conversion efficiency of the xylan preparation was also investigated.     

A modified static respiration assay and its relationship with an enzymatic test to assess compost stability and maturity

Despite the numerous compost stability and maturity tests, no universally accepted compost stability or maturity index exists. The fluorescein di-acetate (FDA) enzymatic assay, originating from soil studies, is examined here as a potential new compost stability test, and is compared to microbial respiration and phytotoxicity indices. Thirteen composts were used in the study from different source materials. Static microbial respiration activity indices calculated were the cumulative O₂ consumptions, O₂ consumption rates, total C-CO₂ production, the respiratory quotient and the bio C/N ratio. Compost phytotoxicity was quantified via a 7-day tomato seed germination assay. Results showed that the net fluorescein release rates correlated with all stability indices. The germination index marginally correlated with the fluorescein release rates, but not with any of the other stability indices. New limits to classify composts regarding their stability were proposed.     

Determination of oxidized and reduced nicotinamide adenine dinucleotide in cell monolayers using a single extraction procedure and a spectrophotometric assay

While many investigations measuring oxidized nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADH) have been carried out on several mammalian tissues and blood cells, few reports have dealt with monolayers of cultured cells. Here we show a novel method to measure NAD+ and NADH in monolayers of a neuroblastoma cell line. The method was established by modifying a single extraction procedure originally developed for erythrocytes and an enzymatic cycling assay using a dye that absorbs in visible range. The following modifications were made. (i) Addition of 0.05% of a detergent, Triton X-100, to carbonate-bicarbonate extraction buffer enabled us to accurately measure cellular [NADH]/([NAD+]+[NADH]). (ii) Addition of N-ethyldibenzopyrazine ethyl sulfate salt (phenazine ethosulfate) immediately before the incubation suppressed the gradual decline of the sensitivity of the assay. The procedure presented here provides a simple and inexpensive measurement of NAD+ and NADH in cell monolayers.     

Convenient enzymatic synthesis of a p-nitrophenyl oligosaccharide series of sialyl N-acetyllactosamine, sialyl Le x and relevant compounds

From the beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (1) prepared by the transglycosylation of beta-galactosidase from Bacillus circulans, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (9) and alpha-D-Neu5Ac-(2-->6)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (10) were effectively synthesized with an equimolar ratio of CMP-Neu5Ac by recombinant rat alpha-(2-->3)-N-sialyltransferase and rat liver alpha-(2-->6)-N-sialyltransferase, respectively. The former enzyme also transferred effectively the Neu5Ac residue from CMP-Neu5Ac to the location of OH-3 in the non-reducing terminal of beta-D-Gal-(1-->4)-beta-D-Gal-OC6H4NO2-p or beta-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p, while the latter enzyme did not. In the case of equimolar ratio of GDP-Fuc/acceptor, 1 and 9 were further fucosylated quantitatively to form beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (14) and alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (13) by recombinant human alpha-(1-->3)-fucosyltransferase VII, respectively.     

Suppression of hypothalamic deiodinase type II activity blunts TRH mRNA decline during fasting

Fasting is characterized by disrupted thyroid feedback, with suppressed levels of thyroid hormones and paraventricular thyrotropin releasing hormone (TRH). We found that third ventricle administration of the deiodinase inhibitor, iopanoic acid, dose-dependently reduced deiodinase type II (DII) activity selectively in the hypothalamus. This suppression of DII by iopanoic acid during fasting prevented elevated DII activity and blunted the decline in hypothalamic TRH mRNA levels. Because fasting-induced elevation in hypothalamic DII activity is paralleled by increased hypothalamic T3 concentration, our study suggests that T3 formation by DII in the hypothalamus is the cause of disrupted thyroid feedback during fasting.     

Synthesis of a novel three-coordinate copper(I) complex: a structural mimic of the reduced form of type 1 site in copper protein

A new calix[4]arene ligand 3 bearing two imidazole and two methylthioethyl groups on the lower rim has been synthesized. The complex with Cu(I) was prepared and characterized by X-ray diffraction analysis and XPS spectrum. Such complex could be as the structural model of the permanently reduced form of type 1 site in human ceruloplasmin and other copper proteins.     

Tuning the redox and enzymatic activity of glucose oxidase in layered organic films and its application in glucose biosensors

Glucose oxidase was embedded in organic films through a layer-by-layer approach, where the enzyme demonstrated significantly enhanced electron-transfer reactivity and finely tuned enzymatic activity. An unmediated, reagentless glucose biosensor was accordingly prepared with two polyethylenimine/glucose oxidase bilayers-modified pyrolytic graphite electrode. A calibration linear range of glucose was 0.5-8.9 mM with a detection limit of 50 microM and sensitivity of 0.76 microA mM(-1).     

High-performance liquid chromatography-based methods of enzymatic analysis: electron transport chain activity in mitochondria from human skeletal muscle

This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as approximately 10 mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40 microg/ml alamethicin and the reaction is terminated by H(2)SO(4), which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1-20 microM. NADH is quantitatively analyzed with a detection limit of 3-5 pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.     

Structure and solution properties of enzymatically synthesized glycogen

Recently, a new enzymatic process for glycogen production was developed. In this process, short-chain amylose is used as a substrate for branching enzymes (BE, EC 2.4.1.18). The molecular weight of the enzymatically synthesized glycogen (ESG) depends on the size and concentration of the substrate. Structural and physicochemical properties of ESG were compared to those of natural source glycogen (NSG). The average chain length, interior chain length, and exterior chain length of ESG were 8.2-11.6, 2.0-3.3, and 4.2-7.6, respectively. These values were within the range of variation of NSG. The appearances of both ESG and NSG in solution were opalescent (milky white and slightly bluish). Furthermore, transmission electron microscopy and atomic force microscopy showed that ESG molecules formed spherical particles, and that there were no differences between ESG and NSG. Viscometric analyses also showed the spherical nature of both glycogens. When ESG and NSG were treated with pullulanase, a glucan-hydrolyzing enzyme known to degrade glycogen only on its surface portion, both glycogens were similarly degraded. These analyses revealed that ESG shares similar molecular shapes and surface properties with NSG.