Nicoletta Curreli

Complete and efficient enzymic hydrolysis of pretreated wheat straw

Abstract A fractionation of wheat straw components in a two-step chemical pretreatment is proposed. Hemicelluloses were hydrolysed by dilute sulphuric acid, allowing a substantial recovery of crystalline xylose. Lignin was removed by means of a mild alkaline/oxidative solubilisation procedure, involving no sulphite or chlorine and its derivatives. The use of diluted reagents and relatively low temperatures, was both cheap and environmentally friendly. The pretreated material was nearly pure cellulose, whose enzymic hydrolysis proceeded fast and with high yields, leading to high glucose syrups of remarkable purity.

Mild alkaline/oxidative pretreatment of wheat straw

A new mild alkaline/oxidative pretreatment of wheat straw prior to enzymic hydrolysis was carried out. It consists of a first alkaline (1% NaOH for 24 h) step, which mainly solubilises hemicellullose and renders the material more accessible to further chemical attack, and a second alkaline/oxidative step (1% NaOH and 0·3% H2O2 for 24 h), which solubilises and oxidises lignin to minor polluting compounds. The entire process was carried out at low temperature (25–40°C) using a low concentration of chemicals, resulting in a relatively low cost and waste liquors containing only trace amounts of dangerous pollutants derived from lignin. Recovery of cellulose after the double pretreatment reached 90% of that contained in the starting material, with a concomitant 81% degradation of lignin. The action of a commercial cellulase on the cellulose obtained produced a syrup with a high concentration of reducing sugars (220 mg/ml), of which a large percentage was glucose.

Effects of plant-derived naphthoquinones on the growth of Pleurotus sajor-caju and degradation of the compounds by fungal cultures

The growth of the white‐rot basidiomycete Pleurotus sajor‐caju in malt‐agar plates was inhibited by three naturally occurring, plant‐derived naphthoquinones: juglone, lawsone, and plumbagin. The latter two compounds exerted the most potent antifungal activity, and lawsone killed the mycelium at concentrations higher than 200 ppm. Plates containing juglone and lawsone presented large decolorized areas extending from area of fungal growth, suggesting an extracellular enzymatic degradation of these quinones. Screening of culture plates for extracellular enzymatic activities revealed the presence of both laccase and veratryl alcohol oxidase in most plates, the diffusion of both enzymes matching the decolorized area. In agitated cultures, the presence of juglone was found to stimulate the production of veratryl alcohol oxidase in a significant manner. This is the first time degradation of plant derived naphthoquinones by a white‐rot fungus is reported.

Effect of 3-hydroxyanthranilic acid on mushroom tyrosinase activity

Tyrosinase is a copper containing protein which catalyzes the hydroxylation of monophenols and the oxidation of diphenols to o-quinones. The monophenolase activity of tyrosinase is characterized by a typical lag time. In this paper the influence of 3-hydroxyanthranilic acid on monophenolase activity of tyrosinase is reported. 3-Hydroxyanthranilic acid reduced the lag time of tyrosinase when the enzyme acted on N-acetyl-L-tyrosine and on 4-tert-butylphenol. In the presence of 3-hydroxyanthranilic acid, the reaction product 4-tert-butyl-o-benzoquinone, derived from 4-tert-butylphenol oxidation, was formed at a higher rate than in its absence. The results reported in this paper indicate that 3-hydroxyanthranilic acid could affect the enzymic activity of mushroom tyrosinase probably by acting as a diphenol substrate. A K(m) value of 0.78 mM was calculated for 3-hydroxyanthranilic acid as substrate. When tyrosinase acted on 4-tert-butylphenol, K(m) for 3-hydroxyanthranilic acid as a cofactor was estimated to be 37.5 microM. No effect was observed on the diphenolase activity of the enzyme acting on 4-tert-butylcatechol in the presence of 3-hydroxyanthranilic acid.

3-hydroxykynurenine as a substrate/activator for mushroom tyrosinase

3-Hydroxykynurenine is a tryptophan metabolite with an o-aminophenol structure. It is both a tyrosinase activator and a substrate, reducing the lag phase, stimulating the monophenolase activity, and being oxidized to xanthommatin. In the early stage of monophenol hydroxylation, catechol accumulation takes place, whereas 3-hydroxykynurenine is substantially unchanged and no significant amounts of the o-quinone are produced. These results suggest an activating action of 3-hydroxykynurenine toward o-hydroxylation of monophenols. 3-Hydroxykynurenine could therefore well act as a physiological device to control phenolics metabolism to catechols and quinonoids.

Novel Diazonium-Functionalized Support for Immobilization Experiments

ABSTRACT: A hydrophilic, water-insoluble polymer was prepared, starting from com-mercial poly(vinyl alcohol) that was crosslinked and functionalized by means of glutar-aldehyde and 4-nitrobenzaldehyde. The resulting beads were then reduced and subse-quently diazotized, and finally contained diazonium moieties capable of covalently cou-pling with electron-rich aromatic systems such as histidine and/or tyrosine residuesof proteins. The described resin is therefore well suitable for protein immobilizationwhenever lysine residues (those involved in covalent coupling with several popularimmobilization procedures) are not available and/or cannot be used unless the biologi-cal activity of the protein is destroyed. q 1997 John Wiley & Sons, Inc. J Appl Polym Sci 66: 1433–1438, 1997 Key words: poly(vinyl alcohol); acetalation; diazotization; immobilization INTRODUCTION In the present work, the preparation of a nitro-aromatic derivative of crosslinked PVA is de-Poly(vinyl alcohol) (PVA) is a hydrophilic, water- scribed that in turn was reduced to the corre-soluble polymer, readily available in a wide range sponding amino aromatic substance and finallyof molecular weights at a very low price.

Dopaquinone hydroxylation through topaquinone cofactor in copper amine oxidases: A simplified chemical model

A simple model, 4‐tert‐butyl‐1,2‐benzoquinone, was chosen to study the hydroxylation step of the tyrosine‐derived Dopaquinone residue at the active site of copper amine oxidases in the self‐catalytic generation of the Topaquinone cofactor. This hydroxylation step was studied both in the presence and absence of free copper(II), and was found to be dependent on pH value but not on the presence of metal ions. It is therefore proposed that, hydroxide ion and not water should be the true reactive species in this key biosynthetic step of the Topaquinone cofactor, and that the active site Cu2+ is implied, at this point of cofactor biosynthesis, in the quinonisation of Topa rather than in the hydroxylation of Dopaquinone.

Improved chromatographic purification of peroxidase and beta-glucosidase from Hordeum vulgare seedlings.

Peroxidases (E.C. 1.11.1.7., hydrogen donor oxidoreductase) utilize hydrogen peroxide or substituted peroxides for the oxidation of a large number of substrates. Peroxidases are widely distributed and have been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance, but the physiological functions and metabolic control of these enzymes are still poorly understood. The simultaneous presence of amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (2,3). Recently we have purified an amine oxidase from Hordeum vulgare (4) and we have attempted to purify the peroxidase in order to study in vitro the reconstituted coupled system. beta-glucosidase (beta-D-glucoside glucohydrolase E.C. 3.2.1.21.) is capable of transforming glucosides in glucose and the corresponding aglycone or disaccharides as cellobiose, sophorose, gentiobiose. This enzyme is widely distributed in plants, fungi, bacteria, yeasts and animals (5,6). In the homogenate of Hordeum vulgare seedlings we also found beta-glucosidase activity and also attempted to purify beta-glucosidase. This enzyme copurified with peroxidase up to the last step. We report here the isolation of peroxidase and beta-glucosidase from Hordeum vulgare seedlings: some molecular and kinetic properties are given.

Degradation of juglone by Pleurotus sajor-caju

The toxic naphthoquinone juglone (5-hydroxy-1,4-naphthoquinone) is efficiently degraded by the ligninolytic fungus Pleurotus sajor-caju, as demonstrated by the total bleaching within 9 d of a conventional liquid culture medium supplemented with 0.6 mM juglone. The oxidative degradation involves the production of hydrogen peroxide arising from both enzymic and non-enzymic oxidation reactions, promoted by the fungus. Juglone is not directly attacked by the oxidative enzymes of the ligninolytic machinery of P. sajor-caju, such as laccase, manganese peroxidase and arylalcohol oxidase. On the other hand, this naphthoquinone is a good substrate for a reductase, which triggers an auto-oxidative process producing reactive oxygen species and leading to juglone degradation. The degradation process continues to completion by means of a direct, presumably non-catalysed reaction with hydrogen peroxide.

Purification and Characterization of an NAD(P)H:Quinone Oxidoreductase from Glycine Max Seedlings

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.