Laura Cantarella

Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF.

Steam‐exploded (SE) poplar wood biomass was hydrolyzed by means of a blend of Celluclast and Novozym cellulase complexes in the presence of the inhibiting compounds produced during the preceding steam‐explosion pretreatment process. The SE temperature and time conditions were 214 °C and 6 min, resulting in a log R0 of 4.13. In enzymatic hydrolysis tests at 45 °C, the biomass loading in the bioreactor was 100 gDW/L (dry weight) and the enzyme‐to‐biomass ratio 0.06 g/gDW. The enzyme activities for endo‐glucanase, exo‐glucanase, and β‐glucosidase were 5.76, 0.55, and 5.98 U/mg, respectively. The inhibiting effects of components released during SE (formic, acetic, and levulinic acids, furfural, 5‐hydroxymethyl furfural (5‐HMF), syringaldehyde, 4‐hydroxy benzaldehyde, and vanillin) were studied at different concentrations in hydrolysis runs performed with rinsed SE biomass as model substrate. Acetic acid (2 g/L), furfural, 5‐HMF, syringaldehyde, 4‐hydroxybenzaldehyde, and vanillin (0.5 g/L) did not significantly effect the enzyme activity, whereas formic acid (11.5 g/L) inactivated the enzymes and levulinic acid (29.0 g/L) partially affected the cellulase. Synergism and cumulative concentration effects of these compounds were not detected. SSF experiments show that untreated SE biomass during the enzymatic attack gives rise to a nonfermentable hydrolysate, which becomes fermentable when rinsed SE biomass is used. The presence of acetic acid, vanillin, and 5‐HMF (0.5 g/L) in SSF of 100 gDW /L biomass gave rise to ethanol yields of 84.0%, 73.5%, and 91.0% respectively, with respective lag phases of 42, 39, and 58 h.

Hydrolytic reactions in two-phase systems. Effect of water-immiscible organic solvents on stability and activity of acid phosphatase, β-glucosidase, and β-fructofuranosidase

The stability and activity of three hydrolytic enzymes, acid phosphatase (EC 3.1.3.2), beta-fructofuranosidase (EC 3.2.1.26), and beta-glucosidase (EC 3.2.1.4), were studied at 30 degrees C in two-phase systems. They were prepared with equal quantities of buffered water and a water-immiscible organic solvent. Low-molecular-weight acetates and paraffins were tested in this investigation. The kinetic constant of storage inactivation was correlated with the logarithm of solvent polarity. Enzyme stability in the presence of organic phases, whose log P value was included in 1.2-2.2, was greater than the one measured in pure buffered aqueous media. On the other hand, a dramatic enzyme denaturation took place making use of solvents at higher log P-value. Experiments carried out during the 24-h operation clarified that the reaction yield does not depend solely on solvent polarity. Acid phosphatase and beta-glucosidase, which are less resistant than beta-fructofuranosidase to temperature and shear in buffered solutions, showed especially significant enhancement of catalytic activity when hydrolysis was performed with the addition of acetates (50% v/v).

High-yield continuous production of nicotinic acid via nitrile hydratase-amidase cascade reactions using cascade CSMRs.

High yields of nicotinic acid from 3-cyanopyridine bioconversion were obtained by exploiting the in situ nitrile hydratase-amidase enzymatic cascade system of Microbacterium imperiale CBS 498-74. Experiments were carried out in continuously stirred tank UF-membrane bioreactors (CSMRs) arranged in series. This reactor configuration enables both enzymes, involved in the cascade reaction, to work with optimized kinetics, without any purification, exploiting their differing temperature dependences. To this end, the first CSMR, optimized for the properties of the NHase, was operated (i) at low temperature (5°C), limiting inactivation of the more fragile enzyme, nitrile hydratase, (ii) with a high residence time (24 h) to overcome reaction rate limitation. The second CSMR, optimized for the properties of the AMase, was operated (i) at a higher temperature (50°C), (ii) with a lower residence time (6h), and (iii) with a lower substrate (3-cyanopyridine) concentration to control excess substrate inhibition. The appropriate choice of operational conditions enabled total conversion of 3-cyanpyridine (up to 200 mM) into nicotinic acid to be achieved at steady-state and for long periods. Higher substrate concentrations required two CSMRs optimized for the properties of the NHase arranged in series to drive the first reaction to completion.

Membrane reactors for the investigation of product inhibition of enzyme activity

Abstract The use of ultrafiltration cells as membrane reactors is extended to the study of enzyme kinetics with product inhibition. This reactor configuration allows the lack of accuracy and instrument limitations typical of differential analysis and of time-course data analysis for experiments performed in batch reactors to be overcome. The hydrolysis of cellobiose to glucose, catalysed by β-glucosidase from Aspergillus niger (E.C. 3.2.1.4), was chosen as model system. The activity of this enzyme is suppressed by glucose according to a mixed-type inhibition pattern. Attention was paid to the possibility of determining the presence of either reversible or irreversible product inhibition. Details of the apparatus, experimental procedure and data correlation are given. Phenomena such as thermal deactivation, mechanical stress by shear and membraneto-enzyme affinity could alter the system response and mask the effects of inhibition.

Inactivating effects of lignin-derived compounds released during lignocellulosic biomass pretreatment on the endo-glucanase catalyzed hydrolysis of carboxymethylcellulose: A study in continuous stirred ultrafiltration-membrane reactor

This study focusses on the reversible/irreversible damage that selected phenolic compounds, released during steam-explosion pretreatment, mandatory for cellulose accessibility, causes on both stability and activity of a commercial cellulase (half-life=173h) during carboxymethyl-cellulose hydrolysis. Long-term experiments performed in continuous stirred UF-membrane bioreactors, operating at steady-state regime, in controlled operational conditions, allowed evaluating the inactivation-constant in the phenol presence (kd1) and after its removal (kd2) from the reactor feed. p-Hydroxybenzoic acid (1 and 2g L(-1)) are the extreme limits in the inactivating effect with enzyme half-lives 99.02 and 14.15h, respectively. The inactivation reversibility was assessed for vanillic acid, p-hydroxybenzoic acid, syringaldehyde, p-coumaric acid, being kd1>kd2. p-Hydroxybenzaldehyde and protocatechuic acid irreversibly affected cellulase stability increasing its inactivation with kd2>kd1. p-Hydroxybenzaldehyde, 1g L(-1), syringaldehyde, and vanillin, at 2gL(-1), had similar kd1÷kd2.

Stability and activity of immobilized hydrolytic enzymes in two-liquid-phase systems: Acid phosphatase, β-glucosidase, and β-fructofuranosidase entrapped in poly(2-hydroxyethyl methacrylate) matrices

Enzyme storage stability and hydrolysis yield were measured in experiments carried out with three model hydrolytic enzymes: acid phosphatase (EC 3.1.3.2), beta-glucosidase (EC 3.2.1.4), and beta-fructofuranosidase (EC 3.2.1.26) entrapped in hydrogels of poly(2-hydroxyethyl methacrylate). Runs were performed at 30 degrees C, under intensive stirring (500 rev min-1), in 50% v/v biphasic media prepared with buffer and organic solvents, whose log P value varied from 0.68 to 8.8. Storage stability was also monitored in the pure solvents. The small average particle size (125-210 microns) and the intensive stirring eliminate hindrances of intra- and interphase mass transfer resistances. The hydrophilic matrix protects the enzymes against thermal and chemical deactivation, thus allowing good production per unit weight of biocatalyst. In biphasic media, storage stability, with the exception of acid phosphatase, was not dependent on solvent polarity. On the contrary, a significant trend was observed when the enzymes were stored in neat organic solvents.

Biosaccharification of cellulosic biomass in immiscible solvent–water mixtures

The enzymatic hydrolysis of wheat straw was carried out in bi-phasic media prepared with acetate esters and Na-acetate buffer. The volume percentage of the organic chemicals was 75%. The biomass was pretreated in a steam explosion plant at 217°C and for 3 min. A cellulase complex from commercial source was utilised and the experiments were run at 45°C and at constant enzyme to biomass weight ratio (0.06). Biomass loadings ranged from 6.25 to 100 g per litre of reactor. The amount of glucose formed per litre of reactor and hour and the glucose yield (grams of product per gram of biomass) were close to the values attained in pure buffer. The glucose concentration in the aqueous phase was in bi-phasic media much higher than in pure buffer and reached the value of 146 g lH2O−1 during 72 h of saccharification. The results were poorly dependent on the physical–chemical properties of the solvents. Nevertheless, butyl acetate could be slightly preferred to propyl and i-amyl acetate. The use of bi-phasic media did not require stirring rate higher than in pure buffer. The presence of acetate ester traces did not alter markedly the production of ethanol in the fermentation stage, but determined the extension of the lag phase.

Nitrile, amide and temperature effects on amidase-kinetics during acrylonitrile bioconversion by nitrile-hydratase/amidase in situ cascade system.

In this study the amidase kinetics of an in situ NHase/AMase cascade system was explored as a function of operational parameters such as temperature, substrate concentration and product formation. The results indicated that controlling amidase inactivation, during acrylonitrile bioconversion, makes it possible to recover the intermediate product of the two-step reaction in almost a pure form, without using purified enzyme. It has been demonstrated, in long-term experiments performed in continuous stirred UF-membrane bioreactors, that amidase is kinetically controlled by its proper substrate, depending on the structure, and by acrylonitrile. Using acrylamide, AMase-stability is temperature dependent (5°C, kd=0.008 h(-1); 30°C kd=0.023 h(-1)). Using benzamide, amidase is thermally stable up to 50°C and no substrate inhibition/inactivation occurs. With acrylonitrile, AMase-activity and -stability remain unchanged at concentrations <200 mM but at 200 mM, 35°C, after 70 h process, 90% irreversible inactivation occurs as no AMase-activity on benzamide revives.

Invertase activity of Saccharomyces cerevisiae cells entrapped in poly (2-hydroxyethyl methacrylate) gels: kinetic and thermostability study in membrane reactors

Films of poly (2-hydroxyethyl methacrylate) with entrapped yeast cells have been prepared and characterized in membrane reactors. Two concentrations, 5 and 10% w/w, of crosslinking agent ethylene dimethacrylate are used. The invertase activity is monitored between 30 and 55 degrees C in the range of sucrose concentration from 40 to 200 mM and during almost 600 h of operation. Comparison is also made with the behaviour of free cells and small size particles (less than 115 mesh) of poly-HEMA immobilized cells. The results show that the whole membrane volume is not involved in substrate permeation and a combined diffusion-reaction rate controlling mechanism holds. An unusual dependence of reaction rate on bulk pH is observed. In the range of pH from 4.0 to 6.0, invertase activity in films continuously increases while two distinctive maxima for free yeast cells (pH 4.75) and for small particles of poly-HEMA-immobilized yeast cells (pH 4.5) are observed.

Immobilised peroxidases from Asparagus acutifolius L. seeds for olive mill waste water treatment

This work compares the main enzymatic parameters of a cationic peroxidase (AaP-1-4), purified and characterized from Asparagus acutifolius L. seeds (Mol. Biotechnol., 2014, 56, 738–746) and its immobilised form (Eup-AaP-1-4), on Eupergit® CM with Horseradish peroxidase. The optimum in the pH-activity profile was pH 4.0 and pH 3.0 for AaP-1-4 and Eup-AaP-1-4, respectively. Ca2+ cation enhanced both enzymatic activities, however, when submitted to a temperature stress (120 min at 50 °C) Eup-AaP-1-4 lost only 20% activity while AaP-1-4 70%. Furthermore, AaP-1-4 was proved to be able to remove (poly)phenols in olive mill waste water (OMW), with hydrogen peroxide electron donor. The Eup-AaP-1-4 kinetic proprieties were investigated and the operational stability evaluated in a continuous stirred membrane bioreactor. AaP-1-4 appears to be a novel non-expensive source of peroxidases suitable for biotechnological applications in the environmental field for the removal of aqueous (poly)phenols produced from several industrial processes.