Francesco Alfani

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.

Comparison of SHF and SSF processes for the bioconversion of steam-exploded wheat straw

Two processes for ethanol production from wheat straw have been evaluated — separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). The study compares the ethanol yield for biomass subjected to varying steam explosion pretreatment conditions: temperature and time of pretreatment was 200°C or 217°C and at 3 or 10 min. A rinsing procedure with water and NaOH solutions was employed for removing lignin residues and the products of hemicellulose degradation from the biomass, resulting in a final structure that facilitated enzymatic hydrolysis. Biomass loading in the bioreactor ranged from 25 to 100 g l−1 (dry weight). The enzyme-to-biomass mass ratio was 0.06. Ethanol yields close to 81% of theoretical were achieved in the two-step process (SHF) at hydrolysis and fermentation temperatures of 45°C and 37°C, respectively. The broth required addition of nutrients. Sterilisation of the biomass hydrolysate in SHF and of reaction medium in SSF can be avoided as can the use of different buffers in the two stages. The optimum temperature for the single-step process (SSF) was found to be 37°C and ethanol yields close to 68% of theoretical were achieved. The SSF process required a much shorter overall process time (≈30 h) than the SHF process (96 h) and resulted in a large increase in ethanol productivity (0.837 g l−1 h−1 for SSF compared to 0.313 g l−1 h−1 for SHF). Journal of Industrial Microbiology & Biotechnology (2000) 25, 184–192.

Models for enzyme superactivity in aqueous solutions of surfactants.

Theoretical models are developed here for enzymic activity in the presence of direct micellar aggregates. An approach similar to that of Bru et al. [Bru, Sánchez-Ferrer and Garcia-Carmona (1989) Biochem. J. 259, 355-361] for reverse micelles has been adopted. The system is considered to consist of three pseudo-phases: free water, bound water and surfactant tails. The substrate concentration in each pseudo-phase is related to the total substrate concentration in the reaction medium. In the absence of interactions between the enzyme and the micelles, the model predicts either monotonically increasing or monotonically decreasing trends in the calculated reaction rate as a function of surfactant concentration. With enzyme-micelle interactions included in the formulation (by introducing an equilibrium relation between the enzyme confined in the free water and in the bound water pseudo-phases, and by allowing for different catalytic behaviours for the two forms), the calculated reaction rate can exhibit a bell-shaped dependence on surfactant concentration. The effect of the partition of enzyme and substrate is described, as is that of enzyme efficiency in the various pseudo-phases.

α-Chymotrypsin superactivity in aqueous solutions of cationic surfactants

Abstract α-Chymotrypsin (α-CT) activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n -dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N -glutaryl- l -phenylalanine p -nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate (at the same free substrate concentration in the medium) was calculated. k cat significantly increased while K m was little changed after correction to free substrate concentration. The ratio of k cat to K m was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of α-CT activity (30%) was less important in the presence of 1×10 −2 M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants.

On the use of chitosan-immobilized β-glucosidase in wine-making: kinetics and enzyme inhibition

Abstract The kinetics of chitosan-immobilized β-glucosidase and enzyme inhibition by several components of wine and must (glucose, fructose and terpenols) were studied. Optimum immobilization conditions were: temperature 25°C, pH between 5·5 and 6·0, polymeric support dimension in the range 38–75 μm, cross-linking time 30 min, glutaraldehyde concentration 0·5–1·0% w/v, 1 g of chitosan per 1000 units of β-glucosidase. The immobilized enzyme retained 29% of the wet biocatalyst activity when freeze-dried and showed good stability (half-life roughly 2 years) when stored at 4°C. Kinetics were tested at 25°C following the hydrolysis of p-nitrophenyl β- d glucopyranoside and obey the Michaelis-Menten rate equation. Km = 1·3 mM and the activation energy, 62·84 kJ mol−1, are close to those of the free enzyme. The operational half-life was roughly 500 h.Glucose only depressed the enzyme activity according to a reversible non-competitive inhibition mechanism with Ki = 11·2 mM.

Operational stability of Brevibacterium imperialis CBS 489-74 nitrile hydratase

Brevibacterium imperialis CBS 489-74 was grown in broths prepared with yeast and malt extract, bacteriological peptone and 2% glucose or differently modified with the addition of Na-phosphate buffer, FeSO 4 , MgSO4 and CoCl 2 . The peak production of nitrile hydratase (NHase) did not change significantly. At the stationary growth phase, the units per milliliter of broth (60 units ml -1 ) were more important than those at the exponential growth phase. The NHase operational stability of whole resting cells was monitored following the bioconversion of acrylonitrile to acrylamide in continuous and stirred UF-membrane reactors. The rate of inactivation was independent on buffer molarity from 25 to 75 mM and on pH from 5.8 to 7.4. Enzyme stability and activity remained unchanged in distilled water. The initial reaction rate increased from 12.8 to 23.8 g acrylamide/g dry cell/h, but NHase half-life dropped from 33 to roughly 7 h when temperature was varied from 4°C to 10°C. The addition of butyric acid up to 20 mM did not improve enzyme operational stability, and largely reduced (94%) enzyme activity. Acrylonitrile caused an irreversible damage to NHase activity. High acrylonitrile conversion (86%) was attained using 0.23 mg cells/ml in a continuously operating reactor.

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).

α-chymotrypsin superactivity in cetyltrialkylammonium bromide-rich media

Abstractα-Chymotrypsin (α-CT) activity was tested with N-glutaryl-l-phenylalanine p-nitroanilide in buffered media with added cationic surfactants. The effect of the commercial cetyltrimethylammonium bromide (CTABr) was compared with that of three other surfactants with ethyl (CTEABr), propyl (CTPABr), and butyl (CTBABr) head groups. These were synthesized and purified in this laboratory. Surfactant head groups provided distinct environments that largely modulated the catalytic performance. Larger alkyl head group hydrophobicity led to a marked enhancement of α-CT activity. CTBABr-rich media induced the highest superactivity.Kinetic measurements were performed in Tris-HCl buffer at a surfactant concentration either below or above CMC, and α-CT superactivity occurred in both media. Positive interactions between the enzyme and surfactants happened independently of thesupramolecular organization of the medium. The reaction followed the Michaelis-Menten kinetics. The substrate to micelle aggregates binding constant was used to calculate the substrate concentration available for catalysis. The kcat to km ratio was in CTBABr-rich media always higher than in pure buffer and depended on the surfactant concentration. α-CT superactivity depended on the pH value of buffer solution. Enzyme inactivation followed a single-step mechanism in pure buffer and a series mechanism in the presence of a surfactant. The rate of activity decay obeyed a first-order kinetics.

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.

Experimental validation of a model for α-chymotrypsin activity in aqueous solutions of surfactant aggregates

Abstract The hydrolysis of N -glutaryl- l -phenylalanine p -nitroanilide and N -succinyl- l -phenylalanine p -nitroanilide catalysed by α-chymotrypsin was studied in the presence of cationic (cetyltrimethylammonium bromide and cetyltripropylammonium bromide) and non-ionic ( t -octylphenoxypolyethoxyethanol and polyoxyethylene-9-lauryl ether) surfactants. The ratio of reaction rates in surfactant rich systems to those in pure buffer solutions was simulated by a previously developed model by Viparelli et al. [Biochem. J. 344 (1999) 765] based on the pseudo-phase approach introduced by Bru et al. [Biochem. J. 259 (1989) 355] for enzymatic reactions in reverse micelles. The system was depicted by three pseudo-phases: free water, bound water and surfactant core. The substrate and enzyme concentration in the pseudo-phase was related to total substrate and enzyme concentrations in the reaction medium. Superactivity occurs only in the presence of cetyltripropylammonium bromide. Plots of reaction rate ratio versus surfactant concentration were bell-shaped. Model simulation indicated that this behaviour could be attributed to the equilibrium between the enzyme confined in the free water and that in the bound water pseudo-phase, and to partition of the substrate in the pseudo-phases. The two enzyme forms must have different catalytic behaviour. The overall reaction rate depends on the two enzymatic reactions. Reasonable agreement was found between experimental results and model predictions.