Maria 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.

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.

Fungal nitrilases as biocatalysts: Recent developments.

Of the numerous putative fungal nitrilases available from protein databases only a few enzymes were purified and characterized. The purified nitrilases from Fusarium solani, Fusarium oxysporum f. sp. melonis and Aspergillus niger share a preference for (hetero)aromatic nitriles, temperature optima between 40 and 50 degrees C and pH optima in the slightly alkaline region. On the other hand, they differ in their chemoselectivity, i.e. their tendency to produce amides as by-products. The production of fungal nitrilases is increased by up to three orders of magnitude on the addition of 2-cyanopyridine to the culture media. The whole-cell and subcellular biocatalysts were immobilized by various methods (LentiKats(R); adsorption on hydrophobic or ion exchange resins; cross-linked enzyme aggregates). Operational stability was examined using continuous stirred membrane bioreactors. Fungal nitrilases appear promising for biocatalytic applications and biodegradation of nitrile environmental contaminants.

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.

Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10

BackgroundNitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.ResultsA nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzymes native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.ConclusionsThe nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.