Paulo Waldir Tardioli

Finding stable cellulase and xylanase: evaluation of the synergistic effect of pH and temperature

Ethanol from lignocellulosic biomass has been recognized as one of the most promising alternatives for the production of renewable and sustainable energy. However, one of the major bottlenecks holding back its commercialization is the high costs of the enzymes needed for biomass conversion. In this work, we studied the enzymes produced from a selected strain of Aspergillus niger under solid state fermentation. The cellulase and xylanase enzymatic cocktail was characterized in terms of pH and temperature by using response surface methodology. Thermostability and kinetic parameters were also determined. The statistical analysis of pH and temperature effects on enzymatic activity showed a synergistic interaction of these two variables, thus enabling to find a pH and temperature range in which the enzymes have a higher activity. The results obtained allowed the construction of mathematical models used to predict endoglucanase, β-glucosidase and xylanase activities under different pH and temperature conditions. Optimum temperature values for all three enzymes were found to be in the range between 35°C and 60°C, and the optimum pH range was found between 4 and 5.5. The methodology employed here was very effective in estimating enzyme behavior under different process conditions.

Hydrolysis of Proteins by Immobilized-Stabilized Alcalase-Glyoxyl Agarose

This paper presents stable Alcalase‐glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing‐stabilizing Alcalase on cross‐linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 °C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half‐lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase‐glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half‐life of ca. 23 h, at pH 8.0 and 63 °C and was ca. 500‐fold more stable than the soluble enzyme. Thermal inactivation of all derivatives followed a single‐step non‐first‐order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc‐Ala‐ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 °C with the most stabilized enzyme was 2‐fold higher than that achieved using soluble enzyme, as a result of the thermal inactivation of the latter. Therefore, the high stability of the new Alcalase‐glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.

Kinetic model for whey protein hydrolysis by alcalase multipoint-immobilized on agarose gel particles

Partial hydrolysis of whey proteins by enzymes immobilized on an inert support can either change or evidence functional properties of the produced peptides, thereby increasing their applications. The hydrolysis of sweet cheese whey proteins by alcalase, which is multipoint-immobilized on agarose gel, is studied here. A Michaelis-Menten model that takes into account competitive inhibition by the product was fitted to experimental data. The influence of pH on the kinetic parameters in the range 6.0 to 11.0 was assessed, at 50oC. Initial reaction-rate assays in a pHstat at different concentrations of substrate were used to estimate kinetic and Michaelis-Menten parameters, k and KM. Experimental data from long-term batch assays were used to quantify the inhibition parameter, KI. The fitting of the model to the experimental data was accurate in the entire pH range.

Design of new immobilized-stabilized carboxypeptidase a derivative for production of aromatic free hydrolysates of proteins.

This paper presents stable carboxypeptidase A (CPA)‐glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced after immobilizing‐stabilizing CPA on cross‐linked 6% agarose beads, activated with low and high concentrations of aldehyde groups, and different immobilization times. The CPA‐glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde as activation reactant. The most stabilized CPA‐glyoxyl derivative was produced using 48 h of immobilization time and high activation grade of the support. This derivative was approximately 260‐fold more stable than the soluble enzyme and presented approximately 42% of the activity of the soluble enzyme for the hydrolysis of long‐chain peptides (e.g., cheese whey proteins previously hydrolyzed with immobilized trypsin and chymotrypsin) and of the small substrate N‐benzoylglycyl‐l‐phenylalanine (hippuryl‐l‐Phe). These results were much better than those achieved using the conventional support, glutaraldehyde‐agarose. Amino acid analysis of the products of the acid hydrolysis of CPA (both soluble and immobilized) showed that approximately four lysine residues were linked on the glyoxyl agarose beads, suggesting the existence of an intense multipoint covalent attachment between the enzyme and the support. The maximum temperature of hydrolysis was increased from 50 °C (soluble enzyme) to 70 °C (most stable CPA‐glyoxyl derivative). The most stable CPA‐glyoxyl derivative could be efficiently used in the hydrolysis of long‐chain peptides at high temperature (e.g., 60 °C), being able to release 2‐fold more aromatic amino acids (Tyr, Phe, and Trp) than the soluble enzyme, under the same operational conditions. This new CPA derivative greatly increased the feasibility of using this protease in the production of protein hydrolysates that must be free of aromatic amino acids.

Enhanced saccharification of sugarcane bagasse using soluble cellulase supplemented with immobilized β-glucosidase

The β-glucosidase (BG) enzyme plays a vital role in the hydrolysis of lignocellulosic biomass. Supplementation of the hydrolysis reaction medium with BG can reduce inhibitory effects, leading to greater conversion. In addition, the inclusion of immobilized BG can be a useful way of increasing enzyme stability and recyclability. BG was adsorbed on polyacrylic resin activated by carboxyl groups (BG-PC) and covalently attached to glyoxyl-agarose (BG-GA). BG-PC exhibited similar behavior to soluble BG in the hydrolysis of cellobiose, while BG-GA hydrolyzed the same substrate at a lower rate. However, the thermal stability of BG-GA was higher than that of free BG. Hydrolysis of pretreated sugarcane bagasse catalyzed by soluble cellulase supplemented with immobilized BG improved the conversion by up to 40% after 96 h of reaction. Both derivatives remained stable up to the third cycle and losses of activity were less than 50% after five cycles.

Production of Cyclodextrins in a Fluidized-Bed Reactor Using Cyclodextrin-Glycosyl-Transferase

Cyclodextrin-glycosyl-transferase (EC2.4.1.19), produced by Wacker (Munich, Germany), was purified by biospecific affinity chromatography with β-cyclodextrin (β-CD) as ligand, and immobilized into controlled pore silica particles (0.42 mm). This immobilized enzyme (IE) had 4.7 mg of protein/g of support and a specific activity of 8.6 μmol of β-CD/(min·gIF) at 50°C, pH 8.0. It was used in a fluidized-bed reactor (FBR) at the same conditions for producing cyclodextrins (CDs) with 10% (w/v) maltodextrin solution as substrate. Bed expansion was modeled by the Richardson and Zaki equation, giving a good fit in two distin ctranges of bed porosities. The minimum fluidization velocity was 0.045 cm/s, the bed expansion coefficient was 3.98, and the particle terminal velocity was 2.4 cm/s. The FBR achieved high productivity, reaching in only 4 min of residence time the same amount of CDs normally achieved in a batch reactor with free enzyme after 24h of reaction, namely, 10.4 mM β-CD and 2.3 mM γ-CD.

Distribution of suspended particles in a Taylor–Poiseuille vortex flow reactor

Vortex flow reactors (VFRs) are specially suited to work with shear-sensitive particles, due to the gentle and efficient stirring characteristics of Taylor vortices. For heterogeneous VFRs, the distribution of the solid phase must be accounted for in detail. This work presents residence-time distributions (RTDs) of agarose gel particles (Φ av =214 μm), suspended in different liquid solutions. Reactor porosity was 90%. The mass transfer coefficients of a lumped-parameter model of the reactor are estimated from residence time distributions of a tracer. The VFR has radius ratio η=0.664 and aspect ratio Γ=14.9. Axial flow rates were selected to provide a mean residence time of 1850 s, adequate for many applications. Minimum rotation rates of the inner cylinder were imposed by suspended particles homogeneity criteria. The VFR operational region was: 0.0645<Re ax <0.592 and 98<Re θ <3820, corresponding to very high values of the ratio Re θ /Re ax (between 5293 and 6236). Under these conditions, the vortices were stationary, and circumvented by a by-pass stream. The results indicate that the gel particles are unevenly distributed in the vortex core and by-pass regions. Mean residence times for the particles are substantially greater than for the liquid. The mathematical model presented in this paper can accommodate this behavior, using a partition coefficient for the solid phase.

Immobilized Lipases on Functionalized Silica Particles as Potential Biocatalysts for the Synthesis of Fructose Oleate in an Organic Solvent/Water System

Lipases from Thermomyces lanuginosus (TLL) and Pseudomonas fluorescens (PFL) were immobilized on functionalized silica particles aiming their use in the synthesis of fructose oleate in a tert-butyl alcohol/water system. Silica particles were chemically modified with octyl (OS), octyl plus glutaraldehyde (OSGlu), octyl plus glyoxyl (OSGlx), and octyl plus epoxy groups (OSEpx). PFL was hyperactivated on all functionalized supports (more than 100% recovered activity) using low protein loading (1 mg/g), however, for TLL, this phenomenon was observed only using octyl-silica (OS). All prepared biocatalysts exhibited high stability by incubating in tert-butyl alcohol (half-lives around 50 h at 65 °C). The biocatalysts prepared using OS and OSGlu as supports showed excellent performance in the synthesis of fructose oleate. High ester synthesis was observed when a small amount of water (1%, v/v) was added to the organic phase, allowing an ester productivity until five times (0.88–0.96 g/L.h) higher than in the absence of water (0.18–0.34 g/L.h) under fixed enzyme concentration (0.51 IU/g of solvent). Maximum ester productivity (16.1–18.1 g/L.h) was achieved for 30 min of reaction catalyzed by immobilized lipases on OS and OSGlu at 8.4 IU/mL of solvent. Operational stability tests showed satisfactory stability after four consecutive cycles of reaction.

Characterization of β -Glucosidase Produced by Aspergillus niger under Solid-State Fermentation and Partially Purified Using MANAE-Agarose.

β-Glucosidase (BGL) is a hydrolytic enzyme with specificity for a wide variety of glycoside substrates, being an enzyme with a large range of biotechnological applications. However, enzyme properties can be different depending both on the microorganism and the cultivation procedure employed. Therefore, in order to explore potential biocatalytical applications of novel enzymes, their characterization is essential. In this work, a BGL synthesized by a selected strain of Aspergillus niger cultivated under solid-state fermentation (SSF) was partially purified and fully characterized in terms of optimum pH, temperature, and thermostability. The single-step purification using MANAE-agarose in a chromatographic column yielded an enzyme solution with specific activity (17.1 IU/mg protein) adequate for the characterization procedures. Electrophoresis SDS-PAGE and size-exclusion chromatography analysis resulted in an estimated molecular mass of 60 kDa. Higher enzyme activities were found in the range between 40 and 65°C and between pH 4 and 5.5, indicating an interesting characteristic for application in the hydrolysis of lignocellulosic biomass for biofuels production. Thermostability studies of purified BGL resulted in half-lives at 37°C of 56.3 h and at 50°C of 5.4 h. These results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

Addition of metal ions to a (hemi)cellulolytic enzymatic cocktail produced in-house improves its activity, thermostability, and efficiency in the saccharification of pretreated sugarcane bagasse.

High activity and stability are essential for (hemi)cellulolytic enzymes used in biomass conversion, while non-productive binding of cellulases to lignin reduces saccharification efficiency and needs to be avoided. One potential strategy is the addition of inexpensive metal ions. This paper describes the influence of divalent metal ions on the activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes produced in-house by Aspergillus niger under solid-state fermentation (SSF). The use of Mn(2+) provided the best (hemi)cellulolytic activity and stability, with an increase in endoglucanase activity of up to 57%. The use of Mn(2+) was then investigated in the saccharification of sugarcane bagasse submitted to acid, steam-explosion, and hydrothermal pretreatments. The addition of Mn(2+) ions at 10mM in the saccharification of acid-pretreated bagasse resulted in a 34% increase in glucose release. These positive effects appeared to be due to a reduction in non-productive enzyme adsorption. The findings suggest that the addition of inexpensive metal ions can help to improve activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes.