Raquel de Lima Camargo Giordano

Improving the Properties of Chitosan as Support for the Covalent Multipoint Immobilization of Chymotrypsin

Changing gel structure and immobilization conditions led to a significant improvement in the covalent multipoint attachment of chymotrypsin on chitosan. The use of sodium alginate, gelatin, or kappa-carrageenan, activation with glutaraldehyde, glycidol, or epichlorohydrin, and addition of microorganisms followed by cellular lysis allowed the modification of the gel structure. Immobilization yields, recovered activities, and stabilization factors at 55 and 65 degrees C were evaluated. Enzyme immobilization for 72 h at pH 10.05, 25 degrees C and reduction with NaBH 4 in chitosan 2.5%-carrageenan 2.5%, with addition of S. cerevisiae 5% and activation with epichlorohydrin led to the best derivative, which was 9900-fold more stable than the soluble enzyme. This support allowed an enzyme load up to 40 mg chymotrypsin x g gel (-1). The number of covalent bonds, formed by active groups in the support and lysine residues of the enzyme, can explain the obtained results. SEM images of the gel structures corroborate these conclusions.

Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan

Abstract - The objective of this work was to study enzyme immobilization on chitosan activated with glutaraldehyde, aiming to produce a cheap biocatalyst. Two different immobilization strategies were studied: one-point and multipoint covalent attachment to the solid matrix. The multipoint covalent attachment derivative had an 82% immobilization yield. It was4.9-fold more stable than the free enzyme at 50°C and 4.5-fold more stable than soluble enzyme at pH 10.0. The one-point derivativehad an 85% immobilization yield. It was 2.7-fold more stable than the free enzyme at 50°C and 3.8-fold more stable than soluble PGA at pH 10.0. Results indicated that chitosan can be loaded with PGA above 330 IU/g. Intraparticle diffusive effects, however,limited hydrolysis of penicillin G catalyzed by those derivatives at 37°C and 25°C. Operational stability assays were performed and the multipoint derivative exhibited a half-life of 40 hours. Keywords : Stabilization of enzymes; Penicillin G acylase; Chitosan and immobilization of enzymes.

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.

Bioelectricity versus bioethanol from sugarcane bagasse: is it worth being flexible?

BackgroundSugarcane is the most efficient crop for production of (1G) ethanol. Additionally, sugarcane bagasse can be used to produce (2G) ethanol. However, the manufacture of 2G ethanol in large scale is not a consolidated process yet. Thus, a detailed economic analysis, based on consistent simulations of the process, is worthwhile. Moreover, both ethanol and electric energy markets have been extremely volatile in Brazil, which suggests that a flexible biorefinery, able to switch between 2G ethanol and electric energy production, could be an option to absorb fluctuations in relative prices. Simulations of three cases were run using the software EMSO: production of 1G ethanol + electric energy, of 1G + 2G ethanol and a flexible biorefinery. Bagasse for 2G ethanol was pretreated with a weak acid solution, followed by enzymatic hydrolysis, while 50% of sugarcane trash (mostly leaves) was used as surplus fuel.ResultsWith maximum diversion of bagasse to 2G ethanol (74% of the total), an increase of 25.8% in ethanol production (reaching 115.2 L/tonne of sugarcane) was achieved. An increase of 21.1% in the current ethanol price would be enough to make all three biorefineries economically viable (11.5% for the 1G + 2G dedicated biorefinery). For 2012 prices, the flexible biorefinery presented a lower Internal Rate of Return (IRR) than the 1G + 2G dedicated biorefinery. The impact of electric energy prices (auction and spot market) and of enzyme costs on the IRR was not as significant as it would be expected.ConclusionsFor current market prices in Brazil, not even production of 1G bioethanol is economically feasible. However, the 1G + 2G dedicated biorefinery is closer to feasibility than the conventional 1G + electric energy industrial plant. Besides, the IRR of the 1G + 2G biorefinery is more sensitive with respect to the price of ethanol, and an increase of 11.5% in this value would be enough to achieve feasibility. The ability of the flexible biorefinery to take advantage of seasonal fluctuations does not make up for its higher investment cost, in the present scenario.

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(®) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).

Analysis of a Taylor-Poiseuille vortex flow reactor - I : Flow patterns and mass transfer characteristics

This paper shows the results of flow visualization and residence time distribution experiments in a Taylor-Poiseuille vortex flow apparatus. It is the first of a series that starts with the identification of flow patterns inside the device and goes up to the assessment of its performance as an enzymatic reactor. The approach is to study in depth one single geometric configuration (radius ratio η=0.677 and aspect ratio Γ=18.30), adequate for use as a heterogeneous reactor and or adsorption system in bio-processes, rather than spanning a range of geometries and proposing empirical expressions for mass transport coefficients. The range of rotations and axial flow rates used in this article correspond to low moderate rotational Reynolds numbers (Re θ from 130 to 615, with 1.6<Re θ /Re θ.c <7.7) and low axial ones (Re ax from 0.172 to 1.067). An unusual behavior of the system was noted in this operational region: the vortex drift velocities are less than one, and decrease continuously with increasing rotations, until a full stop. Except for Re θ close to the critical value, the downstream displacement of vortices is slower than the mean axial velocity. The implications of this fact on the reactor performance are discussed.

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.

Lignocellulose pretreatment technologies affect the level of enzymatic cellulose oxidation by LPMO

Sugarcane bagasse, corn stover, and wheat straw are among the most available resources for the production of cellulosic ethanol. For these biomasses we study the influence of pre-treatment methods on the chemical composition, as well as on the subsequent reactions of enzymatic hydrolysis and oxidation of cellulose. The applied pre-treatment methods are organosolv, hydrothermal, and alkaline. Hydrothermally pretreated wheat straw gave the highest cellulose conversion with 80% glucose yield and 0.8% oxidized cellulose products. Recent studies have shown that lignin is able to boost the activity of the cellulose oxidizing enzyme lytic polysaccharide monooxygenase (LPMO). The highest activity of LPMO was observed for the hydrothermally pretreated biomasses, which also contained the highest level of lignin. All hydrolyses were done at high dry matter levels using a commercial enzyme preparation containing hydrolytic and oxidative enzymes.

Immobilization of trypsin on chitosan gels: Use of different activation protocols and comparison with other supports

Trypsin was immobilized on chitosan gels coagulated with 0.1 or 1 M NaOH and activated with glutaraldehyde or glycidol. The derivatives were characterized by their recovered activity, thermal (40, 55 and 70 degrees C) and alkaline (pH 11) stabilities, amount of enzyme immobilized on gels for several enzyme loads (8-14 mg(protein)/g(Gel)) and compared to agarose derivatives. Enzyme loads higher than 14 mg(protein)/g(Gel) can be immobilized on glutaraldehyde derivatives, which showed 100% immobilization yield and, for loads up to 8 mg(protein)/g(Gel), 100% recovered activity. Activation with glycidol led to lower immobilization yields than the ones obtained with glutaraldehyde, 61% for agarose-glyoxyl (AgGly) with low grade of activation and 16% for the chitosan-glyoxyl (ChGly), but allowed obtaining the most stable derivative (ChGly), that was 660-fold more stable than the soluble enzyme at 55 and 70 degrees C-approximately threefold more stable than AgGly. The ChGly derivative presented also the highest stability during incubation at pH 11. Analyses of lysine residue contents in soluble and immobilized trypsin indicated formation of multipoint bonds between enzyme and support, for glyoxyl derivatives.