Pseudomonas fluorescens lipase immobilization on polysiloxane-polyvinyl alcohol composite chemically modified with epichlorohydrin

The technique based on sol-gel approach was used to generate silica matrices derivatives by hydrolysis of silane compounds. The present work evaluates a hybrid matrix obtained with tetraethoxysilane (TEOS) and polyvinyl alcohol (PVA) on the immobilization yield of lipase from Pseudomonas fluorescens. The resulting polysiloxane-polyvinyl alcohol (POS-PVA) matrix combines the property of PVA as a suitable polymer to retain proteins with an excellent optical, thermal and chemical stability of the host silicon oxide matrix. Aiming to render adequate functional groups to the covalent binding with the enzyme the POS-PVA matrix was chemically modified using epichlorohydrin. The results were compared with immobilized derivative on POS-PVA activated with glutaraldehyde. Immobilization yield based on the recovered lipase activity depended on the activating agent and the highest efficiency (32%) was attained when lipase was immobilized on POS-PVA activated with epichlorohydrin, which, probably, provided more linkage points for the covalent bind of the enzyme on the support. This was confirmed by determining the morphological properties using different techniques as X-ray diffraction and scanning electron microscopy (SEM). Comparative studies were carried out to attain optimal activities for free lipase and immobilized systems. For this purpose, a central composite experimental design with different combinations of pH and temperature was performed. Enzymatic hydrolysis with the immobilized enzyme in the framework of the Michaelis-Menten mechanism was also reported. Under optimum conditions, the immobilized derivative on POS-PVA activated with epichlorohydrin showed to have more affinity for the substrate in the hydrolysis of olive oil, with a Michaelis-Menten constant value (K-m) of 293 mM, compared to the value of 401 mM obtained for the immobilized lipase on support activated with glutaraldehyde. Data generated by DSC showed that both immobilized derivatives have similar thermal stabilities. (C) 2007 Elsevier B.V. All rights reserved.

Response surface methodology as an approach to determine optimal activities of lipase entrapped in sol-gel matrix using different vegetable oils

The conditions for maximization of the enzymatic activity of lipase entrapped in sol-gel matrix were determined for different vegetable oils using an experimental design. The effects of pH, temperature, and biocatalyst loading on lipase activity were verified using a central composite experimental design leading to a set of 13 assays and the surface response analysis. For canola oil and entrapped lipase, statistical analyses showed significant effects for pH and temperature and also the interactions between pH and temperature and temperature and biocatalyst loading. For the olive oil and entrapped lipase, it was verified that the pH was the only variable statistically significant. This study demonstrated that response surface analysis is a methodology appropriate for the maximization of the percentage of hydrolysis, as a function of pH, temperature, and lipase loading.

Performance of different immobilized lipases in palm oil biodiesel synthesis

Performance of different immobilized lipases in palm oil biodiesel synthesis. Optimized conditions for palm oil and ethanol enzymatic biodiesel synthesis were determined with different immobilized lipases SiO(2)-PVA-immobilized lipase from Pseudomonas fluorescens and acrylic resin-immobilized lipase, Novozym (R) 435, from Candida antartica, in solvent-free medium. A full factorial design assessed the influence of temperature (42 - 58 degrees C) and ethanol: palm oil (6:1 - 18:1) molar ratio on the transesterification yield. Main effects were adjusted by multiple regression analysis to linear models and the maximum transesterification yield was obtained at 42 degrees C and 18:1 ethanol: palm oil molar ratio. Mathematical models featuring total yield for each immobilized lipase were suitable to describe the experimental results.

Utilization of pineapple stem juice to enhance enzyme-hydrolytic efficiency for sugarcane bagasse after an optimized pre-treatment with alkaline peroxide

The enzymatic hydrolysis of sugarcane bagasse was investigated by treating a peroxide-alkaline bagasse with a pineapple stem juice, xylanase and cellulase. Pre-treatment procedures of sugarcane bagasse with alkaline hydrogen peroxide were evaluated and compared. Analyses were performed using 2(4) factorial designs, with pre-treatment time, temperature, magnesium sulfate and hydrogen peroxide concentration as factors. The responses evaluated were the yield of cellobiose and glucose released from pretreated bagasse after enzymatic hydrolysis. The results show that the highest enzymatic conversion was obtained for bagasse using 2% hydrogen peroxide at 60 degrees C for 16 h in the presence of 0.5% magnesium sulfate. Bagasse (5%) was treated with pineapple stem extract, which contains mixtures of protease and esterase, in combination with xylanase and cellulase. It was observed that the amount of glucose and cellobiose released from bagasse increased with the mixture of enzymes. It is believed that the enzymes present in pineapple extracts are capable of hydrolyze specific linkages that would facilitate the action of digesting plant cell walls enzymes. This increases the amount of glucose and other hexoses that are released during the enzymatic treatment and also reduces the amount of cellulase necessary in a typical hydrolysis. (C) 2010 Elsevier Ltd. All rights reserved.

Immobilization and stabilization of microbial lipases by multipoint covalent attachment on aldehyde-resin affinity: Application of the biocatalysts in biodiesel synthesis

Microbial lipase preparations from Thermomyces lanuginosus (TLL) and Pseudomonas fluorescens (PFL) were immobilized by multipoint covalent attachment on Toyopearl AF-amino-650M resin and the most active and thermal stable derivatives used to catalyze the transesterificanon reaction of babassu and palm oils with ethanol in solvent-free media For this different activating agents mainly glutaraldehyde glycidol and epichlorohydrin were used and immobilization parameters were estimated based on the hydrolysis of olive oil emulsion and butyl butyrate synthesis ILL immobilized on glyoxyl-resin allowed obtaining derivatives with the highest hydrolytic activity (HA(der)) and thermal stability between 27 and 31 times more stable than the soluble lipase Although PFL derivatives were found to be less active and thermally stables similar formation of butyl butyrate concentrations were found for both ILL and PFL derivatives The highest conversion into biodiesel was found in the transesterification of palm oil catalyzed by both ILL and PFL glyoxyl-derivatives (c) 2010 Elsevier B V All rights reserved

Biological detoxification of different hemicellulosic hydrolysates using Issatchenkia occidentalis CCTCC M 206097 yeast

This work had as its main objective to contribute to the development of a biological detoxification of hemicellulose hydrolysates obtained from different biomass plants using Issatchenkia occidentalis CCTCC M 206097 yeast. Tests with hemicellulosic hydrolysate of sugarcane bagasse in different concentrations were carried out to evaluate the influence of the hydrolysate concentration on the inhibitory compounds removal from the sugarcane bagasse hydrolysate, without reduction of sugar concentration. The highest reduction values of inhibitors concentration and less sugar losses were observed when the fivefold concentrated hydrolysate was treated by the evaluated yeast. In these experiments it was found that the high sugar concentrations favored lower sugar consumption by the yeast. The highest concentration reduction of syringaldehyde (66.67%), ferulic acid (73.33%), furfural (62%), and 5-HMF (85%) was observed when the concentrated hydrolysate was detoxified by using this yeast strain after 24 h of experimentation. The results obtained in this work showed the potential of the yeast Issatchenkia occidentalis CCTCC M 206097 as detoxification agent of hemicellulosic hydrolysate of different biomass plants.

Xylooligosaccharides Production from Alkali-Pretreated Sugarcane Bagasse Using Xylanases from Thermoascus aurantiacus

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of L-arabinose, 4-O-methyl-D-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50 degrees C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5-3.5% (w/v), enzyme load 40-80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 22 factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.

Interesterification of milkfat and soybean oil blends catalyzed by immobilized Rhizopus oryzae lipase

Milkfat (MF)/soybean oil (SBO) blends ranging from 50% to 100% of milkfat (w/w) were enzymatically interesterified with a sn-1,3 specific lipase from Rhizopus oryzae immobilized on polysiloxane-polyvinyl alcohol matrix, in a solvent free medium. Interesterification progress was monitored by following the changes in the relative proportions of 50-carbon triacylglycerols (TAGS) to 44-carbon TAGs (50/44 ratio) in the reaction. The starting materials and products were also analyzed in terms of consistency measured in a texturometer. All reactions gave interesterified (IE) products with lower consistency than non-interesterified (NIE) MF:SBO blends and interesterification degree varied from 0.54 to 2.60 in 48 h reaction. The highest interesterification degree was achieved for 65:35 MF:SBO blends, which gave 76% reduction in the consistency. These results showed the potential of the immobilized lipase to change the TAGs profile of the MF:SBO blend allowing to obtain cold-spreadable milkfat. (C) 2010 Elsevier B.V. All rights reserved.

Screening of lipases for the synthesis of xylitol monoesters by chemoenzymatic esterification and the potential of microwave and ultrasound irradiations to enhance the reaction rate

Lipases from different sources, Pseudomonas fluorescens (AK lipase), Burkholderia cepacia (PS lipase), Penicillium camembertii (lipase G) and Porcine pancreas lipase (PPL), previously immobilized on epoxy SiO(2)-PVA, were screened for the synthesis of xylitol monoesters by esterification of the protected xylitol using oleic acid as acyl donor group. Among all immobilized derivatives, the highest esterification yield was achieved by P. camembertii lipase, showing to be attractive alternative to bulk chemical routes to satisfy increasing commercial demands. Further experiments were performed to determine the influence of fatty acids chain size on the reaction yield and the feasibility of using non-conventional heating systems (microwave and ultrasound irradiations) to enhance the reaction rate. (C) 2010 Elsevier B.V. All rights reserved.

A study on the pretreatment of a sugarcane bagasse sample with dilute sulfuric acid

Experiments based on a 2(3) central composite full factorial design were carried out in 200-ml stainless-steel containers to study the pretreatment, with dilute sulfuric acid, of a sugarcane bagasse sample obtained from a local sugar-alcohol mill. The independent variables selected for study were temperature, varied from 112.5A degrees C to 157.5A degrees C, residence time, varied from 5.0 to 35.0 min, and sulfuric acid concentration, varied from 0.0% to 3.0% (w/v). Bagasse loading of 15% (w/w) was used in all experiments. Statistical analysis of the experimental results showed that all three independent variables significantly influenced the response variables, namely the bagasse solubilization, efficiency of xylose recovery in the hemicellulosic hydrolysate, efficiency of cellulose enzymatic saccharification, and percentages of cellulose, hemicellulose, and lignin in the pretreated solids. Temperature was the factor that influenced the response variables the most, followed by acid concentration and residence time, in that order. Although harsher pretreatment conditions promoted almost complete removal of the hemicellulosic fraction, the amount of xylose recovered in the hemicellulosic hydrolysate did not exceed 61.8% of the maximum theoretical value. Cellulose enzymatic saccharification was favored by more efficient removal of hemicellulose during the pretreatment. However, detoxification of the hemicellulosic hydrolysate was necessary for better bioconversion of the sugars to ethanol.

Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology

Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2(3) full factorial design with six axial points. Temperatures ranged from 132 to 180 degrees C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l(-1) h(-1). The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of beta-glucosidase by P. stipitis. During SSF, free extracellular beta-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g. Published by Elsevier Ltd.

Biomimetic oxidative treatment of spruce wood studied by pyrolysis-molecular beam mass spectrometry coupled with multivariate analysis and (13)C-labeled tetramethylammonium hydroxide thermochemolysis: implications for fungal degradation of wood

In this work, pyrolysis-molecular beam mass spectrometry analysis coupled with principal components analysis and (13)C-labeled tetramethylammonium hydroxide thermochemolysis were used to study lignin oxidation, depolymerization, and demethylation of spruce wood treated by biomimetic oxidative systems. Neat Fenton and chelator-mediated Fenton reaction (CMFR) systems as well as cellulosic enzyme treatments were used to mimic the nonenzymatic process involved in wood brown-rot biodegradation. The results suggest that compared with enzymatic processes, Fenton-based treatment more readily opens the structure of the lignocellulosic matrix, freeing cellulose fibrils from the matrix. The results demonstrate that, under the current treatment conditions, Fenton and CMFR treatment cause limited demethoxylation of lignin in the insoluble wood residue. However, analysis of a water-extractable fraction revealed considerable soluble lignin residue structures that had undergone side chain oxidation as well as demethoxylation upon CMFR treatment. This research has implications for our understanding of nonenzymatic degradation of wood and the diffusion of CMFR agents in the wood cell wall during fungal degradation processes.

Search for optimum conditions of sugarcane bagasse hemicellulose extraction

A process has been elaborated for one-step low lignin content sugarcane bagasse hemicellulose extraction using alkaline solution of hydrogen peroxide. To maximize the hemicellulose yields several extraction conditions were examined applying the 2(4) factorial design: H(2)O(2) concentration from 2 to 6% (w/v), reaction time from 4 to 16 h, temperature from 20 to 60 degrees C, and magnesium sulfate absence or presence (0.5%, w/v). This approach allowed selection of conditions for the extraction of low and high lignin content hemicellulose. At midpoint the yield of hemicellulose was 94.5% with more than 88% of lignin removed. Lignin removal is suppressed at low extraction temperatures and in the absence of magnesium sulfate. Hemicellulose in 86% yield with low lignin content (5.9%) was obtained with 6% H(2)O(2) treatment for 4 h and 20 degrees C. This hemicellulose is much lighter in color than samples obtained at the midpoint condition and was found suitable for subsequent enzymatic hydrolysis. (C) 2009 Elsevier B.V. All rights reserved.

Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with kappa-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA(app)) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g(-1) of gel for GLU, 7.76 mg g(-1) of gel for GLY, and 7.65 mg g(-1) of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g(-1) of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.

Chemoenzymatic synthesis: a strategy to obtain xylitol monoesters

BACKGROUND: Fatty acid sugar esters are used as non-ionic surfactants in cosmetics, foodstuffs and pharmaceuticals. In particular, monoesters of xylitol have attracted industrial interest due to their outstanding biological activities. In this work, xylitol monoesters were obtained by chemoenzymatic synthesis, in which, first, xylitol was made soluble in organic solvent by chemo-protecting reaction, followed by enzymatic esterification reaction using different acyl donors. A commercial immobilized Candida antartica lipase was used as catalyst, and reactions with pure xylitol were carried out to generate data for comparison. RESULTS: t-BuOH was found to be the most suitable solvent to carry out esterification reactions with both pure and protected xylitol. The highest yields were obtained for reactions carried out with pure xylitol, but in this case by-products, such as di- and tri-esters isomers were formed, which required a multi-step purification process. For the systems with protected xylitol, conversions of 86%, 58% and 24% were achieved using oleic, lauric and butyric acids, respectively. The structures of the monoesters were confirmed by (13)C- and (1)H-NMR and microanalysis. CONCLUSION: The chemoenzymatic synthesis of xylitol monoesters avoided laborious downstream processing when compared with reactions performed with pure xylitol. Monoesters production from protected xylitol was shown to be a practical, economical, and clean route for this process, allowing a simple separation, because there are no other products formed besides xylitol monoesters and residual xylitol. (C) 2009 Society of Chemical Industry