Fakher Frikha

Application of response surface methodology to optimize decolourization of dyes by the laccase-mediator system.

Response surface methodology (RSM) was applied to optimize the decolourization of 3 dyes belonging to 3 dye families such as reactive black 5 (diazoic), indigo carmine (indigoid) and aniline blue (anthraquinonic). Crude laccase from Trametes trogii and the laccase-mediator 1-hydroxybenzotriazole (HBT) were used in this study. Box-Behnken design using RSM with six variables namely pH, temperature, enzyme concentration, HBT concentration, dye concentration and incubation time was used in this study to optimize significant correlation between the effects of these variables on the decolourization of reactive black 5 (RB5), indigo carmine (IC) and aniline blue (AB). The optimum of pH, temperature, laccase, HBT, RB5 and reaction time were 4.5, 0.5 U ml(-1), 0.5 mM, 100 mg ml(-1) and 150 min respectively, for a maximum decolourization of RB5 (about 92.92% ± 7.21). Whereas, the optimum decolourization conditions of both IC (99.76% ± 7.75) and AB (98.44% ± 10) were: pH 4.5, temperature of 45 °C, enzyme concentration of 0.1 U ml(-1) and 0.5 U ml(-1), HBT concentration of 0.9 mM and 0.5 mM, dye concentration of 60 mg l(-1) and reaction time of 150 and 90 min, respectively. The experimental values were in good agreement with the predicted ones and the models were highly significant, the correlation coefficient (R(2)) being 0.864, 0.663 and 0.776 for RB5, IC and AB, respectively. In addition, when the kinetic parameters for the three dyes decolourization were calculated according to Hannes-Wolf plot, the following values were obtained: Km of 268.4, 47.94 and 44.64 mg l(-1) then V(max) of 35.58, 10.43 and 9.23 mg l(-1) min for the RB5, IC and AB decolourizations by laccase, respectively.

Sawdust waste as a low-cost support-substrate for laccases production and adsorbent for azo dyes decolorization.

BackgroundLaccases are multicopper oxidases with high potential for environmental and industrial applications. Low-cost laccase production could be achieved by solid state fermentation on agro-industrial by-products.MethodsA number of agro-industrial solid wastes were tested as support-substrate for laccase production by Coriolopsis gallica under solid-state fermentation (SSF) conditions. Response surface methodology (RSM) was used to optimize the medium composition for laccase production. Initial screening by Plackett-Burman design was performed to select the major variables out of 20 tow medium components fellowing this Central composite design (CCD) was employed to optimize the level of the selected variables.ResultsSawdust waste was shown to be the best support-substrate for laccase production by the C. gallica. Peptone as source of organic nitrogen, Cd2+ as laccase inducer and liquid/solid (L/S) ratio were found to have significant effects on laccase production. Operating at optimum concentrations of the most significant variables (peptone, 4.5xa0gxa0L−1, L/S ratio, 5.0 and Cd+2 1.0xa0mM) extracellular laccase activity was enhanced from 1480 U L−1 (60.5 U g−1), to 4880 U L−1 (200 U g−1) which meant a 3.2-fold increase in laccase activity. On the other hand, sawdust waste was studied as a low cost adsorbent to remove the azo dyes Reactive Black 5 (RB5) and Acid Orange 51 (AO51). Decolorization percentages around 67 and 75xa0% were obtained in 24xa0h for RB5 and AO51, respectively.ConclusionWhen used as a support substrate, sawdust yielded the highest laccase production which was increased 3.2 times using RMS optimization.

Decolorization of the azo dye Acid Orange 51 by laccase produced in solid culture of a newly isolated Trametes trogii strain

This study concerns the decolorization and detoxification of the azo dye Acid Orange 51 (AO51) by crude laccase from Trametes trogii produced in solid culture using sawdust as support media. A three-level Box–Behnken factorial design with four factors (enzyme concentration, 1-hydroxybenzotriazole (HBT) concentration, dye concentration and reaction time) combined with response surface methodology was applied to optimize AO51 decolorization. A mathematical model was developed showing the effect of each factor and their interactions on color removal. The model predicted that Acid Orange 51 decolorization above 87.87xa0±xa01.27xa0% could be obtained when enzyme concentration, HBT concentration, dye concentration and reaction time were set at 1xa0U/mL, 0.75xa0mM, 60xa0mg/L and 2xa0days, respectively. The experimental values were in good agreement with the predicted ones and the models were highly significant, the correlation coefficient (R2) being 0.9. Then the desirability function was employed to determine the optimal decolorization condition for each dye and minimize the process cost simultaneously. In addition, germination index assay showed that laccase-treated dye was detoxified; however in the presence of HBT, the phytotoxicity of the treated dye was increased. By using cheap agro-industrial wastes, such as sawdust, a potential laccase was obtained. The low cost of laccase production may further broaden its application in textile wastewater treatment.

Gene cloning and molecular characterization of the Talaromyces thermophilus lipase Catalyzed efficient hydrolysis and synthesis of esters

A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083bp encoding a protein of 269 Aa with an estimated molecular mass of 30kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides.

Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2

BackgroundSecretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be highly expressed in the intestine of mammals. However, no study was reported in birds.ResultsChicken intestinal group IIA phospholipase A2 (ChPLA2-IIA) was obtained after an acidic treatment (pH.3.0), precipitation by ammonium sulphate, followed by sequential column chromatographies on Sephadex G-50 and mono-S ion exchanger. The enzyme was found to be a monomeric protein with a molecular mass of around 14 kDa. The purified enzyme showed a substrate preference for phosphatidylethanolamine and phosphatidylglycerol, and didnt hydrolyse phosphatidylcholine. Under optimal assay conditions, in the presence of 10 mM NaTDC and 10 mM CaCl2, a specific activity of 160 U.mg-1 for purified ChPLA2-IIA was measured using egg yolk as substrate. The fifteen NH2-terminal amino acid residues of ChPLA2-IIA were sequenced and showed a close homology with known intestinal secreted phospholipases A2. The gene encoding the mature ChPLA2-IIA was cloned and sequenced. To further investigate structure-activity relationship, a 3D model of ChPLA2-IIA was built using the human intestinal phospholipase A2 structure as template.ConclusionChPLA2-IIA was purified to homogeneity using only two chromatographic colomns. Sequence analysis of the cloned cDNA indicates that the enzyme is highly basic with a pI of 9.0 and has a high degree of homology with mammalian intestinal PLA2-IIA.

Decolorization and detoxification of two textile industry effluents by the laccase/1-hydroxybenzotriazole system

The aim of this work was to determine the optimal conditions for the decolorization and the detoxification of two effluents from a textile industry—effluent A (the reactive dye bath Bezactive) and effluent B (the direct dye bath Tubantin)—using a laccase mediator system. Response surface methodology (RSM) was applied to optimize textile effluents decolorization. A Box–Behnken design using RSM with the four variables pH, effluent concentration, 1-hydroxybenzotriazole (HBT) concentration, and enzyme (laccase) concentration was used to determine correlations between the effects of these variables on the decolorization of the two effluents. The optimum conditions for pH and concentrations of HBT, effluent and laccase were 5, 1xa0mM, 50xa0% and 0.6xa0U/ml, respectively, for maximum decolorization of effluent A (68xa0%). For effluent B, optima were 4, 1xa0mM, 75xa0%, and 0.6xa0U/ml, respectively, for maximum decolorization of approximately 88xa0%. Both effluents were treated at 30xa0°C for 20xa0h. A quadratic model was obtained for each decolorization through this design. The experimental and predicted values were in good agreement and both models were highly significant. In addition, the toxicity of the two effluents was determined before and after laccase treatment using Saccharomyces cerevisiae, Bacillus cereus, and germination of tomato seeds.

Immobilized Rhizopus oryzae lipase catalyzed synthesis of palm stearin and cetyl alcohol wax esters: Optimization by Response Surface Methodology

BackgroundWaxes are esters of long-chain fatty acids and long-chain alcohols. Their principal natural sources are animals (sperm whale oil) and vegetables (jojoba) which are expensive and not easily available. Wax esters synthesized by enzymatic transesterification, using palm stearin as raw material, can be considered as an alternative to natural ones.ResultsPalm stearin is a solid fraction obtained by fractionation of palm oil. Palm stearin was esterified with cetyl alcohol to produce a mixture of wax esters. A non-commercial immobilized lipase from Rhizopus oryzae was used as biocatalyst. Response surface methodology was employed to determine the effects of the temperature (30-50°C), the enzyme concentration (33.34-300 IU/mL), the alcohol/palm stearin molar ratio (3-7 mol/mol) and the substrate concentration (0.06-0.34 g/mL) on the conversion yield of palm stearin. Under optimal conditions (temperature, 30°C; enzyme concentration, 300 IU/mL; molar ratio 3 and substrate concentration 0.21 g/mL) a high conversion yield of 98.52% was reached within a reaction time of 2 h.ConclusionsResponse surface methodology was successfully applied to determine the optimum operational conditions for synthesis of palm stearin based wax esters. This study may provide useful tools to develop economical and efficient processes for the synthesis of wax esters.

Culture of Staphylococcus xylosus in fish processing by-product-based media for lipase production

Aims:u2002 The objective of this study was to demonstrate that fish‐processing by‐products could be used as sole raw material to sustain the growth of Staphylococcus xylosus for lipase production.

Kinetic and structural characterization of triacylglycerol lipases possessing phospholipase A1 activity.

The pancreatic lipase gene family displays various substrate selectivities for triglycerides and phospholipids. The structural basis for this difference in substrate specificity has not been definitively established. Based on a kinetic comparative study between various pancreatic lipase family members, we showed here that porcine pancreatic lipase (PPL), which was so far classified as classical lipase, was able to hydrolyze phosphatidylcholine (PC). Amino acid sequence alignments revealed that Val260 residue in PPL lid could be critical for the interaction with lipid substrate. Molecular dynamics was applied to investigate PC binding modes within the catalytic cavity of PPL and human pancreatic lipase (HPL), aiming to explain the difference of specificity of these enzymes towards phospholipids. Results showed that with HPL, the oxyanion hole was not able to accommodate the PC molecule, suggesting that no activity could be obtained. With PPL, the formation of a large pocket involving Val260 allowed the PC molecule to come near the catalytic residues, suggesting that it could be hydrolyzed. One more interesting finding is that human pancreatic lipase related protein 2 could hydrolyze phospholipids through its PLA1 and PLA2 activities. Overall, our study shed the light on new structural features of the phospholipase activity of pancreatic lipase family members.

Enzymatic transesterification of palm stearin and olein blends to produce zero-trans margarine fat

BackgroundFood industries aim to replace trans fat in their products by formulations having equivalent functionality and economic viability. Enzymatic transesterification can be a technological option to produce trans free fats targeting commercial applications.ResultsPalm stearin and palm olein blends in different ratios were enzymatically transesterified in a solvent free system using a Rhizopus oryzae lipase immobilised onto CaCO3 to produce a suitable fat for margarine formulation. Slip melting points and triacylglycerols profiles were evaluated upon transesterification. Results indicated that all transesterified blends had lower slip melting points than their non transesterified counterparts. Furthermore, the triacylglycerols profile showed a decrease in the concentration of the high melting point triacylglycerols. The rheological analysis showed that margarine prepared with the transesterified blend showed a better spreadability than that of a control margarine prepared with non transesterified fat. Adding powder of dry bark orange to margarine preparation improved its colour and fairly affected its spreadability and rheological behaviour. The margarine prepared with transesterified fat displayed a rheological behaviour that was comparable to that of commercial sample.ConclusionsThis study is an ecofriendly approach to the utilization of relatively low value bioresources like palm stearin and palm olein for making margarine free of trans fatty acids that are now implicated as risk factor for heart diseases.