The gene encoding Bacillus subtilis α-amylase was cloned into pET-21a (+). The expression level o... more The gene encoding Bacillus subtilis α-amylase was cloned into pET-21a (+). The expression level of the recombinant enzyme was 10.7-fold higher than the expression level of the native one (0.13 mg mL −1). The recombinant enzyme (His 6-rAmyKS) was purified in one step using Ni-NTA column affinity with a specific activity of 664.28 U.mg −1. The biochemical properties of the His 6-rAmyKS were determined and compared to those of the non-tagged enzyme. Interestingly, differences were found between the two enzymes mainly for the optimal temperature and pH. Experimental tests and molecular modeling confirmed that the extra residues (C-terminal His-tag fusion peptide and cleavage thrombin site) could be responsible for the slight increase in total activity and the improvement of biochemical properties of the His-tagged enzyme compared to the native one. The His 6-rAmyKS was used as an additive in breadmaking. It showed a significant effect in improving the dough texture and the bread quality.
This paper concentrates on the biodegradation capability of poly (lactic acid) blends with poly(c... more This paper concentrates on the biodegradation capability of poly (lactic acid) blends with poly(caprolactone) and polyethylene glycol in the solid state and in the liquid phase under aerobic and anaerobic conditions. To this end, blends were processed on a twin-screw extruder with a film die and to determine the efficiency of the biodegradation of polymers, quantitative (mass variations, BOD) and qualitative (DSC and SEM) analyses were made. All biopolymers were degraded in the liquid phase under aerobic conditions and the addition of 5 or 10 wt % PEG mainly increased their biodegradability with values of BOD15 about 38 and 52 mg of O2/mg CO and a weight loss of 0.65 and 1.23 wt %, respectively. Similarly, in anaerobic conditions, biopolymers with 10 wt % PEG displayed the best outcome in terms of biodegradability and the weight loss was around 0.68 wt %. DSC and SEM analyses, on the other hand, showed that PLA/PEG blends were more biodegraded by microorganisms than PLA and PLA/PCL ...
... to substrate 〚5〛 and of lipases inhibited by transition-state analogues [〚6 ... was from Jans... more ... to substrate 〚5〛 and of lipases inhibited by transition-state analogues [〚6 ... was from Jansen (Pantin, France); phosphatidylcholine, sodium deoxycholate (NaDC), sodium taurodeoxycholate (NaTDC), triacetin ... from various origins, was carried out on a solid medium containing ...
... Nature, 227 : 680-5. 17. BEN SALAH A, SAYARI A, VERGER A, GARGOURI Y (2001). Kinetic studies ... more ... Nature, 227 : 680-5. 17. BEN SALAH A, SAYARI A, VERGER A, GARGOURI Y (2001). Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique. ... Biochem Biophys Acta, 530 : 227-35. 23. MOREAU H, MOULIN A, GARGOURI Y, NOEL JP, VERGER R (1991). ...
International Journal of Biological Macromolecules, Nov 1, 2013
Chitosan biofilms, prepared by casting method at various percentage of plasticizer (PEG and glyce... more Chitosan biofilms, prepared by casting method at various percentage of plasticizer (PEG and glycerol), were evaluated for their biological, structural and thermal properties. The addition of PEG at 30% (w/w) and glycerol at 10% (w/w) to chitosan has increased the antioxidant activity of biofilm with the percentages of 22 and 26%, respectively. The increase of ferric reducing power was noted for the mixtures of chitosan-PEG (70-30) and chitosan-GLY (75-25). Additionally, the antibacterial properties of several biofilms were tested against E. coli and S. aureus. Biofilms with 70-30 and 90-10 blends ratio of chitosan-PEG and chitosan-GLY showed the best inhibitory effect against E. coli and S. aureus with 12 and 27%, respectively. All biofilms were degraded in compost in liquid and the addition of plasticizer PEG to chitosan increased his biodegradability with a value of BOD 5 about 2.33 O 2 /mg CO. FT-IR spectra showed that the addition of plasticizer promoted the interactions through hydrogen bonding as reflected on the shifting of main peaks but there is no effect on biodegradation.
Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester... more Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37°C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37°C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65°C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals.
Aerobic biodegradation of biocomposites has been studied in both solid and liquid media. The rese... more Aerobic biodegradation of biocomposites has been studied in both solid and liquid media. The research was concentrated on the biodegradation under aerobic and mesophilic conditions using poly-D-lactic acid (PDLA) and PDLA/cellulose microfibers (CMFs) samples as the sole carbon source. To determine the efficiency of the biodegradation, quantitative (mass variations, optical density (OD)) and qualitative (FTIR, NMR and SEM) analyses have been used to follow the polymer changes after degradation. The weight loss and OD of the biocomposites samples PDLA/CMFs were slower than that of pristine PDLA. The PDLA displayed the most important loss of weight (7.09%, 8.95%) compared to its initial weight and the lowest weight loss was detected in PDLA/CMF300 (1.04%, 2.19%) in solid and liquid mediums respectively. Also, the OD value of PDLA was increased from the seven days (0.381) to the last day (0.969). It appears that the major rate-determining factor affecting material degradation was its crystallinity without or with minimal assistance from abiotic factor because crystalline phases inhibit the diffusion of small water molecules. Otherwise, the Pseudomonas aeruginosa was isolated from Mediterranean soil has been found to be a novel candidate to biodegrade PDLA under mesophilic conditions.
Advances in science, technology & innovation, 2018
Recently, polysaccharides and their derivatives have become a serious alternative polymers and fo... more Recently, polysaccharides and their derivatives have become a serious alternative polymers and found increasing industrial applications.
Numerous researches have paid attention to biocomposites. Biocomposite material can be defined as... more Numerous researches have paid attention to biocomposites. Biocomposite material can be defined as a composite material obtained by products derived from renewable resources such as polylactide acid. The aim of this work was to study biodegradable composites prepared by using cellulose microfibrils (CMFs) as the reinforcement and poly (d-lactic) acid as a matrix. Five average fiber lengths, from 8 to 300 μm, were used for the biocomposite preparation. The thermomechanical properties, crystallization, and the composites morphology were characterized by the means of dynamic mechanical thermal analysis (DMTA), tensile test device, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, and scanning electron microscopy. The effect of fiber length on the thermomechanical properties and crystallinity rate of the composites was investigated. The DMTA results showed that the storage modulus increases with the addition of CMFs. The X-ray diffraction studies, performed on the biocomposites, showed that the crystallinity rate of the blends was improved.
Journal of Molecular Catalysis B-enzymatic, Dec 1, 2010
The ability of a non-commercial immobilized Staphylococcus xylosus lipase to catalyze the esterif... more The ability of a non-commercial immobilized Staphylococcus xylosus lipase to catalyze the esterification of propanol with gallic acid was investigated and the antioxidant as well as the antimicrobial activities of the ester formed were evaluated. The response surface methodology, based on a three variables Box-Behnken design (reaction temperature, enzyme amount and 1-propanol/gallic acid molar ratio), was used to optimize the experimental conditions of propylgallate synthesis. The maximum conversion yield (90% ±3.5) was obtained by using 400 IU of immobilized lipase and a propanol/gallic acid at a molar ratio of 160 at 52 • C. The obtained ester was characterized by spectroscopic methods, NMR and FTIR. The antioxidant activity of propyl gallate was evaluated and compared to the synthetic classical antioxidants, BHA and ascorbic acid, taken as references. In addition, the antimicrobial activity of the propyl gallate was tested against S. xylosus, Escherchia coli and Staphylococcus aureus using disc diffusion and macrodilution methods. Our results show that the synthesized propyl gallate ester presents a higher antioxidant and antimicrobial power than the parent gallic acid as well as the synthetic classical antioxidants.
Using the classical emulsified system and the monomolecular film technique, we compared several i... more Using the classical emulsified system and the monomolecular film technique, we compared several interfacial properties of turkey pancreatic lipase (TPL) and human pancreatic lipase (HPL). TPL, like HPL, presented the interfacial activation phenomenon when vinyl ester was used as substrate. In the absence of colipase and bile salts, using tributyrin emulsion or monomolecular films of dicaprin at low surface pressure, TPL, unlike HPL, hydrolyzes pure tributyrin emulsion as well as dicaprin films maintained at low surface pressures. TPL was also able to hydrolyze triolein emulsion in the absence of any additive and despite the accumulation of long-chain free fatty acids at the interface. The difference of behaviors between TPL and HPL can be explained by the penetration power of each enzyme. The enzyme that presents the maximal p c (TPL) interacts more efficiently with interfaces, and it is not denaturated at high interfacial energy. Turkey pancreatic lipase is more active on rac-dicaprin than HPL; a maximal ratio of 9 was found between the catalytic activities of the two lipases measured at their surface pressure optima (20 mN m À1). A kinetic study on the surface pressure dependency, stereospecificity, and regioselectivity of TPL was performed using enantiopure diglyceride (1,2-sn-dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-dicaprin) that were spread as monomolecular films at the air-water interface. At low surface pressure (15 mN m À1), TPL acts preferentially on primary carboxylic ester groups of the diglyceride isomers (1,3-dicaprin), but at high surface pressure (23 mN m À1), this enzyme prefers both adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). HPL prefers adjacent ester groups of the diglyceride isomers (1,2sn-dicaprin and 2,3-sn-dicaprin). Furthermore, TPL was found to be markedly stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin at low surface pressure (15 mN m À1), while at high surface pressure (23 mN m À1), this lipase presents a stereopreference for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. HPL is stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin both at 15 and 23 mN m À1 .
The present study investigates the biochemical characterization of an extracellular (phospho)lipa... more The present study investigates the biochemical characterization of an extracellular (phospho)lipase from a wood fungus Peziza sp. (medium optimization, inducer concentration and substrate specificity measurements). The strain was identified on the basis of ITS1/ITS4 primers. A 604 bp fragment was amplified by PCR and the obtained nucleotide sequence, showed 99% identity with the ITS region of isolates named as Peziza sp. Interestingly, Peziza sp. has both lipase and phospholipase activities with the same level which require both the presence of Ca 2+ and bile salts. Our result shows that the lipase hydrolyzes preferably the olive oil at 45 °C, pH 8. Whereas, the phospholipase activity was detected on pure PC at 45 °C, pH 9. Lipid extraction from dry biomass using chloroform/methanol (2/1) and quantitative measurement using electron microscope showed that intracellular triglycerides content was significantly high and reaches 20.88%. Gas chromatography analysis shows a majority of C18:1 (76, 98%) and C18:2 (9, 33%). Whereas, saturated fatty acids ranging C16-C20 represent only 11.5% of total lipids composition.
European Journal of Lipid Science and Technology, Oct 1, 2009
ABSTRACT The Staphylococcus xylosus strain produces without induction an original lipase named S.... more ABSTRACT The Staphylococcus xylosus strain produces without induction an original lipase named S. xylosus lipase (SXL). Since considerable interest has been given to microbial lipases for biotechnology applications like detergents, food, drugs and pharmaceutical products, improvement of their production is of great importance to reduce the final cost. This goal could be reached through the optimization of several physicochemical culture conditions. Indeed, an appropriate medium was formulated for SXL production. It was composed of 17 g/L pancreatic digest of casein, 2.5 g/L glucose, 6 g/L yeast extract, 0.75 g/L ammonium sulfate corresponding to a C/N ratio of 6, 1 g/L K2HPO4 and 1 g/L KH2PO4. In such a medium, SXL production reached 42 U/mL. Moreover, the usefulness of such a medium for large-scale production of SXL was also evidenced in an automated fully controlled 2.6-L fermenter. It was shown that aeration of the medium, which strongly affected the growth, regulated the lipase synthesis by the produced cells. It was found that when using a dissolved oxygen saturation of the medium of 50%, the SXL production reached 62 U/mL.
Journal of Colloid and Interface Science, Oct 1, 2010
The interfacial and kinetic properties of wild type, untagged recombinant and tagged recombinant ... more The interfacial and kinetic properties of wild type, untagged recombinant and tagged recombinant forms of three staphylococcal lipases (SSL, SXL and SAL3) were compared using the monomolecular film technique. A kinetic study on the dependence of the stereoselectivity of these nine lipase forms on the surface pressure was performed using the three dicaprin isomers spread in the form of monomolecular films at the air-water interface. New parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC), were introduced. The findings obtained showed that with all the lipases tested, the recombinant expression process and the N-terminal His-tag slightly affect the sn-1 preference for dicaprin enantiomers as well as the penetration capacity into monomolecular films of phosphatidylcholine but significantly decrease the catalytic rate of hydrolysis of three dicaprin isomers. This rate reduction is more pronounced at high surface pressures, i.e. at low interfacial energies. In conclusion, the effects of the heterologous expression process on the catalytic properties of the staphylococcal lipases are three times more deleterious than the presence of an N-terminal tag extension. In the case of the situation most commonly encountered in the literature, i.e. the heterologous expression of a tagged lipase, the rate of catalysis can be decreased by these processes by 42-83% on average in comparison with the values measured with the corresponding wild type form.
Abstract A recombinant Serratia sp. W3 lipase (rSmL) was expressed in E. coli and purified to hom... more Abstract A recombinant Serratia sp. W3 lipase (rSmL) was expressed in E. coli and purified to homogeneity. The kinetic properties, regio-selectivity, and interfacial performances of rSmL were compared with those of the native lipase (nSmL). rSmL has a molecular mass of 67 kDa and a specific activity of 3530 U mg−1 which is around 12-fold higher than that of nSmL (300 U mg−1) when using olive oil as the substrate. Both rSmL and nSmL were able to hydrolyze the ester bond at the sn-1 and sn-3 positions but exhibited a clear regio-preference towards the sn-3 position of the surface-coated triglycerides (TG), which were esterified with α-eleostearic acid at the sn-1/3 position or dicaprin isomers spread as monomolecular films. Molecular modeling and docking of TG into the active site of rSmL indicated that this regio-preference may be due to steric hindrance, created by the residues Ile308 and Trp311, with distances between the sn-1 reactive carbon and the catalytic Serine residue ranging from 3.15 to 5.47 A. In contrast, the sn-3 positioning within the enzyme catalytic pocket resulted in shorter distances, ranging from 2.79 to 4.15 A, therefore facilitating the hydrolysis at the sn-3 position.
The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR t... more The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR technique. The sequence corresponding to the mature lipase was subcloned in the pET-14b expression vector, with a strong T7 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. High level expression of the lipase by Escherichia coli BL21 (DE3) cells harbouring the lipase gene containing expression vector was observed upon induction with 0.4 mM IPTG at 37 degrees C. One-step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified His-tagged SXL was 1500 or 850 U/mg using tributyrin or olive oil emulsion as substrate, respectively. It has been proposed that the region near the residue Asp290 could be involved in the selection of the substrate. Therefore, we also mutated the residue Asp 290 by Ala using site-directed mutagenesis. The mutant SXL-D290A was overexpressed in E. coli BL21 (DE3) and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged SXL-D290A mutant was 1000 U/mg using either tributyrin or olive oil emulsion as substrate. A comparative study of the wild type (His(6)-SXL) and the mutant (His(6)-SXL-D290A) proteins was carried out. Our results confirmed that Asp290 is important for the chain length specificity and catalytic efficiency of the enzyme.
In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the... more In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the concentrated culture medium was stored at 0°C during several months or kept at 6°C during a few days, we noticed the appearance of a second shorter form of ROL32 lacking its N-terminal 28 amino acid (ROL29). ROL29 was purified to homogeneity and its 21 N-terminal amino acid residues were found to be identical to the 29-49 sequence of ROL32. The cleavage of the N-terminal peptide reduced the specific activity of ROL29 by 50% using either triolein or tributyrin as substrates. In order to explain this decrease of the specific activity of ROL29, we measured its critical surface pressure of penetration into phosphatidyl choline from egg yolk films which was found to be 10 mN/m, in contrast to a value of 23 mN/m found in ROL32. A kinetic study on the surface pressure dependency, stereoselectivity and regioselectivity of ROL29 was performed using the three dicaprin isomers spread as monomolecular films at the air-water interface. Our results showed that in contrast to ROL32, ROL29 presented a preference for the distal ester groups of one diglyceride isomer (1,3-sn-dicaprin). Furthermore, ROL32 was markedly more stereoselective than ROL29 for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. A structural explanation of the enhanced penetration capacity as well as the catalytic activity of ROL32 was proposed by molecular modeling. We concluded that the N-terminal peptide of ROL32 can play an important role in the specific activity, the regioselectivity, the stereoselectivity and the binding of the enzyme to its substrate.
Journal of Molecular Catalysis B-enzymatic, Apr 1, 2009
A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture m... more A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture medium. The extracellular lipase from S. aureus (SAL3) is purified to homogeneity. The purified enzyme is a tetrameric protein (180kDa) corresponding to the association of four lipase molecules. The 15 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. The part
The gene encoding Bacillus subtilis α-amylase was cloned into pET-21a (+). The expression level o... more The gene encoding Bacillus subtilis α-amylase was cloned into pET-21a (+). The expression level of the recombinant enzyme was 10.7-fold higher than the expression level of the native one (0.13 mg mL −1). The recombinant enzyme (His 6-rAmyKS) was purified in one step using Ni-NTA column affinity with a specific activity of 664.28 U.mg −1. The biochemical properties of the His 6-rAmyKS were determined and compared to those of the non-tagged enzyme. Interestingly, differences were found between the two enzymes mainly for the optimal temperature and pH. Experimental tests and molecular modeling confirmed that the extra residues (C-terminal His-tag fusion peptide and cleavage thrombin site) could be responsible for the slight increase in total activity and the improvement of biochemical properties of the His-tagged enzyme compared to the native one. The His 6-rAmyKS was used as an additive in breadmaking. It showed a significant effect in improving the dough texture and the bread quality.
This paper concentrates on the biodegradation capability of poly (lactic acid) blends with poly(c... more This paper concentrates on the biodegradation capability of poly (lactic acid) blends with poly(caprolactone) and polyethylene glycol in the solid state and in the liquid phase under aerobic and anaerobic conditions. To this end, blends were processed on a twin-screw extruder with a film die and to determine the efficiency of the biodegradation of polymers, quantitative (mass variations, BOD) and qualitative (DSC and SEM) analyses were made. All biopolymers were degraded in the liquid phase under aerobic conditions and the addition of 5 or 10 wt % PEG mainly increased their biodegradability with values of BOD15 about 38 and 52 mg of O2/mg CO and a weight loss of 0.65 and 1.23 wt %, respectively. Similarly, in anaerobic conditions, biopolymers with 10 wt % PEG displayed the best outcome in terms of biodegradability and the weight loss was around 0.68 wt %. DSC and SEM analyses, on the other hand, showed that PLA/PEG blends were more biodegraded by microorganisms than PLA and PLA/PCL ...
... to substrate 〚5〛 and of lipases inhibited by transition-state analogues [〚6 ... was from Jans... more ... to substrate 〚5〛 and of lipases inhibited by transition-state analogues [〚6 ... was from Jansen (Pantin, France); phosphatidylcholine, sodium deoxycholate (NaDC), sodium taurodeoxycholate (NaTDC), triacetin ... from various origins, was carried out on a solid medium containing ...
... Nature, 227 : 680-5. 17. BEN SALAH A, SAYARI A, VERGER A, GARGOURI Y (2001). Kinetic studies ... more ... Nature, 227 : 680-5. 17. BEN SALAH A, SAYARI A, VERGER A, GARGOURI Y (2001). Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique. ... Biochem Biophys Acta, 530 : 227-35. 23. MOREAU H, MOULIN A, GARGOURI Y, NOEL JP, VERGER R (1991). ...
International Journal of Biological Macromolecules, Nov 1, 2013
Chitosan biofilms, prepared by casting method at various percentage of plasticizer (PEG and glyce... more Chitosan biofilms, prepared by casting method at various percentage of plasticizer (PEG and glycerol), were evaluated for their biological, structural and thermal properties. The addition of PEG at 30% (w/w) and glycerol at 10% (w/w) to chitosan has increased the antioxidant activity of biofilm with the percentages of 22 and 26%, respectively. The increase of ferric reducing power was noted for the mixtures of chitosan-PEG (70-30) and chitosan-GLY (75-25). Additionally, the antibacterial properties of several biofilms were tested against E. coli and S. aureus. Biofilms with 70-30 and 90-10 blends ratio of chitosan-PEG and chitosan-GLY showed the best inhibitory effect against E. coli and S. aureus with 12 and 27%, respectively. All biofilms were degraded in compost in liquid and the addition of plasticizer PEG to chitosan increased his biodegradability with a value of BOD 5 about 2.33 O 2 /mg CO. FT-IR spectra showed that the addition of plasticizer promoted the interactions through hydrogen bonding as reflected on the shifting of main peaks but there is no effect on biodegradation.
Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester... more Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37°C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37°C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65°C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals.
Aerobic biodegradation of biocomposites has been studied in both solid and liquid media. The rese... more Aerobic biodegradation of biocomposites has been studied in both solid and liquid media. The research was concentrated on the biodegradation under aerobic and mesophilic conditions using poly-D-lactic acid (PDLA) and PDLA/cellulose microfibers (CMFs) samples as the sole carbon source. To determine the efficiency of the biodegradation, quantitative (mass variations, optical density (OD)) and qualitative (FTIR, NMR and SEM) analyses have been used to follow the polymer changes after degradation. The weight loss and OD of the biocomposites samples PDLA/CMFs were slower than that of pristine PDLA. The PDLA displayed the most important loss of weight (7.09%, 8.95%) compared to its initial weight and the lowest weight loss was detected in PDLA/CMF300 (1.04%, 2.19%) in solid and liquid mediums respectively. Also, the OD value of PDLA was increased from the seven days (0.381) to the last day (0.969). It appears that the major rate-determining factor affecting material degradation was its crystallinity without or with minimal assistance from abiotic factor because crystalline phases inhibit the diffusion of small water molecules. Otherwise, the Pseudomonas aeruginosa was isolated from Mediterranean soil has been found to be a novel candidate to biodegrade PDLA under mesophilic conditions.
Advances in science, technology & innovation, 2018
Recently, polysaccharides and their derivatives have become a serious alternative polymers and fo... more Recently, polysaccharides and their derivatives have become a serious alternative polymers and found increasing industrial applications.
Numerous researches have paid attention to biocomposites. Biocomposite material can be defined as... more Numerous researches have paid attention to biocomposites. Biocomposite material can be defined as a composite material obtained by products derived from renewable resources such as polylactide acid. The aim of this work was to study biodegradable composites prepared by using cellulose microfibrils (CMFs) as the reinforcement and poly (d-lactic) acid as a matrix. Five average fiber lengths, from 8 to 300 μm, were used for the biocomposite preparation. The thermomechanical properties, crystallization, and the composites morphology were characterized by the means of dynamic mechanical thermal analysis (DMTA), tensile test device, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, and scanning electron microscopy. The effect of fiber length on the thermomechanical properties and crystallinity rate of the composites was investigated. The DMTA results showed that the storage modulus increases with the addition of CMFs. The X-ray diffraction studies, performed on the biocomposites, showed that the crystallinity rate of the blends was improved.
Journal of Molecular Catalysis B-enzymatic, Dec 1, 2010
The ability of a non-commercial immobilized Staphylococcus xylosus lipase to catalyze the esterif... more The ability of a non-commercial immobilized Staphylococcus xylosus lipase to catalyze the esterification of propanol with gallic acid was investigated and the antioxidant as well as the antimicrobial activities of the ester formed were evaluated. The response surface methodology, based on a three variables Box-Behnken design (reaction temperature, enzyme amount and 1-propanol/gallic acid molar ratio), was used to optimize the experimental conditions of propylgallate synthesis. The maximum conversion yield (90% ±3.5) was obtained by using 400 IU of immobilized lipase and a propanol/gallic acid at a molar ratio of 160 at 52 • C. The obtained ester was characterized by spectroscopic methods, NMR and FTIR. The antioxidant activity of propyl gallate was evaluated and compared to the synthetic classical antioxidants, BHA and ascorbic acid, taken as references. In addition, the antimicrobial activity of the propyl gallate was tested against S. xylosus, Escherchia coli and Staphylococcus aureus using disc diffusion and macrodilution methods. Our results show that the synthesized propyl gallate ester presents a higher antioxidant and antimicrobial power than the parent gallic acid as well as the synthetic classical antioxidants.
Using the classical emulsified system and the monomolecular film technique, we compared several i... more Using the classical emulsified system and the monomolecular film technique, we compared several interfacial properties of turkey pancreatic lipase (TPL) and human pancreatic lipase (HPL). TPL, like HPL, presented the interfacial activation phenomenon when vinyl ester was used as substrate. In the absence of colipase and bile salts, using tributyrin emulsion or monomolecular films of dicaprin at low surface pressure, TPL, unlike HPL, hydrolyzes pure tributyrin emulsion as well as dicaprin films maintained at low surface pressures. TPL was also able to hydrolyze triolein emulsion in the absence of any additive and despite the accumulation of long-chain free fatty acids at the interface. The difference of behaviors between TPL and HPL can be explained by the penetration power of each enzyme. The enzyme that presents the maximal p c (TPL) interacts more efficiently with interfaces, and it is not denaturated at high interfacial energy. Turkey pancreatic lipase is more active on rac-dicaprin than HPL; a maximal ratio of 9 was found between the catalytic activities of the two lipases measured at their surface pressure optima (20 mN m À1). A kinetic study on the surface pressure dependency, stereospecificity, and regioselectivity of TPL was performed using enantiopure diglyceride (1,2-sn-dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-dicaprin) that were spread as monomolecular films at the air-water interface. At low surface pressure (15 mN m À1), TPL acts preferentially on primary carboxylic ester groups of the diglyceride isomers (1,3-dicaprin), but at high surface pressure (23 mN m À1), this enzyme prefers both adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). HPL prefers adjacent ester groups of the diglyceride isomers (1,2sn-dicaprin and 2,3-sn-dicaprin). Furthermore, TPL was found to be markedly stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin at low surface pressure (15 mN m À1), while at high surface pressure (23 mN m À1), this lipase presents a stereopreference for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. HPL is stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin both at 15 and 23 mN m À1 .
The present study investigates the biochemical characterization of an extracellular (phospho)lipa... more The present study investigates the biochemical characterization of an extracellular (phospho)lipase from a wood fungus Peziza sp. (medium optimization, inducer concentration and substrate specificity measurements). The strain was identified on the basis of ITS1/ITS4 primers. A 604 bp fragment was amplified by PCR and the obtained nucleotide sequence, showed 99% identity with the ITS region of isolates named as Peziza sp. Interestingly, Peziza sp. has both lipase and phospholipase activities with the same level which require both the presence of Ca 2+ and bile salts. Our result shows that the lipase hydrolyzes preferably the olive oil at 45 °C, pH 8. Whereas, the phospholipase activity was detected on pure PC at 45 °C, pH 9. Lipid extraction from dry biomass using chloroform/methanol (2/1) and quantitative measurement using electron microscope showed that intracellular triglycerides content was significantly high and reaches 20.88%. Gas chromatography analysis shows a majority of C18:1 (76, 98%) and C18:2 (9, 33%). Whereas, saturated fatty acids ranging C16-C20 represent only 11.5% of total lipids composition.
European Journal of Lipid Science and Technology, Oct 1, 2009
ABSTRACT The Staphylococcus xylosus strain produces without induction an original lipase named S.... more ABSTRACT The Staphylococcus xylosus strain produces without induction an original lipase named S. xylosus lipase (SXL). Since considerable interest has been given to microbial lipases for biotechnology applications like detergents, food, drugs and pharmaceutical products, improvement of their production is of great importance to reduce the final cost. This goal could be reached through the optimization of several physicochemical culture conditions. Indeed, an appropriate medium was formulated for SXL production. It was composed of 17 g/L pancreatic digest of casein, 2.5 g/L glucose, 6 g/L yeast extract, 0.75 g/L ammonium sulfate corresponding to a C/N ratio of 6, 1 g/L K2HPO4 and 1 g/L KH2PO4. In such a medium, SXL production reached 42 U/mL. Moreover, the usefulness of such a medium for large-scale production of SXL was also evidenced in an automated fully controlled 2.6-L fermenter. It was shown that aeration of the medium, which strongly affected the growth, regulated the lipase synthesis by the produced cells. It was found that when using a dissolved oxygen saturation of the medium of 50%, the SXL production reached 62 U/mL.
Journal of Colloid and Interface Science, Oct 1, 2010
The interfacial and kinetic properties of wild type, untagged recombinant and tagged recombinant ... more The interfacial and kinetic properties of wild type, untagged recombinant and tagged recombinant forms of three staphylococcal lipases (SSL, SXL and SAL3) were compared using the monomolecular film technique. A kinetic study on the dependence of the stereoselectivity of these nine lipase forms on the surface pressure was performed using the three dicaprin isomers spread in the form of monomolecular films at the air-water interface. New parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC), were introduced. The findings obtained showed that with all the lipases tested, the recombinant expression process and the N-terminal His-tag slightly affect the sn-1 preference for dicaprin enantiomers as well as the penetration capacity into monomolecular films of phosphatidylcholine but significantly decrease the catalytic rate of hydrolysis of three dicaprin isomers. This rate reduction is more pronounced at high surface pressures, i.e. at low interfacial energies. In conclusion, the effects of the heterologous expression process on the catalytic properties of the staphylococcal lipases are three times more deleterious than the presence of an N-terminal tag extension. In the case of the situation most commonly encountered in the literature, i.e. the heterologous expression of a tagged lipase, the rate of catalysis can be decreased by these processes by 42-83% on average in comparison with the values measured with the corresponding wild type form.
Abstract A recombinant Serratia sp. W3 lipase (rSmL) was expressed in E. coli and purified to hom... more Abstract A recombinant Serratia sp. W3 lipase (rSmL) was expressed in E. coli and purified to homogeneity. The kinetic properties, regio-selectivity, and interfacial performances of rSmL were compared with those of the native lipase (nSmL). rSmL has a molecular mass of 67 kDa and a specific activity of 3530 U mg−1 which is around 12-fold higher than that of nSmL (300 U mg−1) when using olive oil as the substrate. Both rSmL and nSmL were able to hydrolyze the ester bond at the sn-1 and sn-3 positions but exhibited a clear regio-preference towards the sn-3 position of the surface-coated triglycerides (TG), which were esterified with α-eleostearic acid at the sn-1/3 position or dicaprin isomers spread as monomolecular films. Molecular modeling and docking of TG into the active site of rSmL indicated that this regio-preference may be due to steric hindrance, created by the residues Ile308 and Trp311, with distances between the sn-1 reactive carbon and the catalytic Serine residue ranging from 3.15 to 5.47 A. In contrast, the sn-3 positioning within the enzyme catalytic pocket resulted in shorter distances, ranging from 2.79 to 4.15 A, therefore facilitating the hydrolysis at the sn-3 position.
The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR t... more The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR technique. The sequence corresponding to the mature lipase was subcloned in the pET-14b expression vector, with a strong T7 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. High level expression of the lipase by Escherichia coli BL21 (DE3) cells harbouring the lipase gene containing expression vector was observed upon induction with 0.4 mM IPTG at 37 degrees C. One-step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified His-tagged SXL was 1500 or 850 U/mg using tributyrin or olive oil emulsion as substrate, respectively. It has been proposed that the region near the residue Asp290 could be involved in the selection of the substrate. Therefore, we also mutated the residue Asp 290 by Ala using site-directed mutagenesis. The mutant SXL-D290A was overexpressed in E. coli BL21 (DE3) and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged SXL-D290A mutant was 1000 U/mg using either tributyrin or olive oil emulsion as substrate. A comparative study of the wild type (His(6)-SXL) and the mutant (His(6)-SXL-D290A) proteins was carried out. Our results confirmed that Asp290 is important for the chain length specificity and catalytic efficiency of the enzyme.
In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the... more In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the concentrated culture medium was stored at 0°C during several months or kept at 6°C during a few days, we noticed the appearance of a second shorter form of ROL32 lacking its N-terminal 28 amino acid (ROL29). ROL29 was purified to homogeneity and its 21 N-terminal amino acid residues were found to be identical to the 29-49 sequence of ROL32. The cleavage of the N-terminal peptide reduced the specific activity of ROL29 by 50% using either triolein or tributyrin as substrates. In order to explain this decrease of the specific activity of ROL29, we measured its critical surface pressure of penetration into phosphatidyl choline from egg yolk films which was found to be 10 mN/m, in contrast to a value of 23 mN/m found in ROL32. A kinetic study on the surface pressure dependency, stereoselectivity and regioselectivity of ROL29 was performed using the three dicaprin isomers spread as monomolecular films at the air-water interface. Our results showed that in contrast to ROL32, ROL29 presented a preference for the distal ester groups of one diglyceride isomer (1,3-sn-dicaprin). Furthermore, ROL32 was markedly more stereoselective than ROL29 for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. A structural explanation of the enhanced penetration capacity as well as the catalytic activity of ROL32 was proposed by molecular modeling. We concluded that the N-terminal peptide of ROL32 can play an important role in the specific activity, the regioselectivity, the stereoselectivity and the binding of the enzyme to its substrate.
Journal of Molecular Catalysis B-enzymatic, Apr 1, 2009
A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture m... more A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture medium. The extracellular lipase from S. aureus (SAL3) is purified to homogeneity. The purified enzyme is a tetrameric protein (180kDa) corresponding to the association of four lipase molecules. The 15 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. The part
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