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). ...
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.
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.
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). ...
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.
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.
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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