Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Optimization of newly isolated Bacillus cereus α-amylase production using orange peels and crab shells and application in wastewater treatment.

A newly isolated amylolytic strain was identified as Bacillus cereus spH1 based on 16S and 16-23S gene sequencing (Accession numbers OP811441.1 and OP819558, respectively), optimization strategies, using one variable at time (OVAT) and Plackett-Burman design, were employed to improve the alpha-amylase (α-amylase) production. Condition inferred revealed that the optimal physical parameters for maximum enzyme production were 30 °C, pH 7.5, and 12 h of incubation, using tryptone, malt extract, orange (Citrus sinensis) peels, crab (Portunus segnis) shells, calcium, and sodium chloride (NaCl) as culture medium. The full factorial design (FFD) model was observed to possess a predicted R2 and adjusted R2 values of 0.9788 and 0.9862, respectively, and it can effectively predict the response variables (p = 0). Following such efforts, α-amylase activity was increased 141.6-folds, ranging from 0.06 to 8.5 U/mL. The ideal temperature and pH for the crude enzyme activity were 65 °C and 7.5, respectively. The enzyme exhibited significant stability, with residual activity over 90% at 55 °C. The maltose was the only product generated during the starch hydrolysis. Moreover, the Bacillus cereus spH1 strain and its α-amylase were used in the treatment of effluents from the pasta industry. Germination index percentages of 143% and 139% were achieved when using the treated effluent with α-amylase and the strain, respectively. This work proposes the valorization of agro-industrial residues to improve enzyme production and to develop a green and sustainable approach that holds great promise for environmental and economic challenges.

Expression and characterization of an organic solvent tolerant recombinant lipase from Staphylococcus capitis SH6 for food wastewater treatment.

The study illustrated here aims on an organic solvent tolerant lipase from Staphylococcus capitis (SCL). The gene part, encoding the mature lipase, was cloned and sequenced. The concluded polypeptide sequence, equivalent to the protein, consist of 388 amino acid residues with a molecular mass of about 45 kDa. A structure-based alignment of the SCL amino acid sequence shows high identities with those many staphylococcal lipases. From this alignment of sequences, the catalytic triad (Ser 117, Asp 308 and His 347) of SCL could be identified. The mature part of the SCL was expressed in Escherichia coli and the recombinant lipase (r-SCL) was purified to homogeneity. The purified r-SCL presented a quite interesting stability at low temperatures (< 30 °C) and the enzyme was found to be highly stable in polar organic solvent and at a pH ranging from 3 to 12. After that, we have demonstrated that the recombinant enzyme may be implicated in the biodegradability of oily wastewater from effluents of fast-food restaurants; the maximum conversion yield into fatty acids obtained at 30 °C, was 65%.

Optimization using response surface methodology of phospholipase C production from Bacillus cereus suitable for soybean oil degumming.

This work deals with the optimization of an extracellular phospholipase C production by Bacillus cereus (PLCBc) using Response Surface Methodology (RMS) and Box-Behnken design. In fact, after optimization, a maximum phospholipase activity (51 U/ml) was obtained after 6 h of cultivation on tryptone (10 g/L), yeast extract (10 g/L), NaCl (8.125 g/L), pH 7.5 with initial OD (0.15). The PLCBc activity, esteemed by the model (51 U) was very approximate to activity gutted experimentally (50 U). The PLCBc can be considered as thermoactive phospholipase since it showed a maximal activity of 50 U/mL at 60 °C using egg yolk or egg phosphatidylcholine (PC) as substrate. In addition, the enzyme was active at pH 7 and is stable after incubation at 55 °C for 30 min. The application of B. cereus phospholipase C in soybean oil degumming was investigated. Our results showed that when using enzymatic degumming, the residual phosphorus decrease more than with water degumming, indeed, it passes from 718 ppm in soybean crude oil to 100 ppm and 52 ppm by degumming using water and enzymatic process, respectively. The diacylgycerol (DAG) yield showed an increase of 1.2% with enzymatic degumming compared to soybean crude oil. This makes our enzyme a potential candidate for food industrial applications such as enzymatic degumming of vegetable oils.

The response surface methodology for optimization of Halomonas sp. C2SS100 lipase immobilization onto CaCO3 for treatment of tuna wash processing wastewater.

An immobilized enzyme could exhibit selectively modified physicochemical properties, and it might offer a better environment for the enzyme activity. In this study, the immobilization yield of crude Halomonas sp. lipase was optimized to improve its stability. Thanks to its high adsorption capacity, CaCO3 has been chosen as support for the immobilization process. Furthermore, response surface methodology (RSM) was used to determine optimal conditions for the immobilization of the bacterial lipase. Five tested factors (enzyme solution, support amount, time, temperature, and acetone volume) were optimized applying a central composite design of RSM. The maximum yield of lipase immobilization was improved to 96%. Furthermore, a biochemical characterization proved a significant improvement of the immobilized lipase stability. The immobilized enzyme is more stable at extreme pH values and high temperatures than the free one. We also tested the reusability of the immobilized lipase by evaluating the recovery of the support using simple filtration. Thanks to its high stability, the immobilized lipase was invested in an effective treatment of tuna wash processing wastewater. The oil biodegradation efficiency was established at 81.5% and was confirmed by Fourier transformation infrared spectrometry. Likewise, the biological oxygen demand values were reduced which makes a possible reduction of the wastewater pollution degree.

Structure elucidation of Staphylococcus capitis lipase. Molecular dynamics simulations to investigate the effects of calcium and zinc ions on the structural stability.

Cold-adapted and organic solvent tolerant lipases have significant potential in a wide range of synthetic reactions in industry. But there are no sufficient studies on how these enzymes interacts with their substrates. Herein, the predicted structure and function of the Staphylococcus capitis lipase (SCL) are studied. Given the high amino acid sequence homology with the Staphylococcus simulans lipase (SSL), 3D structure models of closed and open forms of the S. capitis lipase were built using the structure of SSL as template. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. The SCL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions. By following this model and utilizing molecular dynamics (MD) simulations, the stability of the S. capitis lipase at low temperatures could be explained in the presence and in the absence of calcium and zinc. Due to its thermolability, the SCL is extremely valuable for different biotechnological applications in a wide variety of industries from molecular biology to detergency to food and beverage preparation.Communicated by Ramaswamy H. Sarma.

Fungal lipases as biocatalysts: A promising platform in several industrial biotechnology applications.

Many researchers have found fungi as a reliable source of lipase due to the versatility of their properties, ease of mass production, thermal stability, pH stability, broad substrate specificity, retained activity in organic solvents, and their low-cost extraction procedure. This review paper presents an overview about the main aspects of fungal lipases screened from several types of strains as well as their use as biocatalysts. Additionally, some biochemical properties will be reported. As commonly known, lipases can be produced from animals, plants, and microorganisms. Compared to other lipases, those obtained from fungi have been found to be more productive, a fact that encouraged the massive production of most fungal lipases due to their considerable commercial importance during the past few years. This paper is concerned about some of the major characteristics that made fungal lipases desirable products in the industrial fields. Due to the enantioselective properties of fungal lipases and their ability to remain active under extreme temperature, pH, and organic solvents, enzymes are capable to synthesize esters as well as to catalyze a variety of chemical reactions that include esterification, transesterification, acidolysis and aminolysis in aqueous and nonaqueous media. Furthermore, lipases are considered to have a commercial importance for biotechnological application fields, which makes them increasingly popular in food, detergent, cosmetic, organic synthesis, and pharmaceutical domains. The biotechnological potential of lipases has made the latter a coveted choice in industries for the present and future as biocatalysts. In addition, a classification of these fungal enzymes is also highlighted in this review. Moreover, the impact of an immobilization strategy of these fungal strains to achieve higher yields and to improve their production is discussed. Finally, fungal enzymes have played a crucial role from ancient times to today in different fields using several types of biological systems, which gives them a great interest for the production of these enzymes in large amounts with low cost and easy viability to enlarge their use in many industries. Likewise, some future perspectives on lipase production will also be discussed by focusing on special cases on lipase engineering.

Optimization of an organic solvent-tolerant lipase production by Staphylococcus capitis SH6. Immobilization for biodiesel production and biodegradation of waste greases.

Using the statistical approach, this work seeks to optimize the process parameters to boost the generation of an organic solvent-tolerant lipase by Staphylococcus capitis SH6. The main parameters influencing the enzyme production were identified by using Plackett-Burman's screening design. Among the test variables, only tryptone (25 g/L), malt extract (2.5 g/L), NaCl (10 g/L) and pH (7.0) contributed positively to enzyme production. Then, the crude lipase was immobilized by adsorption on CaCO3 at pH 10. A maximum immobilization efficiency of 82% was obtained by incubating 280 mg of enzyme with CaCO3 (1 g) during 30 min. The immobilized lipase was more stable toward organic solvents than the free enzyme. It retained about 90% of its original activity in the presence of ethanol and methanol. After that, the immobilized enzyme was used for biodiesel production by transesterification process between waste oil and methanol or ethanol during 24 h at 30 °C. Our results show that the lipase can be utilized efficiently in biodiesel industry. Likewise, we have demonstrated that the immobilized enzyme may be implicated in the biodegradability of waste grease; the maximum conversion yield into fatty acids obtained after 12 h at 30 °C, was 57%.

Enzyme Storage and Recycling: Nanoassemblies of α-Amylase and Xylanase Immobilized on Biomimetic Magnetic Nanoparticles.

Immobilization of enzymes has been extensively required in a wide variety of industrial applications as a way to ensure functionality and the potential of enzyme recycling after use. In particular, enzyme immobilization on magnetic nanoparticles (MNPs) could offer reusability by means of magnetic recovery and concentration, along with increased stability and robust activity of the enzyme under different physicochemical conditions. In the present work, microbial α-amylase (AmyKS) and xylanase (XAn11) were both immobilized on different types of MNPs [MamC-mediated biomimetic MNPs (BMNPs) and inorganic MNPs] by using two different strategies (electrostatic interaction and covalent bond). AmyKS immobilization was successful using electrostatic interaction with BMNPs. Instead, the best strategy to immobilize XAn11 was using MNPs through the hetero-crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The immobilization protocols were optimized by varying glutaraldehyde (GA) concentration, enzyme quantity, and reaction time. Under optimal conditions, 92% of AmyKS and 87% of XAn11 were immobilized on BMNPs and MNPs-E/N, respectively (here referred as AmyKS-BMNPs and XAn11-MNPs nanoassemblies). The results show that the immobilization of the enzymes did not extensively alter their functionality and increased enzyme stability compared to that of the free enzyme upon storage at 4 and 20 °C. Interestingly, the immobilized amylase and xylanase were reused for 15 and 8 cycles, respectively, without significant loss of activity upon magnetic recovery of the nanoassemblies. The results suggest the great potential of these nanoassemblies in bioindustry applications.

Spirulina platensis, Punica granatum peel, and moringa leaves extracts in cosmetic formulations: an integrated approach of in vitro biological activities and acceptability studies.

The selection of suitable natural raw materials in the cosmetic research and development is a key point, in order not only to obtain the expected results but also to avoid undesirable side effects. In this study, spirulina platensis, pomegranate (Punica granatum) peel, and moringa leaves alone were evaluated for anti-oxidant and antimicrobial properties. The chemical composition (moisture, dry matter, protein, lipid, and ash) and total polyphenols, flavonoids, and carotenoids content were evaluated in the three extracts. Total antioxidant capacity and ferric reducing activity power of extracts were also studied. Using agar diffusion method, the anti-Micrococcus luteus, Staphylococcus aureus, E. coli, Listeria monocytogenes, Salmonella typhimurium, and Enterococus faecalis activities were measured. Interestingly, after combinations, pomegranate peel/spirulina (A), and moringa/spirulina (B): 25%/75% and 50%/50%, we have found that pomegranate peel can be incorporated into cosmetic formulations as an excellent preservative due to its exceptionally amount of phenolic compounds, powerful antioxidant activity, and its antibacterial activity against pathogenic strains.

Biodegradation study of PDLA/cellulose microfibres biocomposites by Pseudomonas aeruginosa.

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 microfibres (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.

Biochemical characterization and structural insights into the high substrate affinity of a dimeric and Ca2+ independent Bacillus subtilis α-amylase.

An extracellular amylase (AmyKS) produced by a newly isolated Bacillus subtilis strain US572 was purified and characterized. AmyKS showed maximal activity at pH 6 and 60°C with a half-life of 10 min at 70°C. It is a Ca2+ independent enzyme and able to hydrolyze soluble starch into oligosaccharides consisting mainly of maltose and maltotriose. When compared to the studied α-amylases, AmyKS presents a high affinity toward soluble starch with a Km value of 0.252 mg ml-1 . Coupled with the size-exclusion chromatography data, MALDI-TOF/MS analysis indicated that the purified amylase is a dimer with a molecular mass of 136,938.18 Da. It is an unusual feature of a non-maltogenic α-amylase. A 3D model and a dimeric model of AmyKS were generated showing the presence of an additional domain suspected to be involved in the dimerization process. This dimer arrangement could explain the high substrate affinity and catalytic efficiency of this enzyme.

Purification, biochemical and molecular study of lipase producing from a newly thermoalkaliphilic Aeribacillus pallidus for oily wastewater treatment.

Treatment of oily wastewater is constantly a challenge; biological wastewater treatment is an effective, cheap and eco-friendly technology. A newly thermostable, haloalkaline, solvent tolerant and non-induced lipase from Aeribacillus pallidus designated as GPL was purified and characterized of biochemical and molecular study for apply in wastewater treatment. The GPL showed a maximum activity at 65°C and pH 10 after 22 h of incubation, with preference to TC4 substrates. Pure enzyme was picked up after one chromatographic step. It displayed an important resistance at high temperature, pH, NaCl, at the presence of detergents and organic solvents. In fact, GPL exhibited a prominent stability in wide range of organic solvents at 50% (v/v) concentration for 2 h of incubation. The efficiency of the GPL in oil wastewater hydrolysis was established at 50°C for 1 h, the oil removal efficiency was established at 96, 11% and the oil biodegradation was confirmed through fourier transform infrared (FT-IR) spectroscopy. The gene that codes for this lipase was cloned and sequenced and its open reading frame encoded 236 amino acid residues. The deduced amino acids sequence of the GPL shows an important level of identity with Geobacillus lipases.

Studies of crab digestive phospholipase acting on phospholipid monolayers: Activation by temperature.

Secreted phospholipases A2 (sPLA2) are water-soluble lipolytic enzymes that act at the interface of organized lipid substrates, where the catalytic step is coupled to various interfacial phenomena as enzyme penetration, solubilisation of reaction products, lateral packing and loss of mechanical stability of organized assemblies of phospholipid molecule, among others. Using the monomolecular film technique, we compared the interfacial properties of crab digestive sPLA2 (CDPL) with those of the porcine pancreatic one (PPPL). A kinetic study on the surface pressure dependency of the two sPLA2 was performed using monomolecular films of three different substrates: di C12-PC (1.2-dilauroyl-sn-glycerol-3-phosphocholine); di C12-PG (1.2-dilauroyl-sn-glycerol-3-phosphoglycerol) and di C12-PE (1.2-dilauroyl-sn-glycerol-3-phosphoethanolamine). The use of a substrate in monolayer state, during the catalytic reactions, allows us to monitor the effect of several physicochemical parameters by altering the "quality of interface". The effect of temperature on the hydrolysis rate of these substrates was also checked. Our results show that activities of both phospholipases were affected by the variation of the subphase temperature. CDPL was irreversibly inactivated by p-bromo-phenacyl bromide, the specific inhibitor of sPLA2. The hyperbolic catalytic behaviour observed was coherent with hopping mode of action, one of the two characteristic mechanisms of interfacial catalysis of sPLA2.

Biological properties and biodegradation studies of chitosan biofilms plasticized with PEG and glycerol.

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 BOD5 about 2.33 O2/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.

Purification, biochemical and kinetic properties of recombinant Staphylococcus aureus lipase.

We have compared the purification procedures as well as the biochemical and kinetic properties of wild type (wt-SAL3), untagged recombinant (rec(-His)SAL3), and tagged recombinant (rec(+His)SAL3) purified forms of Staphylococcus aureus lipase (SAL3). We used the pH-stat method (with emulsified tributyrin and olive oil as substrates) and the monomolecular film technique (with the three dicaprin isomers spread in the form of monomolecular films at the air-water interface). The data obtained showed that the recombinant expression process as well as the presence of a his-tag at the N-terminus of recombinant SAL3 affects significantly many biochemical and catalytic properties. 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.

Heterologous expression and N-terminal His-tagging processes affect the catalytic properties of staphylococcal lipases: a monolayer study.

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.

Staphylococcal lipases stereoselectively hydrolyse the sn-2 position of monomolecular films of diglyceride analogs. Application to sn-2 hydrolysis of triolein.

Using the monomolecular film technique, a kinetic study on the stereoselectivity of nine staphylococcal lipase forms was carried out with three pairs of enantiomers from diglyceride analogs (didecanoyl-deoxyamino-O-methyl glycerol, DDG) containing a single hydrolysable decanoyl ester group and two lipase-resistant groups. Our results show that the kinetic profiles of the wild type, the recombinant untagged and the recombinant tagged forms of staphylococcal lipases are significantly different. As with most of the lipases investigated so far, these staphylococcal lipases showed higher catalytic rates with primary esters than with secondary esters. However, it is noteworthy that all these staphylococcal lipases were found to significantly hydrolyse the secondary ester group of diglyceride analogs, with a strong preference for the R configuration. This stereopreference, which was predicted on the basis of Kazlauskas' rule, was comparable to that of Candida rugosa and Pseudomonas glumae lipases. As was to be expected, all the staphylococcal lipases tested efficiently hydrolysed triolein at the sn-2 position. This hydrolytic activity was quantified by performing thin-layer chromatography to analyse the hydrolytic products of triolein. From the qualitative point of view, the sn-2 preferences observed with triolein and diglyceride analogs bearing a secondary ester function were in good agreement. Diglyceride analogs might therefore provide useful initial screening tools for use in future searches for strictly sn-2 specific lipases.

Enzymatic synthesis of eugenol benzoate by immobilized Staphylococcus aureus lipase: optimization using response surface methodology and determination of antioxidant activity.

The ability of a non-commercial immobilized Staphylococcus aureus lipase to catalyze the esterification of eugenol with benzoic acid was checked and the antioxidant power of the ester formed was evaluated. Response surface methodology based on four variables (the reaction temperature, the amount of lipase, the benzoic acid/eugenol molar ratio and the volume of solvent) was used to optimize the experimental conditions of eugenol benzoate synthesis. The maximum conversion yield (75%) was obtained using 240 IU of immobilized lipase, a benzoic acid/eugenol molar ratio of 1.22 dissolved in 4.6 ml chloroform at 41 degrees Celsius. The antioxidant activities of eugenol and its ester were evaluated. Compared to BHT, used as a model synthetic antioxidant, the eugenol benzoate showed a higher antioxidative activity. The IC(50) value for 1,1-diphenyl-2-picrylhydrazyl was found to be 18.2 microg/ml versus 20.2 microg/ml for eugenol and eugenol benzoate.

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli.

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.