Purification and characterization of a thermostable alkaline protease from alkalophilic Thermoactinomyces sp. HS682.

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatographies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25,000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37 degrees C for 60 min. The optimum pH was pH 11.5-13.0 at 37 degrees C and the optimum temperature was 70 degrees C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl2, it showed maximum proteolytic activity at 80 degrees C and stability from pH 4-12.5 at 60 degrees C and below 75 degrees C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of microbial serine protease, although alanine of the NH2-terminal amino acid was deleted.     

Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium

An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and alpha-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 degrees C. The purifed enzyme hydrolyzed synthetic peptides preferentially at the carboxy terminus of phenylalanine or leucine and was almost completely inhibited by PMSF, TPCK, and chymostatin, similarly to a chymotrypsin-like serine protease. Kinetic analysis of the Tp. volcanium protease reaction performed using N-succinyl-L-phenylalanine-p-nitroanilide as substrate revealed a Km value of 2.2 mM and a Vmax value of 0.045 micromol(-1) ml(-1) min(-1). Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca2+ and Mg2+ at 2-12 mM concentration. The serine protease is a monomer with a molecular weight of 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram activity staining.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT-1

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH2-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin I was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80 degrees C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine.HCl and 1% SDS) at 23 degrees C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.     

Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction

Protease La is an ATP-dependent protease that catalyzes the rapid degradation of abnormal proteins and certain normal polypeptides in Escherichia coli. In order to learn more about its specificity and the role of ATP, we tested whether small fluorogenic peptides might serve as substrates. In the presence of ATP and Mg2+, protease La hydrolyzes two oligopeptides that are also substrates for chymotrypsin, glutaryl-Ala-Ala-Phe-methoxynaphthylamine (MNA) and succinyl-Phe-Leu-Phe-MNA. Methylation or removal of the acidic blocking group prevented hydrolysis. Closely related peptides (glutaryl-Gly-Gly-Phe-MNA and glutaryl-Ala-Ala-Ala-MNA) are cleaved only slightly, and substrates of trypsin-like proteases are not hydrolyzed. Furthermore, several peptide chloromethyl ketone derivatives that inhibit chymotrypsin and cathepsin G (especially benzyloxycarbonyl-Gly-Leu-Phe-chloro-methyl ketone), inhibited protease La. Thus its active site prefers peptides containing large hydrophobic residues, and amino acids beyond the cleavage site influence rates of hydrolysis. Peptide hydrolysis resembles protein breakdown by protease La in many respects: 1) ADP inhibits this process rapidly, 2) DNA stimulates it, 3) heparin, diisopropyl fluorophosphate, and benzoyl-Arg-Gly-Phe-Phe-Leu-MNA inhibit hydrolysis, 4) the reaction is maximal at pH 9.0-9.5, 5) the protein purified from lon- E. coli or Salmonella typhymurium showed no activity against the peptide, and that from lonR9 inhibited peptide hydrolysis by the wild-type enzyme. With partially purified enzyme, peptide hydrolysis was completely dependent on ATP. The pure protease hydrolyzed the peptide slowly when only Mg2+, Ca2+, or Mn2+ were present, and ATP enhanced this activity 6-15-fold (Km = 3 microM). Since these peptides cannot undergo phosphorylation, adenylylation, modification of amino groups, or denaturation, these mechanisms cannot account for the stimulation by ATP. Most likely, ATP and Mg2+ affect the conformation of the enzyme, rather than that of the substrate.     

Biochemical and molecular characterization of PepR, a dipeptidase, from Lactobacillus helveticus CNRZ32.

A dipeptidase with prolinase activity from Lactobacillus helveticus CNRZ32, which was designated PepR, was purified to gel electrophoretic homogeneity and characterized. The NH2-terminal amino acid sequence of the purified protein had 96% identity to the deduced NH2-terminal amino acid sequence of the pepR gene, which was previously designated pepPN, from L. helveticus CNRZ32. The purified enzyme hydrolyzed Pro-Met, Thr-Leu, and Ser-Phe as well as dipeptides containing neutral, nonpolar amino acid residues at the amino terminus. Purified PepR was determined to have a molecular mass of 125 kDa with subunits of 33 kDa. The isoelectric point of the enzyme was determined to be 4.5. The optimal reaction conditions, as determined with Pro-Leu as substrate, were pH 6.0 to 6.5 and 45 to 50 degrees C. The purified PepR had a Km of 4.9 to 5.2 mM and a Vmax of 260 to 270 mumol of protein per min/mg at pH 6.5 and 37 degrees C. The activity of purified PepR was inhibited by Zn2+ but not by other cations or cysteine, serine, aspartic, or metal-containing protease inhibitors or reducing agents. Results obtained by site-directed mutagenesis indicated that PepR is a serine-dependent protease. Gene replacement was employed to construct a PepR-deficient derivative of CNRZ32. This mutant did not differ from the wild-type strain in its ability to acidify milk. However, the PepR-deficient construct was determined to have reduced dipeptidase activity compared to the wild-type strain with all dipeptide substrates examined.     

A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713

An alkaline protease was isolated from culture filtrate of B. subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C. The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1). The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr. During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+. Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C. About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C. DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained. However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+. The results indicated that this is a serine protease.     

Characterization of a keratinolytic protease produced by the feather-degrading Amazonian bacterium Bacillus sp. P45

Abstract

An extracellular keratinolytic protease produced by Bacillus sp. P45 was purified and characterized. The keratinase had a molecular weight of approximately 26 kDa and was active over wide pH and temperature ranges, with optimal activity at 55°C and pH 8.0. However, this enzyme displayed low thermostability, being completely inactivated after 10 min at 50°C. Keratinase activity increased with Ca²⁺, Mg²⁺, Triton X-100, ethanol and DMSO, was stable in the presence of the reducing agent 2-mercaptoethanol, and was inactivated by SDS. PMSF (phenylmethylsulfonyl fluoride) completely inactivated and EDTA strongly inhibited the enzyme, indicating that the keratinase is a serine protease depending on metal ions for optimal activity and/or stability. Accordingly, analysis of tryptic peptides revealed sequence homologies which characterize the keratinase as a subtilisin-like serine protease. The purified enzyme was able to hydrolyze azokeratin and keratin azure. Casein was hydrolyzed at higher rates than keratinous substrates, and 2-mercaptoethanol tended to enhance keratin hydrolysis. With synthetic substrates, the keratinase showed a preference for aromatic and hydrophobic residues at the P1 position of tetrapeptides; the enzyme was not active, or the activity was drastically diminished, towards shorter peptides. Keratinase from Bacillus sp. P45 might potentially be employed in the production of protein hydrolysates at moderate temperatures, being suitable for the bioconversion of protein-rich wastes through an environmentally friendly process requiring low energy inputs.     

Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease

Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.     

Purification and characterization of a metalloprotease from Chryseobacterium indologenes Ix9a and determination of the amino acid specificity with electrospray mass spectrometry

The heat-stable protease from Chryseobacterium indologenes Ix9a was purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction.     

Degradation of keratinous materials by the plant pathogenic fungus <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis>

In this paper it is described for the first time the capability of Myrothecium verrucaria to grow in submerged and solid state cultures using poultry feathers as the only substrate. The fungus produced a protease with an unusual keratinolytic activity among plant pathogenic fungi. Its crude protease hydrolyzed keratinous substrates at pH 9.0 and 40 °C in the following order: poultry feather keratin > sheep wool keratin > human nail keratin > human hair keratin. Protease activity was highly sensitive to phenylmethyl sulphonyl fluoride (PMSF) indicating that the enzyme belonged to the serine protease family.     

Purification and characterization of a basic amino acid-specific peptidase from seeds of jack bean (Canavalia ensiformis)

A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60 degrees C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.     

Purification and characterization of an alkaline protease from Oerskovia xanthineolytica TK-1

Alkaline protease from Oerskovia xanthineolytica TK-1 was purified to an electrophoretically homogeneous state by phenyl-Sepharose CL-4B and DEAE-Sephacel. The molecular mass of the enzyme was 20,000 Da by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 9.5–11.0 and 50°C. It was inhibited by inhibitors of serine protease. The enzyme preferentially hydrolyzed the ester of phenylalanine among N-CBZ amino acid p-nitrophenol esters. These results indicate that the protease can be classified as an alkaline serine protease.     

Isolation of a cadmium-releasing bacterium and characterization of its novel protease

Microorganisms were screened for their ability to release cadmium from scallop hepatopancreas, which is the main residue after removing of the edible parts of scallop. The isolated strain, 23-0-11, identified as Arthrobacter nicotinovorans, secreted a protease which released cadmium from scallop hepatopancreas into the liquid medium. The molecular mass of the enzyme was estimated to be 27 kDa. The sequence of the 15 N-terminal amino acids of the protease showed no close similarity with any other protein. Compared with a commercial enzyme, the purified protease had greater ability to release cadmium. The enzyme activity was greatest at 50 degrees C and pH 7.0, and was enhanced in the presence of Ca(2+), Mg(2+) and Mn(2+), while being strongly inhibited by Co(2+). The inhibition profile by the serine protease inhibitor, phenylmethylsulphonyl fluoride (PMSF), confirmed that the protease belonged to the serine protease family.     

Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis

A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca²⁺ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca²⁺ ions and gramicidin S; its pH and temperature optima were 9.0 and 37°C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.     

Calcium-ion transport by intact Ehrlich ascites-tumour cells. Role of respiratory substrates, Pi and temperature.

1. The interaction of intact Ehrlich ascites-tumour cells with Ca2+ at 37 degrees C consists of Ca2+ uptake followed by efflux from the cells. Under optimum conditions, two or three cycles of uptake and efflux are observed in the first 15 min after Ca2+ addition. 2. The respiratory substrates malate, succinate and ascorbate plus p-phenylenediamine support Ca2+ uptake. Ca2+ uptake at 37 degrees C is sensitive to the respiratory inhibitors rotenone and antimycin A when appropriate substrates are present. Ca2+ uptake and retention are inhibited by the uncoupler S-13. 3. Increasing extracellular Pi (12 to 30 mM) stimulates uncoupler-sensitive Ca2+ uptake, which reaches a maximum extent of 15 nmol/mg of protein when supported by succinate respiration. Ca2+ efflux is partially inhibited at 30 mM-Pi. 4. Optimum Ca2+ uptake occurs in the presence of succinate and Pi, suggesting that availability of substrate and Pi are rate-limiting. K. Ca2+ uptake occurs at 4 degrees C and is sensitive to uncouplers and oligomycin. Ca2+ efflux at this temperature is minimal. These data are consistent with a model in which passive diffusion of Ca2+ through the plasma membrane is followed by active uptake by the mitochondria. Ca2+ uptake is supported by substrates entering respiration at all three energy-coupling sites. Ca2+ efflux appears to be an active process with a high temperature coefficient.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and 37 degrees , respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin alpha-chain followed by the gamma-gamma chains. It also hydrolyzed the beta-chain, but more slowly. The Aalpha, Bbeta, and gamma chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by Cu2+ and Co2+, but enhanced by the additions of Ca2+ and Mg2+ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it 's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.     

Identification and primary characterization of specific proteases in the digestive juice of Archachatina ventricosa

The profile of sedimentation on a 4-20% (w/v) linear sucrose gradient of the digestive juice of the mollusk Archachatina ventricosa revealed the presence of at least four specific proteases. A first peak, corresponding to a sedimentation coefficient of 3.9 S, contained two endoproteases that could be assayed, one with Leu-pNA and the other with Met-pNA. Their activity was maximal at pH 8.0 and increased in the presence of Ca(2+) ions. Both enzymes were inhibited by the chelating agent 1,10-phenanthroline but their thermal inactivation kinetics were different. A second protease peak was observed at 6.8 S and corresponded to a metallo-endoprotease that hydrolyzed with a maximal activity at pH 8.0 only the amide bonds of peptide substrates having a threonine residue at the P1' position. A last protease peak identified at 9.0 S contained a protease that preferentially acted on tripeptides, such as Val-Pro-Leu (diprotin B) and Thr-Val-Leu, releasing the C-terminal residue. Unlike the proteases identified in the two other peaks, its activity was maximal at acid pH (5.0) and was inhibited by the serine protease inhibitors. Together these results show the potential of A. ventricosa as a source of specific proteases.     

Biochemical and molecular characterization of Staphylococcus xylosus lipase

The Staphylococcus xylosus strain secretes a non-induced lipase in culture medium: S. xylosus lipase (SXL). Pure SXL is a monomeric protein (43 kDa). The 23 N-terminal amino acid residues were sequenced. This sequence is identical to that of Staphylococcus simulans lipase (SSL); in addition, it exhibits a high degree of homology with Staphylococcus aureus lipase (SAL NCTC 8530) sequences. The cloning and sequencing of gene part encoding the mature lipase shows one nucleotide difference with SSL, which corresponds to the change of one residue at a position 311. The lipase activity is maximal at pH 8.2 and 45 degrees C. SXL is able to hydrolyse triacylglycerols without chain length specificity. The specific activity of about 1900 U/mg was measured using tributyrin or triolein as substrate at pH 8.2 and at 45 degrees C in the presence of 2 mM CaCl2. In contrast to some previously characterized staphylococcal lipases, Ca2+ is not required to trigger the activity of SXL. SXL was found to be stable between pH 5 and pH 8.5. The enzyme maintains 50% of its activity after a 15-min incubation at 60 degrees C. Using tripropionin or vinyl esters as substrates, SXL does not present the interfacial activation phenomenon. Unlike many lipases, SXL is able to hydrolyse its substrate in the presence of bile salts or amphiphilic proteins. SXL is a serine enzyme, which is inhibited by THL.