Molecular Cloning and Expression of a Cold-Adapted Lipase Gene from an Antarctic Deep Sea Psychrotrophic Bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. 7323

A psychrotrophic bacterium producing a cold-adapted lipase was isolated from the deep-sea sediment of Prydz Bay, Antarctic and identified as a Pseudomonas strain. Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. 7323 (lipA) revealed that LipA is composed of 617 amino acid residues with a calculated molecular weight of 64,466 Da. LipA has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The lipase purified from the Escherichia coli transformant (rLipA) by metal-chelating chromatography exhibited the same electrophoretic mobility as did the wild-type lipase (wLipA) purified from strain 7323, and both enzymes were quite similar in physicochemical properties. The optimal temperature and pH value for the lipases activity were 30 degrees C and 9.0, respectively. They were unstable at temperatures above 25 degrees C and only retained half of their highest activity after incubation at 60 degrees C for 5 min. These results indicated that the enzymes were typical alkaline cold-adapted enzymes. Both enzymes were particularly activated by Ca(2+). Additionally, the enzymes hydrolyzed p-nitrophenyl caprate and tributyrin at the highest velocity among the other p-nitrophenyl esters and triglycerides.     

Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterisation

Thermus thermophilus HB27 produces important levels of extracellular lipolytic activity when grown for 30 h at 70 degrees C in a complex medium. A method to detect esterase activity in these samples after non-reducing SDS-polyacrylamide electrophoresis was developed. The method, that implies the renaturalisation of the enzymes in the SDS-gels by washing with Triton X-100 at high temperatures, allowed detecting three esterases with different molecular weights (108, 62 and 34 kDa, respectively). The electrophoretic mobility determined under different experimental conditions suggested that the 34- and 108 kDa-esterases might correspond with two oligomeric states of a sole enzyme (monomer and trimer). Dissociation of the trimer into the monomer started when the samples were heated at temperatures higher than 60 degrees C in the presence of sodium dodecyl sulphate. Evidences were found that indicated the independent nature of the 62 kDa-esterase. A method to purify these enzymes from postincubates of T. thermophilus HB27 was developed following three steps: sodium cholate treatment, ethanol/ether precipitation and hydrophobic chromatography. In this way, an enzyme solution was obtained that contained the identified esterases/lipases. The partially purified enzymes showed an optimum of activity for the hydrolysis of p-nitrophenyl laurate at alkaline pH values and 80 degrees C, a high thermal stability and were very stable in the presence of high concentrations of isopropanol.     

Novel minor lipase from Rhizopus chinensis during solid-state fermentation: Biochemical characterization and its esterification potential for ester synthesis

Rhizopus chinensis produces two lipases that catalyze ester synthesis when cultured under solid-state fermentation. The Lip2 was purified to homogeneity by ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. It has an apparent molecular weight of 33 kDa estimated from SDS–PAGE and 32 kDa calculated from analytical gel permeation, with synthetic activity and purification fold of 96.8 U/mg and 138.3, respectively. Maximum hydrolytic activity was obtained at pH 8.0–8.5 and 40 °C using pNPP as substrate. Slight activation of the enzyme was observed when Mn²⁺ is present. The enzyme was most active on p-nitrophenyl laurate (C12). The purified lipase exhibited maximum synthetic activity at pH memory of 6.0 and 30 ^oC. Most of ethyl esters synthesized by lyophilized enzyme achieved good yields (>90%), and caprylic acid served as the best acyl donor. The enzyme presented a particular affinity for ethanol, n-propanol and n-hexanol, with conversion of 92%, 93% and 92%, respectively, after 20 h incubation.     

Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases

The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

Purification and some properties of a thermostable acid esterase from Acidiphilium sp. AIU 409

Extracellular and cell-bound esterases produced by Acidiphilium sp. AIU 409 were homogeneously purified from culture broth and cells, respectively, and some properties were investigated. Both esterases more rapidly hydrolyzed p-nitrophenyl acyl esters containing long-chain fatty acids from C 8:0 to C 18:0 than those containing short-chain fatty acids from C 2:0 to C 6:0. The Km values for p-nitrophenyl long-chain fatty acid esters from C 8:0 to C 18:0 were approximately 1.3-1.5 mM. The enzymes were stable at 50 degrees C for 2 days between pH 3.0 and 6.5, and optimum pH and temperature were 5.0 and 70 degrees C, respectively. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride and SDS. The molecular mass of both enzymes was estimated to be approximately 64 kDa by SDS-PAGE. The 23 amino acid sequence from the NH(2)-terminus was also the same in both enzymes. These results suggest that extracellular esterase might be composed of the same components as cell-bound esterase.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

Characterization of a new extracellular hydrolase from Thermobifida fusca degrading aliphatic-aromatic copolyesters

The paper describes the purification, biochemical characterization, sequence determination, and classification of a novel thermophilic hydrolase from Thermobifida fusca (TfH) which is highly active in hydrolyzing aliphatic-aromatic copolyesters. The secretion of the extracellular enzyme is induced by the presence of aliphatic-aromatic copolyesters but also by adding several other esters to the medium. The hydrophobic enzyme could be purified applying a combination of (NH(4))SO(4)-precipitation, cation-exchange chromatography, and hydrophobic interaction chromatography. The 28 kDa enzyme exhibits a temperature maximum of activity between 65 and 70 degrees C and a pH maximum between pH 6 and 7 depending on the ion strength of the solution. According to the amino sequence determination, the enzyme consists of 261 amino acids and was classified as a serine hydrolase showing high sequence similarity to a triacylglycerol lipase from Streptomyces albus G and triacylglycerol-aclyhydrolase from Streptomyces sp. M11. The comparison with other lipases and esterases revealed the TfH exhibits a catalytic behavior between a lipase and an esterase. Such enzymes often are named as cutinases. However, the results obtained here show, that classifying enzymes as cutinases seems to be generally questionable.     

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.     

Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45 degrees C and the half-life was 1 h at 64 degrees C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chain fatty acid esters. The enzyme had a KM of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37 degrees C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Various properties of cathepsin G and elastase from swine peripheral blood neutrophils

Using gel filtration through Sephadex G-100 and bioaffinity chromatography on contrical-Sepharose, cathepsin G and elastase were isolated from pig peripheral blood neutrophil granules and purified to homogeneity. Both enzymes hydrolyzed the total histone from calf thymus as well as synthetic substrates--tert-butoxy-L-alanine p-nitrophenyl ester (elastase) and benzoyltyrosine ethyl ester (cathepsin G). The use of natural and synthetic protease inhibitors showed that both enzymes were related to the group of serine proteases. The molecular mass of the cathepsin G subunit as determined by SDS polyacrylamide gel electrophoresis is 28-29 kD, that of elastase--30-31 kD. The pH optima for the hydrolysis of proteinaceous and synthetic substrates for cathepsin G and elastase are 8.0-8.5 and 7.0-7.5, respectively. The isoelectric points for elastase and cathepsin G are 9.7-10.0 and greater than 10, respectively; the temperature optima--30-40 degrees C and 50-60 degrees C, respectively. The amino acid composition of the two enzymes from pig granulocytes revealed a high content of arginine and was similar to that of human granulocytes.     

Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon

We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (est(Pc)) from strain VA1. est(Pc) consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As est(Pc) showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90 degrees C), but also at ambient temperature (1,050 U/mg at 30 degrees C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p-nitrophenyl caproate (C(6)) the best substrate among the p-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.     

Synthesis of lactose monolaurate as influenced by various lipases and solvents

Fatty acid sugar esters are non-ionic detergents with multiple uses in the cosmetic, food, and pharmaceutical industries. Of the many different sugar esters synthesized, lactose, a by-product of cheese manufacture, has not been investigated. The objective of this research was to investigate the synthesis of novel lactose monolaurate (LML) and sucrose monolaurate (as a comparison) (SML) using four different immobilized lipases in three different solvents at constant sugar, vinyl laurate, temperature, and enzyme concentrations. Overall, the solvent 2-methyl-2-butanol gave the highest yields and reactions rates for the synthesis of both LML and SML. Of the immobilized lipases, those from Pseudomonas cepacia, Mucor miehei and Thermomyces lanuginosus were effective depending on the sugar/solvent combination. Higher overall yields were obtained for the synthesis of LML with the differences in yields presumably due to the decreased solubility of sucrose as compared to lactose in 3 of the solvents used. Response surface methodology was used to determine the optimal temperature, enzyme concentration and ratio of reactants for LML synthesis using the immobilized lipase from M. miehei in 2-methyl-2-butanol. Based on the analysis of ridge max, the optimal synthesis conditions were predicted to occur at 61 °C, with an enzyme amount of 32 mg/mL, and a molar ratio of lactose to vinyl laurate of 1:3.8; and the optimal actual yield was 99.3%.     

Biochemical and molecular characterization of Staphylococcus simulans lipase.

Staphylococcus simulans strain secretes a non-induced lipase in the culture medium. Staphylococcus simulans lipase (SSL), purified to homogeneity, is a tetrameric protein (160 kDa) corresponding to the association of four lipase molecules. The 30 N-terminal amino acid residues were sequenced. This sequence is identical to the one of Staphylococcus aureus PS54 lipase (SAL PS54) and exhibits a high degree of homology with Staphylococcus aureus NCTC8530 lipase (SAL NCTC8530), Staphylococcus hyicus lipase (SHL) and Staphylococcus epidermis RP62A lipase (SEL RP62A) sequences. But the cloning and sequencing of the part of the gene encoding the mature lipase show some differences from SAL PS54 sequence, which suggest that it is a new sequence. The lipase activity was maximal at pH 8.5 and 37 degrees C. SSL is able to hydrolyze triacylglycerols without chain length specificity. A specific activity of about 1000 U/mg was measured on tributyrin or triolein as substrate at 37 degrees C and at pH 8.5 in the presence of 3 mM CaCl(2). In contrast to other staphylococcal lipases previously characterized, Ca(2+) is not required to express the activity of SSL. SSL was found to be stable between pH 4 and pH 9. The enzyme is inactivated after a few minutes when incubated at 60 degrees C. Using tripropionin as substrate, SSL does not present the interfacial activation phenomenon. In contrast to many lipases, SSL is able to hydrolyze its substrate in the presence of bile salts or amphiphilic proteins.     

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.     

Screening and characterization of a novel esterase from a metagenomic library

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

Esterases from Bacillus subtilis and B. stearothermophilus share high sequence homology but differ substantially in their properties

A novel esterase from Bacillus subtilis (BsubE) was cloned, functionally expressed in Escherichia coli and biochemically characterized. BsubE shows high homology (74% identity, >95% homology) to an esterase from the thermophilic B. stearothermophilus (BsteE). Both enzymes were efficiently expressed in E. coli, using a L-rhamnose-expression system [11,500 units/l (BsteE), 3,400 units/l (BsubE)] and were purified by Ni-nitrilotriacetic acid chromatography, yielding specific activities of 70 units/mg (BsteE) and 40 units/mg (BsubE), as determined by the hydrolysis of p-nitrophenyl acetate. Despite the high homology, both esterases revealed remarkable differences in their properties. As expected, the esterase from the thermophilic organism showed significantly higher temperature stability. Whereas BsteE showed highest activity at 65-70 degrees C, BsubE was almost inactivated at 50 degrees C. Moreover, both enzymes showed quite different substrate patterns in the hydrolysis of various esters. Whilst the B. subtilis esterase accepted esters with a branched alcohol moiety well, the B. stearothermophilus esterase was more useful in the hydrolysis of substrates with a sterically demanding carboxylic acid group. BsteE showed excellent enantioselectivity ( E>100) in the kinetic resolution of menthyl acetate and even accepted the bulky menthyl benzoate as substrate ( E=19). In contrast, BsubE converted 1-phenethylacetate with higher selectivity ( E>150 vs E=8).     

Enhancement of ethyl propionate synthesis by poly (AAc-co-HPMA-cl-MBAm)-immobilized Pseudomonas aeruginosa MTCC-4713, exposed to Hg2+ and NH4+ ions

A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa MTCC-4713 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel. The hydrogel-bound lipase achieved 93.6% esterification of ethanol and propionic acid (300 mM: 100 mM) into ethyl propionate at temperature 65 degrees C in 3 h in the presence of a molecular sieve (3 angstroms). In contrast, hydrogel-immobilized lipase pre-exposed to 5 mM of HgCl2 orNH4Cl resulted in approximately 97% conversion of reactants in 3 h into ethyl propionate under identical conditions. The salt-exposed hydrogel was relatively more efficient in repetitive esterification than the hydrogel-bound lipase not exposed to any of the cations. Moreover, bound lipase exposed Hg2+ or NH4+ ions showed altered specificity towards p-nitrophenyl esters and was more hydrolytic towards higher C-chain p-nitrophenyl esters (p-nitrophenyl laurate and p-nitrophenyl palmitate with C 12 and C 16 chain) than the immobilized lipase not exposed to any of the salts. The later showed greater specificity towards p-nitrophenyl caprylate (C 8).     

Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen

Two novel lipase genes RlipE1 and RlipE2 which encoded 361- and 265-amino acid peptides, respectively, were recovered from a metagenomic library of the rumen microbiota of Chinese Holstein cows. A BLAST search revealed a high similarity (90%) between RlipE2 and a carboxylesterase from Thermosinus carboxydivorans Nor1, while there was a low similarity (below 50%) between RlipE1 and other lipases. Phylogenetic analysis indicated that RlipE2 clustered with the lipolytic enzymes from family V while RlipE1 clustered with six other putative bacterial lipases which might constitute a new subfamily. The recombinant lipases were thermally unstable and retained 60% activity over a pH range of 6.5-8.5. Substrate specificity assay indicated that both enzymes had higher hydrolytic activity toward laurate (C₁₂), palmitate (C₁₆) and stearate (C₁₈). The novel phylogenetic affiliation and high specificity of both enzymes for long-chain fatty acid make them interesting targets for manipulation of rumen lipid metabolism.