Bacterial Lipolytic Enzymes Research Articles

Cloning, expression and characterization of two thermostable esterases from Aquifex aeolicus VF5

Thermophilic enzymes have attracted widespread attention due to their unique characteristics such as temperature, solvent, and pH stability. In this study, two novel esterases were identified in hyperthermophilic bacterium Aquifex aeolicus VF5 based on the typical characteristics of lipolytic enzymes. Two genes, Aaeo1 and Aaeo2 encoded 207 and 226 amino acids respectively, were synthesized after optimization according to the P. pastoris codon preferences. Using sequence alignment and phylogenetic analysis, Aaeo1 and Aaeo2, were classified into the bacterial lipolytic enzyme family VIII and V, respectively. Two genes were cloned and expressed in P. pastoris cells, and the recombinant enzymes were purified from culture supernatants by Ni2+-NTA affinity chromatography and characterized. The optimum temperatures of Aaeo1 and Aaeo2 were 80°C and 85°C, respectively, and both of the optimal pH was 8.0. Aaeo1 had the activity preference both to short and long chain p-nitrophenyl esters and triacylglycerides, and the Km and kcat of the best substrate, p-nitrophenyl butyrate (p-NPC4) were 5.11±0.31mM and 16.12±2.32s−1, respectively. While Aaeo2 only displayed high activity towards short chain p-nitrophenyl esters, p-nitrophenyl butyrate (p-NPC4), and exhibited Km and kcat of 0.79±0.03mM and 3.59±1.15s−1, respectively. The activities of both esterases were inhibited by PMSF, DEPC, and EDTA, but were activated by DTT. The two esterases exhibited certain tolerance to various organic solvents and some detergents. These characteristics make them very promising for the industrial application.

Isolation and Characterization of a Novel Cold-Adapted Esterase, MtEst45, from Microbulbifer thermotolerans DAU221.

A novel esterase, MtEst45, was isolated from a fosmid genomic library of Microbulbifer thermotolerans DAU221. The encoding gene is predicted to have a mass of 45,564 Da and encodes 495 amino acids, excluding a 21 amino acid signal peptide. MtEst45 showed a low amino acid identity (approximately 23–24%) compared with other lipolytic enzymes belonging to Family III, a closely related bacterial lipolytic enzyme family. MtEst45 also showed a conserved GXSXG motif, G131IS133YG135, which was reported as active site of known lipolytic enzymes, and the putative catalytic triad composed of D237 and H265. Because these mutants of MtEst45, which was S133A, D237N, and H265L, had no activity, these catalytic triad is deemed essential for the enzyme catalysis. MtEst45 was overexpressed in Escherichia coli BL21 (DE3) and purified via His-tag affinity chromatography. The optimal pH and temperature of MtEst45 were estimated to be 8.17 and 46.27°C by response surface methodology, respectively. Additionally, MtEst45 was also active between 1 and 15°C. The optimal hydrolysis substrate for MtEst45 among p-nitrophenyl esters (C2–C18) was p-nitrophenyl butyrate, and the Km and Vmax values were 0.0998 mM and 550 μmol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg2+, Zn2+, and Cu2+ ions; by phenylmethanesulfonyl fluoride; and by β-mercaptoethanol. Ca2+ did not affect the enzyme's activity. These biochemical properties, sequence identity, and phylogenetic analysis suggest that MtEst45 represents a novel and valuable bacterial lipolytic enzyme family and is useful for biotechnological applications.

A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters.

BackgroundMarine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnological purposes. However, since most microbes cannot be cultured in the laboratory, a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. Therefore, with the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extracted from marine mud.ResultsBased on functional heterologous expression, 34 clones that showed lipolytic activity were isolated. The five clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Molecular analysis revealed that these encoded proteins showed 48–79 % similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree analysis classified these five protein sequences as new members of known families of bacterial lipolytic enzymes. Among them, EST4, which has 316 amino acids with a predicted molecular weight of 33.8 kDa, was further studied in detail due to its strong hydrolytic activity. Characterization of EST4 indicated that it is an alkaline esterase that exhibits highest hydrolytic activity towards p-nitrophenyl butyrate (specific activity: 1389 U mg−1) at 45 °C and pH 8.0. The half-life of EST4 is 55 and 46 h at 40 and 45 °C, respectively, indicating a relatively high thermostability. EST4 also showed remarkable stability in organic solvents, retaining 90 % of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. Furthermore, EST4 was used as an efficient whole-cell biocatalyst for the synthesis of short-chain flavor esters, showing high conversion rate and good tolerance for high substrate concentrations (up to 3.0 M). These results demonstrate a promising potential for industrial scaling-up to produce short-chain flavor esters at high substrate concentrations in non-aqueous media.ConclusionsThis manuscript reports unprecedented alcohol tolerance and conversion of an esterase biocatalyst identified from a marine mud metagenomic library. The high organic solvent tolerance and thermostability of EST4 suggest that it has great potential as a biocatalyst.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0435-5) contains supplementary material, which is available to authorized users.

Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

Unusual carboxylesterase bearing a GGG(A)X-type oxyanion hole discovered in Paenibacillus barcinonensis BP-23.

Strain Paenibacillus barcinonensis BP-23, previously isolated from Ebro's river delta (Spain), bears a complex hydrolytic system showing the presence of at least two enzymes with activity on lipidic substrates. EstA, a cell-bound B-type carboxylesterase from the strain was previously isolated and characterized. The gene coding for a second putative lipase, located upstream cellulase Cel5A, was obtained using a genome walking strategy and cloned in Escherichia coli for further characterization. The recombinant clone obtained displayed high activity on medium/short-chain fatty acid-derivative substrates. The enzyme, named Est23, was purified and characterized, showing maximum activity on pNP-caprylate (C8:0) or MUF-heptanoate (C7:0) under conditions of moderate temperature and pH. Although Est23 displays a GGG(A)X-type oxyanion hole, described as an important motif for tertiary alcohol ester resolution, neither conversion nor enantiomeric resolution of tertiary alcohols could be detected. Amino acid sequence alignment of Est23 with those of known bacterial lipase families and with closely related proteins suggests that the cloned enzyme does not belong to any of the described bacterial lipase families. A phylogenetic tree including Est23 and similar amino acid sequences showed that the enzyme belongs to a differentiated sequence cluster which probably constitutes a new family of bacterial lipolytic enzymes.

An Essential Esterase (BroH) for the Mineralization of Bromoxynil Octanoate by a Natural Consortium of Sphingopyxis sp. Strain OB-3 and Comamonas sp. Strain 7D-2

A natural consortium of two bacterial strains ( Sphingopyxis sp. OB-3 and Comamonas sp. 7D-2) was capable of utilizing bromoxynil octanoate as the sole source of carbon for its growth. Strain OB-3 was able to convert bromoxynil octanoate to bromoxynil but could not use the eight-carbon side chain as its sole carbon source. Strain 7D-2 could not degrade bromoxynil octanoate, although it was able to mineralize bromoxynil. An esterase (BroH) that is involved in the conversion of bromoxynil octanoate into bromoxynil and is essential for the mineralization of bromoxynil octanoate by the consortium was isolated from strain OB-3 and molecularly characterized. BroH encodes 304 amino acids and resembles α/β-hydrolase fold proteins. Recombinant BroH was overexpressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. BroH was able to transform p-nitrophenyl esters (C2-C14) and showed the highest activity toward p-nitrophenyl caproate (C6) on the basis of the catalytic efficiency value (Vmax/Km). Additionally, BroH activity decreased when the aliphatic chain length increased. The optimal temperature and pH for BroH activity was found to be 35 °C and 7.5, respectively. On the basis of a phylogenetic analysis, BroH belongs to subfamily V of bacterial lipolytic enzymes.

Role of key salt bridges in thermostability of G. thermodenitrificans EstGtA2: distinctive patterns within the new bacterial lipolytic enzyme subfamily XIII.2 [corrected

Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15th family (family XV) and suggest criteria for the assignation of protein sequences to the N’ subfamily. Five selected salt bridges, hallmarks of the N’ subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/K→A) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing T m by 5 to 14°C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N’ subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for optimization of EstGtA2 for specific biotechnological purposes.

Isolation and characterization of a metagenome-derived thermoalkaliphilic esterase with high stability over a broad pH range

A novel alkaliphilic esterase (EstJ) was identified from a soil metagenome of Jeju Island, Korea, using a 96-well plate-based functional assay for determination of pH dependence of activity. The amino acid sequence of EstJ showed low similarity (32-45%) to putative α/β hydrolases derived from whole-genome sequencing studies. EstJ, although not belonging to any of the known families of bacterial lipolytic enzymes, however, it showed closest sequence identity to the family IV enzymes that are related to the mammalian hormone-sensitive lipases. The highly conserved motifs of family IV enzymes were found in EstJ, but the corresponding sequences of each motif in EstJ were unique; most particularly the -(F/Y)(F/Y/L)HGGG- motif was represented by -WMVSGG-. The purified EstJ was highly active from pH 8.5 to 10.5. More than 90% of maximum activity was also retained over a wide pH range of 5.5-0.5 after prolonged incubation. EstJ was also moderately thermophilic with an optimum temperature of 55°C. Therefore, EstJ is the first metagenome-derived bacterial family IV esterase possessing both highly alkaliphilic and moderately thermophilic properties.

Isolation and characterization of a novel organic solvent-tolerant and halotolerant esterase from a soil metagenomic library

Abstract Soil metagenome conceals a great variety of unexploited genes for industrially important enzymes. To identify novel genes conferring lipolytic activity, one metagenomic library comprising of 200,000 transformants were constructed. Among the 48,000 clones screened, 19 clones which exhibited lipolytic activity were obtained. After sequence analysis, 19 different lipolytic genes were identified. One of these genes, designated as estWSD, consisted of 1152 nucleotides, encoding a 383-amino-acid protein. Multiple sequence alignment and phylogenetic analysis indicated that EstWSD and its closest homologues may constitute a new family of bacterial lipolytic enzymes. The best substrate for the purified EstWSD among the ρ-nitrophenol esters examined was ρ-nitrophenol butyrate. Recombinant EstWSD displayed a pH optimum of 7.0 and a temperature optimum of 50 °С. This enzyme retained 52% of maximal activity after incubation at 50 °C for 3 h. Furthermore, EstWSD also exhibited salt tolerance with over 51% of its initial activity in the presence of up to 4.5 M NaCl for 1 h. In particular, this enzyme showed remarkable stability in 15% and 30% dimethylsulfoxide, ρ-xylene, hexane, heptane, and octane even after incubation for 72 h. To our knowledge, it is the first report to find a novel esterase belonging to a new lipolytic family and possessing such variety of excellent features. All these characteristics suggest that EstWSD may be a potential candidate for application in industrial processes.

A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

BackgroundMicrobial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities.Methodology/Principal FindingsBacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220.ConclusionEstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.

A thermostable esterase from Thermoanaerobacter tengcongensis opening up a new family of bacterial lipolytic enzymes

An unidentified α/β hydrolase gene lipA3 from thermostable eubacterium species Thermoanaerobacter tengcongensis MB4 was cloned and heterologously expressed by Escherichia coli BL21(DE3)pLysS. The purified recombinant enzyme EstA3 turned out to be a monomeric thermostable esterase with optimal activity at 70°C and pH 9.5. The enzyme showed lipolytic activity towards a wide range of ester substrates including p-nitrophenyl esters and triacylglycerides, with the highest activity being observed for p-nitrophenyl caproate at 150 U/mg and for Triacetin at 126U/mg, respectively. Phylogenetic analysis revealed that EstA3 did not show homology to any identified bacterial lipolytic hydrolases. Sequence alignment showed that there was a common pentapeptide CHSMG with a cysteine replacing the first glycine in most esterase and lipase conserved motif GXSXG. The catalytic triad of EstA3 is Ser92, Asp269 and His292, which was confirmed by site directed mutagenesis. Based on the enzymatic properties and sequence alignment we concluded that the esterase EstA3 represented a novel bacterial lipolytic enzyme group and in chorological order this group was assigned as Family XIV.

Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment

To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50-57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.

Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome

Functional screening for lipolytic enzymes at low temperatures resulted in the isolation of the novel cold-active esterases, EstM-N1 and EstM-N2, from a metagenomic DNA library of arctic soil samples. EstM-N1 and EstM-N2 were 395 and 407 amino acids in length, respectively, and showed the highest similarity to class C β-lactamases. However, they shared a relatively low level of sequence similarity (30%) with each other. Phylogenetic analysis of bacterial lipolytic enzymes confirmed that EstM-N1 and EstM-N2 belonged to family VIII of bacterial esterases/lipases. The (His)(6)-tagged esterases were purified to about 99% homogeneity from the soluble fraction of recombinant Escherichia coli cultures. The purified EstM-N1 and EstM-N2 retained more than 50% of maximal activity in the temperature range of 0-35 °C, with optimal temperatures of 20 °C and 30 °C, respectively. Both enzymes preferred the short acyl chains of p-nitrophenyl esters and exhibited very narrow substrate specificity, indicating that they are typical esterases. The β-lactamase activity of EstM-N1 and EstM-N2 was also detected and reached about 31% and 13% of the positive control enzyme, Bacillus cereus β-lactamase, respectively. These first cold-active esterases belonging to family VIII are expected to be useful for potential biotechnological applications as interesting biocatalysts.

English

Lipases play an important role in lipid metabolism and are produced by a variety of species. All lipases are members of the a/â hydrolase fold super-family. Also, lipases share a conserved active site signature, the Gly-Xaa-Ser-Xaa-Gly motif. To obtain an overview of this industrially and very important class of enzymes and their characteristics, we collected and classified bacterial lipases sequences available from protein databases. Here we proposed an updated and revised classification of family I bacterial true lipases based mainly on a comparison of their amino acid sequences and some fundamental physicochemical and biological properties. The result of this work has identified 11 subfamilies of “true” lipases. This work will therefore contribute to a faster identification and to an easier characterization and classification of novel bacterial lipolytic enzymes. Key words : Lipases, phylogenetic analysis, lipolytic enzymes.

Cloning and heterologous expression of two cold-active lipases from the Antarctic bacterium Psychrobacter sp. G

Antarctic bacteria producing extracellular lipolytic enzymes with activity at low temperature were isolated, and the most promising strain, named G, was identified as a Psychrobacter species based on 16S rDNA sequence alignment. The genomic DNA of this bacterium was used to construct its plasmid genomic library into pUC118 plasmid vectors, and to screen the cold-active lipolytic enzyme genes. Two genes encoding for cold-active lipolytic enzymes, Lip-1452 (with an open reading frame of 1452 bp in length) and Lip-948 (with an open reading frame of 948 bp in length), were screened. The primary structure of the two lipases deduced from the nucleotide sequence showed a consensus pentapeptide containing the active serine (Lip-1452, GDSAG, and Lip-948, GNSMG) and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. Protein sequence alignment and conserved regions analysis indicated that the two lipases probably belonged to family IV and family V of the bacterial lipolytic enzymes, respectively. The upstream and downstream sequences of the two lipolytic lipases were also obtained. The two lipase genes were cloned into the expression vector pCold III and integrated into Escherichia coli BL21 (DE3). The functional expression of both lipase genes by E. coli BL21 (DE3) cells was observed as the formation of clear haloes around colonies on a 1% (vol/vol) tributyrin plate upon induction with isopropyl-b-Dthiogalactopyranoside at 5°C. A lipase activity assay showed that the specific activity of the pCold III+Lip-948 expression system was up to 3.7 U ml-1, whereas that of pCold III+Lip-1452 was very low.

The Pseudomonas aeruginosa patatin‐like protein PlpD is the archetype of a novel Type V secretion system

We discovered a novel secreted protein by Pseudomonas aeruginosa, PlpD, as a member of the bacterial lipolytic enzyme family of patatin-like proteins (PLPs). PlpD is synthesized as a single molecule consisting of a secreted domain fused to a transporter domain. The N-terminus of PlpD includes a classical signal peptide followed by the four PLP conserved blocks that account for its lipase activity. The C-terminus consists of a POTRA (polypeptide transport-associated) motif preceding a putative 16-stranded beta-barrel similar to those of TpsB transporters of Type Vb secretion system. We showed that the C-terminus remains inserted into the outer membrane while the patatin moiety is secreted. The association between a TpsB component and a passenger protein is a unique hybrid organization that we propose to classify as Type Vd. More than 200 PlpD orthologues exist among pathogenic and environmental bacteria, which suggests that bacteria secrete numerous PLPs using this newly defined mechanism.

Novel lipolytic genes from the microbial metagenomic library of the South China Sea marine sediment

Metagenomic cloning is a powerful tool for the discovery of novel genes and biocatalysts from environmental microorganisms. Based on activity screening of a marine sediment microbial metagenomic library, a total of 19 fosmid clones showing lipolytic activity were identified. After subcloning, 15 different lipolytic genes were obtained; their encoded proteins showed 32-68% amino acid identity with proteins in the database. Multiple sequence alignment and phylogenetic tree analysis demonstrated that most of these predicted proteins are new members of known families of bacterial lipolytic enzymes. However, two proteins, FLS18C and FLS18D, could not be assigned to any known family, thus probably representing a novel family of the bacterial lipolytic enzyme. The activity assay results indicated that most of these lipolytic enzymes showed optimum temperature for hydrolysis at 40-50 degrees C with p-nitrophenol butyrate as a substrate. The lipolytic gene fls18D was overexpressed, and the resulting protein FLS18D was characterized as an alkaline esterase. Furthermore, the whole sequence of fosmid pFL18 containing FLS18C and FLS18D was shotgun sequenced, and a total of 26 ORFs on it were analyzed and annotated.

Cloning and heterologous expression of two cold-active lipases from the Antarctic bacterium Psychrobacter sp. G

Antarctic bacteria producing extracellular lipolytic enzymes with activity at low temperature were isolated, and the most promising strain, named G, was identified as a Psychrobacter species based on 16S rDNA sequence alignment. The genomic DNA of this bacterium was used to construct its plasmid genomic library into pUC118 plasmid vectors, and to screen the cold-active lipolytic enzyme genes. Two genes encoding for cold-active lipolytic enzymes, Lip-1452 (with an open reading frame of 1452 bp in length) and Lip-948 (with an open reading frame of 948 bp in length), were screened. The primary structure of the two lipases deduced from the nucleotide sequence showed a consensus pentapeptide containing the active serine (Lip-1452, GDSAG, and Lip-948, GNSMG) and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. Protein sequence alignment and conserved regions analysis indicated that the two lipases probably belonged to family IV and family V of the bacterial lipolytic enzymes, respectively. The upstream and downstream sequences of the two lipolytic lipases were also obtained. The two lipase genes were cloned into the expression vector pCold III and integrated into Escherichia coli BL21 (DE3). The functional expression of both lipase genes by E. coli BL21 (DE3) cells was observed as the formation of clear haloes around colonies on a 1% (vol/vol) tributyrin plate upon induction with isopropyl-b-Dthiogalactopyranoside at 5°C. A lipase activity assay showed that the specific activity of the pCold III+Lip-948 expression system was up to 3.7 U ml-1, whereas that of pCold III+Lip-1452 was very low.

The cold-active Lip1 lipase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is a member of a new bacterial lipolytic enzyme family

The genome of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 was searched for the presence of genes encoding ester-hydrolysing enzymes. Amongst the others, the gene PSHAa0051 coding for a putative secreted esterase/lipase was selected. The psychrophilic gene was cloned, functionally over-expressed in P. haloplanktis TAC125, and the recombinant product (after named PhTAC125 Lip1) was purified. PhTAC125 Lip1 was found to be associated to the outer membrane and exhibited higher enzymatic activity towards synthetic substrates with long acyl chains. A structural model was constructed using the structure of carboxylesterase Est30 from Geobacillus stearothermophilus as template. The model covered the central part of the protein with the exceptions of PhTAC125 Lip1 N- and C-terminal regions, where the psychrophilic protein displays extra-domains. The constructed model showed a typical alpha/beta-hydrolase fold, and confirmed the presence of a canonical catalytic triad consisting of Ser, Asp and His. The sequence analysis showed that PhTAC125 Lip1 is distantly related to other lipolytic enzymes, but closely related to other putative psychrophilic esterases/lipases. The aligned proteins share common features, such as: (1) a conserved new active-site pentapeptide motif (LGG(F/L/Y)STG); (2) the likely extra-cytoplasmic localization, (3) the absence of a typical calcium-binding pocket, and (4) the absence of a canonical lid. These observations strongly suggest that aligned proteins constitute a novel lipase family, typical of psychrophilic marine gamma-proteobacteria, and PhTAC125 Lip1 could be considered the first characterised member of this family.

Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7–9 and 8–10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50–75 °C, with optimal activity at 70 °C and were stable at 70 °C for at least 120 min. Both PLP and Est1 exhibited high V max toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C 4 and C 5) and long chain (C 14 and C 16) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C 10. We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.