Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Biochemical and molecular characterisation of a thermoactive, alkaline and detergent-stable lipase from a newly isolated Staphylococcus aureus strain

A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture medium. The extracellular lipase from S. aureus (SAL3) is purified to homogeneity. The purified enzyme is a tetrameric protein (180 kDa) corresponding to the association of four lipase molecules. The 15 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. The part of the gene encoding the mature SAL3 is cloned and sequenced. The deduced polypeptide sequence, corresponding to the mature SAL3, was very similar to the mature Staphylococcus simulans lipase sequence with two additional amino acid residues (LK) at the N-terminus of SAL3. The lipase activity is maximal at pH 9.5 and 55 °C. The specific activity of about 4200 U/mg or 3500 U/mg was measured using tributyrin or olive oil emulsion as substrate, respectively, at pH 9.5 and 55 °C.In contrast to other staphylococcal lipases previously characterised, SAL3 is found to be stable between pH 5 and 12 after 24 h incubation. The enzyme retained 50% of its activity after 60 min incubation at 60 °C. This novel lipase is able to hydrolyse its substrate in presence of various oxidizing agents as well as some surfactants and some commercial detergents, then SAL3 can be considered as a good candidate for industrial and biotechnological applications.     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124

By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10–90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (ee_p 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.

     

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. GMD509

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.     

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K _m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.     

Optimization of a Thermostable Lipase from Bacillus stearothermophilus P1: Overexpression, Purification, and Characterization

An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55°C. It was highly stable in the temperature range of 30–65°C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37°C in 0.1% Chaps and Triton X-100.     

Characterization of two soil metagenome-derived lipases with high specificity for <Emphasis Type="Italic">p</Emphasis>-nitrophenyl palmitate

Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters. P. Wei and L. Bai contributed equally to this work.     

Cloning,expression and characterization of a new lipase from <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Bioinformatic analysis of the Yarrowia lipolytica CLIB122 genome has revealed 18 putative lipase genes all of which were expressed in Escherichia coli and screened for hydrolyzing activities against p-nitrophenyl-palmitate. One positive transformant containing an ORF of 1,098 bp encoding a protein of 365 amino acids was obtained. To characterize its enzymatic properties, the lipase gene was functionally expressed in Pichia pastoris. The resulting lipase exhibited the highest activity towards p-NP-decanoate at pH 7 and 35°C. In addition, the new lipase had a lower optimal temperature and pH compared to other Y. lipolytica lipases. It was noticeably enhanced by Ca²⁺, but was inhibited by PMSF, Hg²⁺ and Ni²⁺. The new lipase displayed the 1,3-specificity for triolein.     

Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader <Emphasis Type="Italic">Bacillus</Emphasis> sp. HH-01

An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4–11 and up to 30°C, and its optimum pH and temperature were 8–9 and 30°C, respectively. The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly, enhanced by supplementing with 1 mM CoCl₂, in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil.     

Novel thermostable lipase from <Emphasis Type="Italic">Bacillus circulans</Emphasis> IIIB153: comparison with the mesostable homologue at sequence and structure level

Thermophilic Bacillus circulans IIIB153 isolated from hot springs of North West Himalayas, India, produced an extracellular lipase, which exhibited significant biofilm disruption property on the static biofilm disruption model with a single species of Actinomyces viscosous. The gene encoding the lipase was cloned and overexpressed in Escherichia coli. Recombinant Bacillus circulans lipase (BCL), a monomer with molecular mass of 43 kDa also exhibited significant biofilm disruption activity. The enzyme was optimally active at 60°C, pH 8.5 and retained >70% of its original activity after 1 h incubation at 60°C. 3D structure of BCL developed by homology modeling showed a typical α/β hydrolase fold, a characteristic feature of lipolytic enzymes. Comparison of thermostable BCL with mesostable lipase from Chromobacterium viscosum at the sequence and structure level showed distinct variations in the structural features, with the presence of a high content of proline residues, aromatic amino acids and salt bridges. These features along with the presence of zinc-binding site observed in BCL structure could have a potential role in thermal stability of the enzyme.     

Surface display of active lipase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using Cwp2 as an anchor protein

Lipase Lip2 from Yarrowia lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm that the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 ± 0.4 U/g (dry cell) when using (G₄S)₃ sequence as the linker, with an optimal temperature and pH at 40°C and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free Lip2 as reported previously.     

Purification and Biochemical Characterization of Digestive Lipase in Whiteleg Shrimp

Penaeus vannamei lipase was purified from midgut gland of whiteleg shrimp. Pure lipase (E.C. 3.1.1.3) was obtained after Superdex 200 gel filtration and Resource Q anionic exchange. The pure lipase, which is a glycosylated molecule, is a monomer having a molecular mass of about 44.8 kDa, as determined by SDS-PAGE analysis. The lipase hydrolyses short and long-chain triacylglycerols and naphthol derivates at comparable rates. A specific activity of 1787 U mg⁻¹ and 475 U mg⁻¹ was measured with triolein and tributyrin as substrates, respectively, at pH 8.0 and 30°C in the absence of colipase. The lipase showed a K _{m, app} of 3.22 mM and k _{cat, app}/K _{m, app} of 0.303 × 10³ mM⁻¹ s⁻¹ using triolein as substrate. Natural detergents, such as sodium deoxycholate, act as potent inhibitors of the lipase. This inhibition can be reversed by adding fresh oil emulsion. Result with tetrahydrolipstatin, an irreversible inhibitor, suggests that the lipase is a serine enzyme. Peptide sequences of the lipase were determined and compared with the full-length sequence of lipase which was obtained by the rapid amplification of cDNA ends method. The full cDNA of the pvl was 1,186 bp, with a deduced protein of 362 amino acids that includes a consensus sequence (GXSXG) of the lipase superfamily of α/β-hydrolase. The gene exhibits features of conserved catalytic residues and high homology with various mammalian and insect lipase genes. A potential lid sequence is suggested for pvl.     

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.     

Enantioselective hydrolysis of β-hydroxy nitriles using the whole cell biocatalyst Rhodococcus rhodochrous ATCC BAA-870

Candida rugosa lipase was covalently immobilized onto silica gel in two different ways: via glutaraldehyde (L_GAL) and via hydrophobic spacer arm (1,6 diamino hexane) (L_SA). Free lipase, L_GAL and L_SA were used to investigate the hydrolysis of two different substrates, namely p-nitrophenyl palmytate (pNPP) and p-nitrophenyl acetate (pNPA), both in aqueous medium. In addition, these lipase samples were used to synthesize the pNPP from p-nitrophenol (pNP) and palmytic acid (PA) and pNPA from pNP and acetic acid (AA), both in hexane medium. Hydrolytic and synthetic activities of L_SA were higher than those of free lipase and L_GAL. Synthetic activities of free lipase, L_GAL and L_SA for pNPA in the presence of pNP and AA within hexane medium were higher than those of hydrolytic activities for pNPA in aqueous medium. The same tendency was also observed with pNPP. The effects of pH and temperature on hydrolytic and synthetic activities were investigated for all lipase preparations. Operational stability was the highest for L_GAL and L_SA when these enzymes were used for pNPP synthesis and in hexane medium, after 100 repeated uses, 68% and 51% of initial activities remained, respectively, at the end of 100 repeated cycles. Free lipase lost all of its activity within 15 and 20 days when stored at 25 °C and 5 °C, respectively. However, L_GAL showed 54% and 70% of initial activity at the end of 60 storage days at 25 °C and 5 °C, respectively, while these values were observed as 36% and 60% for L_SA.     

Cloning and Characterization of a Carboxylesterase from Bacillus coagulans 81-11

A genomic library of Bacillus coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27 528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50°C, although the enzyme displayed stability at temperatures up to 60°C.     

<Emphasis Type="Italic">Fervidobacterium changbaicum</Emphasis> Lip1: identification,cloning, and characterization of the thermophilic lipase as a new member of bacterial lipase family V

A novel lipase gene encoded 315 amino acid residues was obtained using lipase-prospecting primers and genome walking from hyperthermophilic bacterium Fervidobacterium changbaicum CBS-1. Sequence alignment and phylogenetic analysis revealed this novel lipase is a new member of bacterial lipase family V. The recombinant enzyme F. changbaicum lipase 1 (FCLip1) showed maximum activity at 78°C and pH 7.8. It displayed extreme thermostability at 70°C and was also stable across a wide pH range from 6.0 to 12.0. Kinetic study demonstrated FCLip1 preferentially hydrolyzed middle-length acyl chains, especially p-nitrophenyl caprate and tricaprylin. With p-nitrophenyl caprate as a substrate, the enzyme exhibited a K _m and k _cat of 4.67 μM and 22.7/s, respectively. In addition, FCLip1 was resistant to various detergents and organic solvents. This enzyme is the first reported thermophilic lipase from bacterial family Thermotogaceae. Its extreme stability with respect to temperature and pH, along with its triglyceride hydrolysis activity, indicate that FCLip1 has high potential for future application.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

Production of a novel extracellular acidic lipase from Pseudomonas gessardii using slaughterhouse waste as a substrate

An isolate exhibiting high extracellular lipolytic activity was identified as Pseudomonas gessardii by 16S rDNA gene sequence analysis. The slaughterhouse waste, goat tallow, was used as a lipid substrate for the production of acidic lipase by P. gessardii. The maximum lipase activity of 156 U/ml was observed at an acidic pH of 3.5 and at 0.31 g substrate concentration. The purification steps resulted in the isolation of acidic lipase with a specific activity of 1,473 U/mg and a molecular weight of 94 kDa. One interesting feature of this purified lipase is its stability at highly acidic pH ranging from 2.0 to 5.5 with a high molecular weight. The amino acid composition was determined using HPLC. This acidic lipase has potential applications in the medicinal field as a substitute for pancreatic lipases for enzyme therapy, oleochemical and in biotechnological industries.