Acidic lipase Lip I.3 from a Pseudomonas fluorescens-like strain displays unusual properties and shows activity on secondary alcohols



Paola Panizza, Department of Microbiology, Faculty of Biology, University of Barcelona, Av. Diagonal 645, 08028-Barcelona, Spain. E-mail:



Identification, cloning, expression and characterization of a novel lipase – Lip I.3 – from strain Pseudomonas CR-611.

Methods and Results

The corresponding gene was identified and isolated by PCR-amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0-1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C-terminal type I secretion signal and several putative Ca2+ binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca2+ ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols.


A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts.

Significance and Impact of the Study

The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis.


Lipases (EC are enzymes that hydrolyze the carboxyl ester bonds in acyl-glycerides. Microbial lipases are the second largest group of industrial biocatalysts after bacterial amylolytic enzymes (Guncheva and Zhiryakova 2011). They have found numerous applications within biotechnology industries such as food technology, detergent formulation, chemical industry and biomedical sciences (Hasan et al. 2006; Guncheva and Zhiryakova 2011). More interestingly, some lipases show high regio and stereoselectivity, rendering them important tools in the synthesis of chiral compounds for the pharmaceutical industry (Patel 2006; Ghanem 2007; Turner 2010). The increasing demand for environmentally benign industrial processes, as well as the rising need for stereoselective synthetic routes, has prompted the search for novel enzymes to broaden the scope of biocatalytic tools and applications (Steele et al. 2009; Zhang et al. 2009).

Most lipases used in biocatalysis are microbial enzymes isolated from both, fungi and bacteria. Amongst the fungal lipases, those isolated from Candida antarctica, Candida rugosa, Geotrichum and Rhizopus have found extensive applications in organic synthesis (Lambusta et al. 2003; Domínguez de María et al. 2005, 2006; Ghanem 2007). Amongst the bacterial lipases, the most widely explored are those belonging to the genus Pseudomonas (Lambusta et al. 2003; Grogan 2008; Turner 2010) or those of Bacillus-related species (Ruiz et al. 2002, 2003, 2005; Sanchez et al. 2002). However, most reports on lipases from Pseudomonas have centred on enzymes belonging to Families I.1 and I.2 (Arpigny and Jaeger 1999; Bofill et al. 2010). On the contrary, lipase Family I.3 has scarcely been explored from the biocatalytic point of view (Angkawidjaja and Kanaya 2006). Therefore, the study of different members of this family can provide an insight to their potential applications in biocatalysis.

Family I.3 lipase is a subfamily of gram negative bacterial true lipases secreted by a type I secretion system (Angkawidjaja and Kanaya 2006). A few members of this family have been reported so far, with Pseudomonas sp. MIS38 (PML) and Serratia marcescens (SML) lipases being the most largely explored from a structural and biochemical perspective (Li et al. 1995; Amada et al. 2000; Hyun-Ju et al. 2000; Angkawidjaja et al. 2007; Meier et al. 2007), the latter showing promising results for the preparation of a key intermediate in the synthesis of diltiazem, a major pharmaceutical used as a coronary vasodilator (Jaeger et al. 1999; Jaeger and Eggert 2002). Thus, characterization of other members of this lipase family can provide an insight on the usefulness of this type of lipases for further biotechnological applications.

Previously reported members of bacterial family I.3 lipases have optimum temperatures ranging 35–55°C, and a mildly alkaline to optimum pH (pH 7·5–8·5) (Angkawidjaja and Kanaya 2006). The structure of Pseudomonas sp. MIS38 (PML) lipase reveals an N-terminal domain containing the lipase catalytic triad, a C-terminal domain displaying a β-roll structure containing the Ca2+-binding motif and a C-terminal secretion signal (Angkawidjaja et al. 2007, 2010). As most true lipases, PML shows ‘interfacial activation’, with two lids covering the active site (Angkawidjaja et al. 2010). Here, we describe the cloning, expression, purification and characterization of a novel lipase LipI.3 from Pseudomonas CR-611 belonging to Family I.3 (Ruiz et al. 2005; Prim et al. 2006). The new enzyme differs from the previously reported enzymes of this family in the optimal pH, being the first acidophilic member described. A peculiar finding was the lack of interfacial activation in this enzyme, an unexpected phenomenon amongst true lipases (Ghanem 2007; Meier et al. 2007).

Materials and methods

Strains and growth conditions

Strain Pseudomonas sp. CR-611 (CECT 8156) (Ruiz et al. 2005) was grown in Luria–Bertani (LB) broth or on LB agar plates at 30°C. Strains Escherichia coli 5K or DH5α were cultivated at 37°C in LB broth or on LB agar plates and supplemented with ampicillin (200 μg ml−1) when harbouring plasmids.

DNA manipulation and cloning

Standard procedures were used for DNA manipulation (Sambrook and Russell 2001). Plasmid DNA was purified using commercial chromatography kits (Qiagen-tip 100 MidiPrep; Qiagen, Hilden, Germany). Restriction nucleases (Roche, Rotkreuz, Switzerland) and thermostable polymerase Taq (Biotools, Madrid, Spain) were used according to the manufacturers' instructions. PCR amplifications were performed in a GeneAMP PCR system 2400 (Perkin Elmer, Waltham, MA, USA) using adequate cycling periods. To obtain the nucleotide sequences, isolated DNA fragments were analyzed using the ABI Prism® BigDye® Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), and the analytical system CEQTM 8000 (Beckman-Coulter, Brea, CA, USA), available at the Serveis Científico Tècnics of the Universitat de Barcelona. DNA samples were routinely analyzed by agarose gel electrophoresis (Sambrook and Russell 2001). Nucleic acid concentration and purity was measured using a NanoDrop® ND-1000 spectrophotometer (Thermo-Scientific, Wilminton, DE, USA).

Primers FWPSLIP (5′-TAT CCC ATC GTG CTG GCC CAC GG-3′) and BKPSLIP (5′-GGC TGT GGC CGA TCA GAT TGA C-3′) were designed based on conserved regions from reported lipases belonging to the Pseudomonas genus (Bofill et al. 2010). Amplified DNA bands were purified by gel electrophoresis using QIAquick Gel Extraction Kit (Qiagen), and their sequence determined. Blast search (Altschul et al. 1997) was performed at the National Center for Biotechnology Information (NCBI) webpage ( Sequences of different Pseudomonas lipases (accession numbers CP000094, AY623009, AY721617, AY694785, AF307943, AF202538, AB025596, AB109040 and D11455) were aligned using Clustal W MultalignX. Specific primers FwLip1-3 (5′-AAG TAG TAC TGA AAG CTT CCG-3′) and BwLip1-3 (5′-GTA TCT AGA TCA GGC GAT CAC RATY-3′), designed based on the consensus regions of the former Family I.3 sequences, were used to amplify a 1950 bp fragment corresponding to Lip I.3 gene and its associated regulation regions (accession no. JX073030). The amplified fragment was ligated to pGEM-T vector (Promega, Fitchburg, WI, USA) and cloned into E. coli DH5α and 5K.

Analysis of Lip I.3 sequence

DNA sequences were analyzed and translated using Vector NTi software package (Invitrogen, Carlsbad, CA, USA). The search and alignment of the nucleotide and amino acid sequences was performed using Blast (Altschul et al. 1997) or Clustal W MultalignX (Thompson et al. 1994; Larkin et al. 2007). A 3D model of Lip I.3 was constructed with Swiss-model (Arnold et al. 2006) using reported 3D structures of Pseudomonas sp. MIS38 lipase (pdb|2Z8Z|A or pdb|2ZVD|A) as templates. For comparison, crystal and modelled structures were visualized using Cn3D (NCBI).

Production and purification of lipase Lip I.3

The cloned Lip I.3 lipase was purified from E. coli cell extracts, using a previously described method for inclusion body solubilization (Kojima et al. 2003) with minor modifications. Cells from 400 ml cultures were collected by centrifugation, suspended in 25 ml 50 mmol l−1 sodium phosphate buffer (pH = 7) and disrupted with a French press (1000 psi, 1 min, twice for every sample). Crude extracts obtained by this procedure were directly used for assays, or further purified by ion exchange fast protein liquid chromatography using a Tricorn MonoQ 5/50 column in an ÄKTA protein purifier (Amersham Biosciences, Amersham, UK). Tris-HCl 20 mmol l−1 pH 7 with 0·5% Triton X-100 was used as running buffer, and purification was achieved by applying a NaCl concentration gradient from 0 to 1 mol l−1. Lipolytic activity was detected on filter paper or by zymogram (Diaz et al. 1999), using methylumbeliferone (MUF)-butyrate or MUF-heptanoate as substrates.

Activity assays

General procedure

The activity was determined using either esters of p-nitrophenol (pNP) (Lesuisse et al. 1993; Prim et al. 2000) or esters of 4-MUF (Diaz et al. 1999; Prim et al. 2003) as model substrates. Unless otherwise specified, all assays were done on microtitre plates, performing three repetitions in each replicate and at least two replicates for assay. Three blanks were always done for each set of conditions (reaction mixture without enzyme, without substrate and without either the enzyme or substrate). The catalytic unit was defined as the amount of enzyme that released 1 μmol of pNP or MUF in 1 min under the conditions used. For enzyme characterization assays, the General procedure was used with the modifications stated in Table 1.

Table 1. Changes made to the General procedure for activity determination
  1. a

    50 mmol l−1 ATB buffer: 50 mmol l−1 acetic acid, 50 mmol l−1 Tris·HCl, 50 mmol l−1 boric acid.

Optimum pH50 mmol l−1 ATB buffera adjusted to each pH was used instead of Tris or Acetate buffer
Optimum temperatureVariable temperatures instead of 30°C
Calcium concentrationVariable calcium concentrations in the reaction mix
Kinetic parametersVariable substrate concentration in the reaction mix
Inhibitors/activatorsInhibitors/activators were included in the enzyme mix at a concentration of 1 or 10 mmol l−1, and 0·5 U ml−1 Lip I.3 were used
Thermal stability (short term)Enzyme mix was pre-incubated for 1 h at each temperature and then assayed under optimum conditions
Thermal stability (long term)Enzyme was maintained for 1 h at the tested temperatures. Assays were then performed as indicated in the General procedure
pH resistanceEnzyme was pre-incubated for 1 h at room temperature in ATB buffera adjusted to each pH prior to assay at optimum conditions

Activity assays using pNP derivatives

One volume of enzyme mix (20 mmol l−1 Tris buffer pH 7, 20 mmol l−1 CaCl2 and 11–18 U Lip I.3) and one volume of substrate mix (20 mmol l−1 Tris buffer pH 7, 1·2% Triton X-100, and 2 mmol l−1 pNP-derivative) were pre-incubated for 15 min at 30°C before mixing them at a 1 : 1 ratio in a final volume of 100 μl. Reactions were incubated at 30°C for 15 min, and absorbance at 405 nm was measured in a microtitre plate reader (Bio-Rad Model 3550; Bio-Rad, Hercules, CA, USA) for activity calculation based on a calibration plot previously obtained for free pNP. For pNP-laurate, -palmitate and -stearate, 1 mmol l−1 concentration was used in the substrate mix due to their low solubility, and results were corrected by a factor of 2 with respect to those of shorter substrates, for comparison.

Activity assays using MUF derivatives

Enzyme mix solution (50 mmol l−1 acetate buffer pH 5·5, 20 mmol l−1 CaCl2, and 0·10–0·25 U Lip I.3) and substrate mix solution (50 mmol l−1 acetate buffer pH 5·5, 0·8% Triton X-100 and 0·8 mmol l−1 MUF-derivative) were pre-incubated for 15 min at 30°C, and further mixed at a 1 : 1 ratio in a final volume of 100 μl. Reactions were incubated at 30°C for 30 min and the released fluorescence was measured in a Varian Cary Eclipse spectrofluorometer (λex = 323 nm, λem = 448 nm) for activity determination based on a previously obtained MUF calibration curve.

Secondary alcohol hydrolysis assays

Hydrolytic activity of Lip I.3 on substrates 14 (Fig. 1) was evaluated based on a previously reported method (Naik et al. 2010). Reaction media for hydrolysis assays contained 50 mmol l−1 Tris (pH 7), 5 mmol l−1 CaCl2, 0·4% Triton X-100 and 40 mmol l−1 of each substrate. A total volume of 6 μl of Lip I.3 crude cell extract, 3 mg of CAL-B (Novozyme 435; Novozyme, Bagsværd, Denmark) or 3 mg of CAL-A (Fluka, Buchs, Switzerland) were used in 5 ml reaction assays. Blanks without enzyme were also performed. After incubation in a linear shaker at 30°C for 48 h, the reaction medium was extracted with 5 ml CH2Cl2, the organic layer was dried with Na2SO4 and the extract analyzed by gas chromatography using the conditions described in Table 2.

Table 2. Gas chromatography analysis conditions for the compounds assayed
SubstrateColumnTemperature program
1 Carbowax 20 (0·25 mm × 30 m × 0·25 μm)60°C|40°C min−1|110°C (5 min)|3°C min−1|160°C|25°C min−1|240°C (5 min)
Megadex_DET_TBS_Beta (0·25 mm × 25 m × 0·25 μm)60°C|40°C min−1|100°|1°C min−1|125°C|45°C min−1|200°C (5 min)
2 Carbowax 20 (0·25 mm × 30 m × 0·25 μm)60°C|25°C min−1|130°C|7°C min−1|240°C (5 min)
3 Carbowax 20 (0·25 mm × 30 m × 0·25 μm)60°C|40°C min−1|130°C (5 min)|3°C min−1|180°C|25°C min−1|240°C (5 min)
Megadex_DET_TBS_Beta (0·25 mm × 25 m × 0·25 μm)60°C|40°C min−1|100°C|1·5°C min−1|140°C|45°C min|200°C (5 min)
4 Carbowax 20 (0·25 mm × 30 m × 0·25 μm)60°C|40°C min−1|80°C (5 min)|3°C min−1|120°C|25°C min−1|240°C (5 min)
Figure 1.

Compounds used to map substrate specificity of Lip I.3.


Lip I.3 isolation and cloning

Previous analysis of Pseudomonas CR-611 lipolytic system revealed the presence of a family VI intracellular esterase, already characterized (Prim et al. 2006) plus an unknown secreted lipase (Ruiz et al. 2005). To isolate the gene coding for such lipase, PCR amplification using consensus primers FWPSLIP and BKPSLIP derived from lipase Families I.1 and I.2 was performed. Pseudomonas CR-611 genome rendered a 450-bp amplicon showing homology to Family I.3 lipases. A new pair of primers (FwLip1-3 and BwLip1-3) was designed based on alignment of the nearest Pseudomonas lipase gene sequences deposited in the databanks. This allowed amplification of a 1950-bp DNA fragment including the whole gene, the putative −10 and −35 promoter regions and the ribosome binding site (accession no. JX073030). The amplified DNA fragment was subsequently ligated to pGEM-T and cloned in E. coli DH5α, obtaining recombinant clones bearing activity on MUF-butyrate on the screening assay (Diaz et al. 1999). Sequencing results confirmed the presence of the correct gene and regulation regions in the cloned construction, which were different from the previously isolated est6 gene (Prim et al. 2006). Due to the high lipolytic background shown by E. coli DH5α itself, the newly constructed recombinant plasmid was transferred to strain E. coli 5K, displaying no lipolytic background. The new 5K recombinant clone was used to produce and purify active Lip I.3 lipase.

Sequence and structure analysis

Analysis of Lip I.3 gene sequence revealed a GC-content of 62·6%. The −10 and −35 promoters plus a canonical Shine-Dalgarno region could be identified upstream the start codon, but no evident termination signals could be located on the cloned sequence. According to in silico translation of the gene, it encodes for a protein of 617 aa, with a predicted molecular mass of 65 kDa and a deduced pI of 4·63. This protein has no cysteine residues and thus, no disulfide bonds would be expected. As in other subfamily I.3 lipases, a five-residue conserved motif VTLVG (599–603), located at the C-terminal region, plus a C-terminal motif DGIVIA (612–617), apparently involved in type I secretion system (Kuwahara et al. 2011), were found. These motifs are preceded by three tandems of the repetitive nine-residue motif GGxGxDxux, that constitutes a calcium-binding domain (Kuwahara et al. 2011). Secondary structure analysis of LipI.3 revealed the typical α/β fold of lipases, together with an important hydrophobic region, and the presence of the characteristic nucleophilic elbow, constituted by the pentapeptide Gly-His-Ser-Leu-Gly, with the embedded catalytic serine residue at position 207. Surprisingly, two additional G-X-S-X-G pentapeptide-like motifs were found at positions 152–156 (Gly-Asp-Ser-Ile-Gly) and 268–272 (Gly-Ala-Ser-Leu-Gly), an unfrequent trait amongst lipases. Lip I.3 amino acid sequence shows a high identity with previously reported lipases belonging to subfamily I.3, particularly those of Pseudomonas fluorescens Pf0-1 (YP_348417) and Pseudomonas sp. MIS38 (pdb|2Z8Z|A or pdb|2ZVD|A). A 3D homology model of LipI.3 was constructed using the crystal structure of Pseudomonas sp. MIS38 lipase as a template. An excellent match of Lip I.3 and that of Pseudomonas sp. MIS38 was obtained in terms of structure and folding (not shown). This allowed unambiguous identification of the catalytic triad Ser207, Asp255 and His313 and discarded a role for Ser154 and Ser 270 in catalysis. Like in other subfamily I.3 lipases, no signal peptide could be identified in Pseudomonas CR-611 Lip I.3, suggesting that secretion occurs through the type I secretion system (Angkawidjaja and Kanaya 2006). Homology studies indicate that the catalytic domain is located at the most proximal amino acids of the sequence, whereas the C-terminal region of the protein is occupied by a well-defined β-roll structure constituted by several antiparallel β-sheets acting as calcium-binding sites (Angkawidjaja et al. 2007).

Enzyme production

Lipase I.3 was produced as inclusion bodies in strain E. coli 5K (pGEM-T Lip I.3). For enzyme recovery and purification, a previously reported method (Kojima et al. 2003) was used with minor modifications. Further purification from urea-solubilized inclusion bodies was achieved by ion-exchange fast protein liquid chromatography. A single band was obtained in SDS-PAGE that could be visualized by both, zymogram with MUF-butyrate and Coomassie Blue stains (Fig. 2). The purified protein displayed a specific activity of 1·14 U mg−1 prot when measured on pNP-caprilate and a specific activity of 13·2 U mg−1 prot when measured on MUF-heptanoate.

Figure 2.

Purification of Lip I.3 by ion exchange fast protein liquid chromatography, after inclusion body solubilization with urea. SDS-PAGE gel stained with Coomassie Blue (a) and zymogram of the same gel (b). Lane 1: molecular mass marker, Lane 2: crude cell extract applied to the column, Lane 3: fraction obtained after purification.

Lip I.3 characterization

Characterization of Pseudomonas CR-611 Lip I.3 was performed using pNP and MUF derivatives as model substrates. Lip I.3 showed the highest activity on medium-chain substrates like pNP-caprate or MUF-heptanoate (Fig. 3), whereas lower activity was found on longer chain-length substrates like pNP- or MUF-stearate. MUF derivatives with chain lengths between 8 and 16 are not commercially available and could not be tested for activity. Lipase Lip I.3 displayed maximum activity at 30°C, showing good catalytic activity between 20 and 40°C when assayed on MUF-heptanoate and pNP-caprilate (not shown). Using espectrofluorometry and MUF-heptanoate as substrate, optimum pH for Lip I.3 was determined to be 5·5 (Fig. 4a), an unusual trait amongst Family I.3 lipases. Lip I.3 activity was greatly affected by calcium concentration (data not shown). In the absence of calcium, Lip I.3 displayed only basal activity on pNP-caprate when assayed under optimum pH and temperature conditions. On the contrary, at 1 mmol l−1 calcium concentration, the activity was enhanced by 38-fold, and above that value, activity continued to increase until concentrations of 20 mmol l−1. Sodium dodecyl sulphate (SDS) dramatically inhibited Lip I.3 activity. Nevertheless, this inhibition was reversible and the activity could easily be restored by the addition of Triton X-100, as shown by the zymogram results obtained from denaturing gels. Urea also contributed to reduce Lip I.3 activity. 1 mol l−1 urea caused 40% loss of activity, whereas concentrations above 2 mol l−1 produced almost complete enzyme inhibition. However, this inhibition could be restored after urea removal, as confirmed during the purification process. Triton X-100 also displayed a negative effect on Lip I.3 activity at concentrations higher than 0·5 mol l−1. However, at low Triton X-100 concentrations, this effect was positive, contributing to enhance Lip I.3 activity. Phenylmethylsulphonyl fluoride (PMSF) was also assayed, showing no influence on Lip I.3 activity, even at 5 mmol l−1 concentration. In addition to the previous compounds, the effect of 17 inorganic ions on Lip I.3 activity was also tested. No significant effect was observed for most of the ions assayed at 1 mmol l−1 except for Fe and Cu (Fig. 4b). Nevertheless, several ions produced a drastic activity reduction (less than 50% residual activity) when assayed at 10 mmol l−1 (Fig. 4b). Analysis of the kinetic parameters of Lip I.3 was performed using MUF-heptanoate under optimum pH (5·5) and temperature (30°C) conditions and in the presence of 10 mmol l−1 CaCl2, revealing a Michaelis-Menten plot with no interfacial activation (not shown). The calculated apparent Vmax and Km values found for the enzyme on MUF-heptanoate were 2·48 ± 0·75 × 103 U mg−1 and 382 ± 47 μmol l−1 respectively.

Figure 3.

Substrate profile of Pseudomonas CR-611 Lip I.3.

Figure 4.

Optimum pH determination (a) and effect of different inorganic salts on Lip I.3 activity (b), measured at 1 and 10 mmol l−1 concentrations. Maximum activity was obtained at pH 5·5. A 100% corresponds to control activity without any added inorganic salt. (image_n/jam12089-gra-0001.png) 1 mmol l−1 and (image_n/jam12089-gra-0002.png) 10 mmol l−1

Stability of Lip I.3

Short- and long-term thermal stability assays were performed by measuring activity in a spectrofluorometer, using MUF-heptanoate as a substrate. Short-term thermal stability of Lip I.3 (Fig. 5a) was determined by measuring the residual activity under optimum conditions after 1 h incubation at temperatures ranging from 0 to 70°C. In the absence of Triton X-100, Lip I.3 lost activity when pre-incubated for 1 h at all the temperatures tested. A 40% decrease in the activity was observed when Lip I.3 was incubated for 1 h at 30°C, whereas at 60°C, the enzyme was almost completely inactivated after 1 h incubation. Stability of Lip I.3 on long-term storage was studied at −20°C, 4°C and room temperature, both in the presence or absence of Triton X-100. To measure the influence of Triton X-100 on storage, a solution of 20 mmol l−1 Tris·HCl (pH 7) was used as storage buffer with three different concentrations of surfactant (0, 0·2 and 0·5% v/v) (Fig. 5b). In the absence of Triton X-100, Lip I.3 activity decreased during storage at the three temperatures tested. However, in the presence of Triton X-100, the enzyme retained almost 100% activity for at least 22 days at 4 and −20°C. Room temperature-storage resulted in complete loss of activity after 7 days even in the presence of Triton X-100.

Figure 5.

Short (a) and long-term (b) thermal stability of Lip I.3. A 100% corresponds to activity before pre-incubation, measured under optimum conditions at time zero. Without Triton X-100: Enzyme was stored in 20 mmol l−1 Tris pH 7. With Triton X-100: Enzyme was stored in 20 mmol l−1 Tris pH 7 with 0·2 or 0·5% Triton X-100. (image_n/jam12089-gra-0003.png) −20°C; (image_n/jam12089-gra-0004.png) 4°C; (image_n/jam12089-gra-0005.png) RT; (image_n/jam12089-gra-0003.png) −20°C Triton 0·2%; (image_n/jam12089-gra-0004.png) 4°C Triton 0·2%; (image_n/jam12089-gra-0005.png) RT Triton 0·2%; (image_n/jam12089-gra-0006.png) −20°C Triton 0·5%; (image_n/jam12089-gra-0007.png) 4°C Triton 0·5% and (image_n/jam12089-gra-0008.png) RT Triton 0·5%.

Stability of Lip I.3, when exposed for 1 h to buffers with different pH values at room temperature was also studied. Residual activity was determined under optimum conditions, using MUF-heptanoate as substrate. A pH of 4 or lower caused more than 80% reduction of Lip I.3 activity. Nevertheless, Lip I.3 retained 80% activity after 1 h incubation at pH from 5 to 9 (data not shown).

Mapping the substrate specificity of Lip I.3

Naik and collaborators (Naik et al. 2010) reported the classification of different lipases based on their hydrolytic activity on eight substrates comprising two categories: (a) substrates either branched/large on the acidic residue or (b) substrates either branched/large on the alcohol part. Two substrates of each of such categories (Fig. 1) were used to test the hydrolytic activity of Lip I.3 on nonmodel substrates, providing the results shown in Table 3. Although the hydrolytic activity of Lip I.3 on such secondary alcohols was poor, a considerably high ee value was obtained for substrate 3, suggesting a better performance of the enzyme on aromatic alcohols rather than on alkyl alcohols.

Table 3. Biotransformation of substrates 14 using Pseudomonas CR-611 Lip I.3
SubstrateCategoryConversion (%)% ee
1 Ethyl-2-methyl butyratea480
2 Ethyl benzoatea1
3 Styrallyl butyrateb799
4 Cyclohexyl acetateb2


As expected from previous observations (Ruiz et al. 2005; Prim et al. 2006), lipase I.3 gene was identified by molecular methods and amplified using consensus primers FwLip1-3 and BwLip1-3. The amplified gene was successfully ligated to pGEMT and cloned in E. coli 5K to obtain a lipase-producing strain without significant lipolytic background. Lip I.3 was produced as inclusion bodies, the lipase remaining in the cellular debris after cell lysis. Kojima method (Kojima et al. 2003) proved to be useful to obtain the enzyme from inclusion bodies, with a high rate recovery of activity. This method not only allowed recovery of the enzyme in an active form, but also allowed separation of the cloned lipase from the intrinsic lipolytic enzyme of E. coli. Further purification by ion-exchange chromatography rendered the enzyme with a high degree of purity.

Amino acid sequence analysis and comparison indicates that the cloned enzyme belongs to Family I.3, a family that comprises few reported enzymes, most of them belonging to the genus Pseudomonas or Serratia, although enzymes belonging to Families I.1 and I.2 are more common in applied biocatalysis amongst Pseudomonas species (Arpigny and Jaeger 1999; Bornscheuer et al. 2002). According to the 3D structural model obtained based on homologous lipases, the active site was located at position Ser207. Contrary to what is expected for a Serine-protein, PMSF did not inhibit Lip I.3, a fact that has previously been described for other lipases (Das et al. 2000; Falcocchio et al. 2006; Bofill et al. 2010). This could be associated to the structural features that preclude PMSF from entering the active site (Das et al. 2000; Côté and Shareck 2008).

Surprisingly, Lip I.3 displayed typical Michaelis-Menten kinetics without any significant interfacial activation. However, the closest lipase from Pseudomonas MIS38, showing a 93% identity with Lip I.3, displayed interfacial activation (Amada et al. 2000). A deeper 3D modelling and analysis of Lip I.3 structure seems to indicate that the two lids proposed for Pseudomonas MIS38 lipase (PML) would also be present in Lip I.3. Nevertheless, the amino acid variations detected between Lip I.3 and PML at positions 149 (Asn in PML and Ile in Lip I.3) and 152 (Leu in PML and Gly in Lip I.3), both located in Lid 1 of PML, could be responsible for the lack of interfacial activation observed for Lip I.3. A similar amino acid variation was found in Serratia family I.3 lipase (SML), bearing Ser and Gly at those positions respectively (Meier et al. 2007). The side chain of Leu152 in PML points outward, probably favouring the observed bending of the α-helix (Angkawidjaja et al. 2007). In SML, a straight α-helix forms the lid, and a Ca2+ ion (Ca1) contributes to anchor the lid. The differences in amino acids between SML and Lip I.3 in the Lid 1 region are conservative, and this would result in a Lip I.3 Lid 1 more similar to SML Lid 1 than to PML Lid 1. Further crystal and X-ray studies of Lip I.3 may allow comparison of the three structures and enlighten this hypothesis.

In agreement with the Calcium-binding sites detected on Lip I.3, activity of the enzyme was strongly affected by Calcium concentration. The requirement of Calcium by subfamily I.3 lipases has been previously reported (Amada et al. 2000; Kojima et al. 2003; Bae et al. 2006) and has been explained based on structural data due to the presence of the β-roll structure located at the C-terminus of the protein (Angkawidjaja et al. 2007; Meier et al. 2007; Kuwahara et al. 2008). On the contrary, none of the other divalent cations tested had a positive effect on Lip I.3 activity, which was strongly inhibited by Zn2+, Hg2+, Fe2+ and Cu2+. Lip I.3 behaves as a robust enzyme when adequate conditions are provided. The presence of Triton X-100 has a positive effect; in the absence of this surfactant, low temperatures accelerate the aggregation process, leading to a faster decrease of activity. Presence of 0·2–0·5% Triton X-100 enhances Lip I.3 activity, probably because it avoids aggregation of the enzyme and thus, inactivation. Triton X-100 also enhances long-term stability of the enzyme, although at room temperature, denaturing and degradation surpass this effect. But when Triton X-100 is present at 0·2% concentration, the enzyme maintains its activity for at least 22 days at 4 and −20°C. As reported before for other members of subfamily I.3 (Angkawidjaja and Kanaya 2006), Lip I.3 showed preference for medium-chain substrates. Maximum activity was found at 30°C, a slightly lower temperature than those previously reported for other subfamily I.3 lipases. Optimum pH for Lip I.3 was determined to be 5·5, making Lip I.3 from Pseudomonas sp. CR-611 the first acidic lipase reported that belongs to this subfamily (Gupta et al. 2004; Angkawidjaja and Kanaya 2006). Lip I.3 displayed high activity towards pNP and MUF derivatives, also displaying activity on secondary alcohols. Nevertheless, activity towards substrates 1, 2, 3 and 4 was very low (Table 3). The fact that Lip I.3 showed better activity towards substrate 3, along with the results obtained for pNP and MUF derivatives, suggests that compounds containing an aromatic alcohol portion are better substrates for this enzyme. This could provide an interesting biocatalyst for fine chemicals since many drugs include chiral aromatic alcohols. Further substrate testing and enzyme modification will be performed to assess this hypothesis.


We thank the Serveis Cientifico-Tècnics of the University of Barcelona for technical assistance in sequencing. The present study was financed by the Scientific and Technological Research Council (CICYT, Spain), grant ref. CTQ2010-21183-C02-02/PPQ, by the IV Pla de Recerca de Catalunya (Generalitat de Catalunya), grant ref. 2009SGR-819, by the PCI-AECID, grant ref. A203563511, by the Agencia Nacional de Investigación e Innovación (ANII, Uruguay), grant ref. FMV 2009_1_2074, and by the Generalitat de Catalunya to the ‘Xarxa de Referència en Biotecnologia’ (XRB). M. Sc. Paola Panizza was a recipient of an MAEC-AECID fellowship (no. 0000309207), a graduate student scholarship from ANII (Uruguay) and currently holds a scholarship from CSIC, Universidad de la República, (Uruguay). P. Panizza thanks PEDECIBA Química for their support.