Sequence analysis of lip5-wt from C. albicans showed no introns in the lip5-wt gene sequence, and a putative N terminus signal sequence includes 14 amino acid residues, which exports the Lip5 outside the cells. lip5-wt gene with the deleted signal sequence was cloned from C. albicans genome DNA and sequenced. It was found that two non-universal Ser codons (CTG) exist in the lip5-wt gene, located in amino acid site 154 and 293 respectively. Triplet codon CTG was usually mistranslated into leucine in other organisms instead of serine [14]. To investigate the influence of codon CTG on the expression and characterization of lip5-wt, three mutants, which are lip5-m₁₅₄, lip5-m₂₉₃ and lip5-dm, were constructed. lip5-m₁₅₄, lip5-m₂₉₃ indicates that the corresponding gene codon of 154 and 293 amino acid were modified as TCT, respectively. lip5-dm was the double mutant at above two sites. The primers for gene mutation were listed in Table 1.

A search of Genbank using the deduced Lip5 amino acid sequence (HM581681) as a query by Blastp program revealed that Lip5 shared significant sequence identity with those of lipases from other Candidas species. Lip5 has 87%, 84%, 81%, 58% and 34% amino acid sequence identity with those of lipases from Candida dubliniensis (CDLip), Candida albiacans (CALip8 and CALip4) [15,16], Candida parapsilosis (CPLip2) [17] and Candida Antarctica (CALA) [18], respectively. It was also found that Lip5 shows low sequence identity with those of known lipases from psychrophilic organisms, including Photobacterium sp. M37 [8], Pseudomonas sp. 7323 [19], Pseudomonas fragi [20] and Psychrobacter sp. 2–17 [21] (data not shown). In addition, sequence alignment showed that all the lipase sequences contained a highly conserved pentapeptide GYSGG (Figure 1), which is consistent with the GXSXG motif found in the active site of most of the lipolytic enzyme [22]. Based on the alignment results, the putative catalytic triad of Lip5 is composed of Ser-194, Asp-343 and His-376 (Figure 1).

Lipases with different gene sequences, which included lip5-wt, lip5-m₁₅₄, lip5-m₂₉₃ and lip5-dm, have been expressed secretively in P. pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase (GAP) constitutive promoter, and their activities were investigated and the results were shown in Figure 2. Lip5-M₁₅₄ and Lip5-DM was expressed at high level with a molecular weight of about 50 kDa (Figure 2A), however, no obvious expression was observed from Lip5-WT and Lip5-M₂₉₃. It suggested that mistranslation of 154-Ser to leucine resulted in loss of expression of Lip5-WT, and that 154-Ser might either play an important role in the stability of the Lip5-WT, or have a negative effect on the expression of Lip5-WT. Roustan et al. have reported that a lipase from C. albicans (CaLip4) could be overproduced in Saccharomyces cerevisiae only after site mutation of its CUG codon (159-Ser) into a universal one [15]. Lip5-DM, which has double mutagenesis of 154 and 293 amino acids, showed better activity than Lip5-M₁₅₄ (Figure 2B). The specific activity of pure Lip5-DM was also higher than that of Lip5-M₁₅₄ (data not shown). It indicated that 293-Ser might be a critical amino acid which contributes to the enzymatic activity. Similar results were found for lipases from Candida rugosa, which required replacement of all the non-universal condons by universal serine codons with site-directed mutagenesis or whole gene synthesis methods to obtain functional lipase in universal host such as P. pastoris and E. coli [23–27].

The recombinant Lip5-DM was separated from the culture media by two purification steps according to the method described in the experimental section. The crude lipase protein was precipitated with the increment of mass fraction of (NH₄)₂SO₄, and the maximum protein precipitation was obtained when the mass fraction of (NH₄)₂SO₄ was more than 70%. The crude lipase, which was precipitated completely by 80% of (NH₄)₂SO₄, was dissolved in Tris-HCl buffer (20 mM, pH 8.0), and was purified further by anion-exchange chromatography. The recombinant Lip5-DM could be eluted when the concentration of NaCl increased to 200 mM, and the fractionated eluant was analyzed by SDS-PAGE (Figure 3A). Zymogram analysis shows that the Lip5-DM was purified to homogeneity (Figure 3B). The pure lipase was collected for further analysis of enzymatic characterization.

To determine the optimal temperature of the recombinant lipase, lipolytic activity of Lip5-DM to p-NP-caprylate was measured under different temperatures (5–55 °C). The relative activities at various temperatures are shown in Figure 4A, taking the activity at 15 °C as 100%. It is interesting to observe that the recombinant lipase displayed the highest activity at 15 and 25 °C, and maintained more than 70% of the maximum activity at 35 °C. Even at a lower temperature of 5 °C, the recombinant lipase still retained more than 50% of the maximal activity (Figure 4A). The optimum temperatures for Lip5 was lower than those of lipase CALip4, CPLip2 and CALA, which were 50–60 °C [15], 50 °C [17] and 50–70 °C [28], respectively. The Lip5-DM exhibited high hydrolytic activity at low temperature, and this property was similar to that of cold active lipase from psychrophilic organisms with high activity in a temperature range of 0–30 °C [6,29]. The reactions catalyzed by the enzymes derived from cold-adapted organisms are usually lower than those catalyzed by the corresponding enzymes from their mesophilic counterparts [30]. Therefore, we further determined the activation energy for hydrolysis of p-NP-caprylate catalyzed by Lip5-DM. The value of activation energy was about 8.5 kcal/mol in the temperature range from 5 to 25 °C (Figure 4B). This value was lower than that of a cold-adapted lipase LipP from an Alaskan Psychrotroph, Pseudomonas sp. Strain B11-1, with an activation energy of 11.2 kcal/mol [31] and it was higher than that of cold active lipases from Psychrobacter sp. and a deep-sea sediment metagenome, with activation energy of 5.5 kcal/mol for MBP-lipase and 3.28 kcal/mol for rEML1, respectively [9,10]. Thermostability of Lip5-DM was also investigated by measuring the residual activities during incubation at various temperatures for 2 h at pH 6.0. The Lip5-DM showed good thermostability over a range of temperature from 15 °C to 45 °C, with more than 60% of maximal activity retained after 2 h incubation (Figure 5). However, the stability of recombinant lipase decreased at 55 °C, with 23.86% residual activity after incubation for 2 h. These thermal properties were similar to that displayed by a cold active lipase from Aspergillus nidulans, which sharply lost its activity when the temperature exceeded 40 °C [32].

To investigate the influence of pH on Lip5-DM, the activity of lipase was measured at various pHs. The recombinant lipase was active in a pH range of 5.0–7.0, with the maximal activity at pH 5.0 and 6.0 (Figure 6A). The pH stability of lipase was explored by pre-incubating Lip5-DM in buffer with different pH at 4 °C for 12 h. The results are shown in Figure 6B. The Lip5-DM activity exhibited stable within the pH range of 5–9, and it remained more than 60% of maximal activity. The recombinant lipase sharply lost its activity when incubated at pH 4.0, and only preserved 26% activity.

Effect of metal ions on the recombinant Lip5-DM hydrolysis activities was determined. The metal ion concentration was selected as 1 mM and 5 mM respectively. The results are shown in Table 2. Among the metals ions tested, Zn²⁺ increased the lipase activity at either low (1 mM) or high concentration (5 mM) with respect to the blank control, possibly by involving stabilizing the active conformation of the enzyme. The structural stabilizing role of Zn²⁺ on lipase from Geobacillus stearothermophilus has been observed by Choi et al. [33]. Fe²⁺, Fe³⁺ and Hg²⁺ strongly inhibit the lipase activity, and the residual activities of lipase were 20%, 35% and 13% respectively when their concentrations were 5 mM. A similar inhibitory effect of these ions on the activity of microbial lipases has been reported by other authors [34–36]. The activity of lipase was inhibited by Hg²⁺, suggesting that the thiol-group harboring amino acids might be involved in activation of the lipase. Ethylenediaminetetraacetic acid (EDTA) inhibited the activity of lipase by about 38% (1 mM), which showed that certain metal ions might contribute to the catalysis mechanism of lipase. Moreover, Phenylmethanesulfonyl fluoride (PMSF) also decreased the recombinant lipase activity, indicating that the Ser residues played an important role in the biocatalysis function of the lipase. Surfactants such as Tween 20, Tween 80 and TritonX-100 showed inhibitory effect in the lipase activity (Table 3). Sodium dodecyl sulfate (SDS) sharply decreased the lipase activity by 85% with a low concentration of 0.1%. Methanol, ethanol, and acetone showed negative effect on the lipase activity, and the residual activities of Lip5-DM remained at 48%, 24% and 44% respectively after incubation for 1 h. Lipase activity was almost lost in the presence of isopropanol alcohol. These properties differ from those displayed by lipase from Fusarium heterosporum, which was activated by pre-incubation in organic solvent [37].

A series of p-NP esters with a variety of carbon chain lengths was employed to determine the substrate specificity of recombinant Lip5-DM. It was observed that Lip5-DM exhibited a preference for the short and medium chain length p-NP (C4 and C8 acyl group) esters rather than the long chain length p-NP esters (C12, C16 and C18 acyl group), with relative activity of 100, 59, 39, 22 and 14% for the C8, C4, C16, C12 and C18 derivatives, respectively (Figure 7). Similar substrate preference property was found for CALip4, which displays maximum hydrolysis activity to the C6 and C8 p-NP derivatives [15]. The kinetic constant were determined and summarized in Table 4. The highest K_cat value was observed with p-NP-caprylate (C8), whereas p-NP-laurate (C12) showed the lowest K_cat value. Among the p-nitrophenyl esters tested, p-NP-caprylate (C8) was observed to have the highest specificity constant (K_cat/K_m), and the K_m and K_cat values were 0.27 mM and 55.12 S⁻¹, respectively. Therefore, p-NP-caprylate (C8) was the most suitable substrate for the recombinant enzyme.

To determine the lipase activity of recombinant Lip5-DM, triacylglycerols such as olive oil and safflower oil were hydrolyzed by recombinant enzyme, and the hydrolysis activities were 3200 U/L and 3800 U/L, respectively. This showed that the Lip5-DM was a lipase and displayed lipolytic activity to lipid.

Strain C. albicans ATCC 10231 was collected from Guangdong Microbial Culture Collection Center. E. coli DH5α, P. pastoris X33 (Invitrogen), plasmid pBluescript SK vector (Stratagene) and pGAPZαA (Invitrogen) were used for gene cloning and protein expression. The substrates (p-nitrophenyl (pNP) esters derivates) were purchased from Sigma. Other reagents and solvents were of analytical grade.

C. albicans genomic DNA was prepared using a fungal genome extraction kit (Omega) according to the manufacturer’s instruction. PCR was performed to amplify the lip5-wt gene sequence (without the signal peptide) using C. albicans genome DNA as template. The primers were Lip5-FP and Lip5-RP (Table 1). The amplified fragments were inserted into pBluescript vector, resulting plasmid pBluescript-lip5-wt, and completely sequenced. The nucleotide sequence of lip5 reported in this work has been deposited in the GenBank under the accession number HM581681. Multiple alignments of amino acid sequences of lip5-wt with other lipases from other Candida species were performed by the program ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2).

Triplet codon CTG in the lip5-wt gene sequence, which is translated into serine in C. albicans, was mutated into TCT by overlap extension PCR method. The primers are shown in Table 1. Three DNA fragments (lip5-A, lip5-B and lip5-C) were produced by PCR using pBluescript-lip5-wt as template. lip5-dm, which has double mutant, was produced using the mixture of the three purified DNA fragments of (lip5-A, lip5-B and lip5-C) as template with primer Lip5-FP and Lip5-RP. To obtain lip5-m₁₅₄ gene containing only a single mutation in site 154 amino acid, PCR was performed with primer Lip5-CTG₁₅₄-FP and Lip5-RP using pBluescript-Lip5-wt as template to yield DNA fragment lip5-BC. Then fusion of lip5-A and lip5-BC was performed to generate lip5-m₁₅₄ with primers lip5-FP and lip5-RP. Similarly, lip5-AB was produced by PCR with primers lip5-FP and lip5-CTG₂₉₃-RP. And then, lip5-m₂₉₃ gene was obtained by fusion of DNA fragment lip5-AB and lip5-C. All resulting DNA fragments lip5-dm, lip5-m₁₅₄ and lip5-m₂₉₃ were inserted into pBluescript vector at the site of Kpn I and Xho I to produce pBluescript-lip5-dm, pBluescript-lip5-m₁₅₄ and pBluescript-lip5-m₂₉₃, respectively. The plasmids containing targeted genes were fully sequenced. And then, subcloning of these genes to the expression vector pGAPZαA was performed to generate vector pGAPZαA-lip5-dm, pGAPZαA-lip5-m₁₅₄ and pGAPZαA-lip5-m₂₉₃.

Vectors pGAPZαA-lip5-dm, pGAPZαA-lip5-m₁₅₄, pGAPZαA-lip5-m₂₉₃ and pGAPZαA were linearized by AvrII digestion. The purified digestion products were transformed into P. pastoris X-33 by electroporation, and the transformants were screened on YPD plates (1% yeast extract, 2% peptone, 2% dextrose and 2% agar) containing 100 μg/mL Zeocin. The transformed strains were grown in YPD liquid medium for protein expression. The single transformed colony was picked into 3 mL YPD medium containing 100 μg/mL Zeocin in a tube and grown at 30 °C and 200 rpm until the OD₆₀₀ reached 2–6. Then the seed culture was inoculated into 100 mL YPD and cultured at 30 °C for 72 h.

All the purification steps were performed at 4 °C. The supernatant was clarified by centrifugation (12,000 × g, 4 min, 4 °C) to remove cells. The crude enzyme solution was concentrated by ammonium sulfate precipitation (50–80%) and the precipitated fractions were collected by centrifugation (12,000 × g, 30 min), and then the crude lipases precipitation was dissolved with dilution buffer (20 mM Tris-HCl, pH 8.0) for further purification by anion-exchange chromatography with a Q Sepharose™ Fast Flow column (0.8 × 15 cm, GE Healthcare). The samples were applied onto the column which was pre-equilibrated with 20 mM Tris-HCl (pH 8.0), and then washed with the buffer (20 mM Tris-HCl, pH 8.0) and the buffer (20 mM Tris-HCl, 100 mM NaCl, pH 8.0) respectively. The recombinant Lip5-DM was eluted with the buffer (20 mM Tris-HCl, 200 mM NaCl, pH 8.0) at a flow rate of 3 mL/min, and fractionated eluant from the column were analyzed by SDS-PAGE.

Esterase activity was measured by spectrophotometric method using p-nitrophenyl caprylate (p-NP-caprylate) as substrate. The reaction mixture consisted of 100 mM Na₂HPO₄-NaH₂PO₄ buffer (pH 6.0), 2 mM p-NP-caprylate and an appropriate amount of the lipase. The reaction was carried out at 25 °C for 5 min. The amount of released p-nitrophenol was quantified by its absorbance at 405 nm by a spectrophotometer. One unit of activity was defined as the amount of enzyme needed to release 1 μmol of p-nitrophenol per minute under the assay conditions.

Lipase activity was quantified at pH 6.0 by free fatty acid titration with 20 mM NaOH after incubated for 10 min at 25 °C in a vessel. The assay mixture consisted of 4 mL 100 mM Na₂HPO₄-NaH₂PO₄ buffer, 5 mL emulsified olive oil or sufflower oil and 1ml enzyme solution. One unit of lipase activity was defined as the amount of enzyme releasing 1μmol of free fatty acids per minute. Protein samples were mixed with loading buffer and heated to 95 °C for 5 min and loaded onto 12% SDS-PAGE gels. Proteins were stained by Coomassie blue as instructions. Protein concentration was determined using a BCA Protein Assay Kit (Sangon, Shanghai, China) according to the manufacturer’s instruction.

Zymogram analysis was done by running the purified lipase sample on a 12% native PAGE gel. After electrophoresis, the gel was incubated for 30 min in 50 mM phosphate buffer (pH 7.2) containing 1% β-naphthyl acetate at room temperature. After addition of 0.5% Fast Blue RR solution, enzyme activity could be detected by the appearance of brown colored bands in the gel.

Effect of temperature on the lipase activity was determined by measuring the hydrolytic activity at different temperatures in 100 mM Na₂HPO₄-NaH₂PO₄ buffer (pH 6.0). The temperature was set as 5 °C, 15 °C, 25 °C, 35 °C, 45 °C and 55 °C. For investigation of the thermostability, the purified lipase was incubated at different temperature for 2 h, and activity measurements were performed every 30 min for each temperature.

The optimum pH was investigated by measuring the hydrolytic activity of lipase at various pH value. The pH was set as 4, 5, 6, 7, 8, 9. The buffer was used as following: 100 mM sodium acetate (pH 4.0, pH 5.0), 0.1 M Na₂HPO₄-NaH₂PO₄ (pH 6.0, pH 7.0), 50 mM Tris-HCl (pH 8.0) and 50 mM Gly-NaOH (pH 9.0). The reaction was performed at 25 °C. To analyze pH stability of lipase, the pure Lip5-DM was pre-incubated in buffers within the different pH value at 4 °C for 12 h. Then the residual activity was analyzed.

The influence of various chemicals, surfactants, inhibitors and water-miscible solvent on the hydrolytic activity was determined by detecting the residual activity at 25 °C after incubating the pure Lip5-DM in 100 mM Na₂HPO₄-NaH₂PO₄ (pH 6.0) buffer containing each of various metal ions (1 mM and 5 mM), each of surfactants (0.1% v/v) and each of water-miscible solvents (30% v/v) at 4 °C for 1 h. The control was performed without metal ions, detergents and water-miscible solvents. The metal ions include ZnSO₄, CuSO₄, MgSO₄, FeCl₃, CaCl₂, MnSO₄, HgCl₂, LiCl, FeSO₄, NiCl₂, BaCl₂, CoCl₂. Inhibitors include Ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF). The detergents include Tween 20, Tween 80, Triton X-100 and SDS. The water-miscible solvents include methanol, ethanol, acetone and isopropanol alcohol.

The substrate specificity of Lip5-DM was investigated using p-nitrophenyl fatty acid esters with various acyl chain lengths. The substrates are as following: p-nitrophenyl butyrate (p-NP-butyrate, C4), p-nitrophenyl caprylate (p-NP-caprylate, C8), p-nitrophenyl laurate (p-NP-laurate, C12), p-nitrophenyl palmitate (p-NP-palmitate, C16) and p-nitrophenyl stearate (p-NP-stearate, C18) were used. Determination of kinetic constants and activation energy Kinetic constants of Lip5-DM were determined by using p-NP esters as substrate. The initial rate of p-NP esters hydrolysis was measured at various substrate concentrations over the range 0.125 mM to 5 mM. The reaction was performed at 5, 15 and 25 °C according to the procedure described above. The V_max and K_m values were determined by Lineweaver-Burk plots method. K_cat was calculated subsequently with enzyme concentration. The activation energy (E_a) was determined by using the slope of the Arrhenius plot and Arrhenius equation.