There were two non-universal CUG-Ser codons (residue number 210 and 378) in the C. albicans lip10 gene (Figure 1a). Both the CUG codons were converted into universal serine codons TCT by using overlap extension PCR (Figure 1b) with the primers in Table 1. The mutated lip10 DNA sequence, termed as lip10-dm, was confirmed by full-length DNA sequencing.

P. pastoris transformants containing lip10 and lip10-dm were screened using 1% tributyrin-emulsion YPD plates. The clear zone on the opaque tributyrin emulsion identified the lipase-secreting transformants. It can be seen from Figure 2 that a clear zone was formed around wild type lip10 and mutated lip10-dm, and the diameter of the transparent ring formed by the mutant was larger than that formed by the wild type. Transformants, which contained wild lip10 and mutant lip10-dm, were cultured in YPD medium to investigate the enzymatic activity. After 72 h, the lipase activities were 2.05 U/mL, and 4.92 U/mL, respectively. These results indicated that both residue Ser-210 and Ser-378 played an important role in lipolytic activity. The reason may be that Ser-210 and Ser-378 are key amino acids which are involved in the active domain, or they have impact effect on the correct folding of protein. Roustan et al. reported that the mutagenesis of CUG codons in one of the lipases from C. albicans (CaLIP4) resulted in a ten-fold lipase activity increase [11].

The CCD was predicted to obtain the possible culture conditions for CaLIP10 production from recombinant P. pastoris. Experiments 1–30 were performed with different combinations and those from 10 to 15 were under the same conditions (Table 2). The actual lipase activity obtained in the experiments and predicted lipase activity produced by the model are also given in Table 2. By analysis of the experimental data, the quadratic equation model was as follows:

where y is the predicted response; x₁, x₂, x₃ and x₄ are the uncoded values: Temperature, initial pH of culture, concentration of yeast extract content and concentration of glucose. The analysis of variables for the model and factors are shown in Table 3 which lists the regression coefficients calculated by the model for β_i, β_ii, β_ij along with significance levels of the terms. From p-values of terms, it can be seen that the linear term of temperature, yeast extract concentration and glucose concentration as well as the square term for temperature, pH and glucose concentration, were highly significant on CaLIP10 activity; the linear term of initial pH (0.0126) was the least significant. The interactions of initial pH and glucose concentration (0.0014), temperature and glucose concentration (0.0009) have a minor effect on the CaLIP10 activity while the interaction of initial pH and yeast extract (0.8225) has the most significance on the activity of CaLIP10.

The coefficients of correlation and variation and F-value are given in Table 4. The mathematical model was very reliable with an R² value of 0.9814. The closer the R² is to 1, the better the model fits the experimental data, the less the difference between the predicted and the observed values. The compared F-value of 56.64214 was greater than the F (14, 15) value in the statistic table at 1% level. It reflected the significance of the model. In summary, those values indicated a satisfactory fitness of the quadratic model and were adequate to represent the actual relationship between the responses (lipolytic activity) and the significant variables.

The three-dimensional response surface and contour curves for the four variables are shown in (Figure 3). The response surface representing CaLIP10 activity was a function of two tested variables with the other two variables being maintained at their optimal level. It was clear and obvious to understand the interaction between two factors and to predict their optimal levels.

From the response surface, it indicated that the optimal initial pH of the culture was 6.86 for CaLIP10 activity (Figure 3a,d,e) since a lower or higher initial pH decreased not only the activity of CaLIP10 but had a negative effect on the cell growth. The corresponding cell density (OD₆₀₀) was 13.5, 16.8 and 26 when the initial pH of the culture medium was selected to be 3, 11 and 7 respectively with other process parameters being the same. The reason why recombinant CaLIP10 production was related to the cell growth was that the constitutive promoter was used in the vector system. Culture temperature made an impact on the CaLIP10 production (Figure 3a–c) with temperatures over 30 °C being detrimental to the cell growth (data not shown) and protein production while a lower temperature (25.53 °C) favored maximal lipase production, probably because the higher specific growth rates caused much more metabolic burden to be placed on the cell. Yeast extract concentration of 1.32% and a glucose concentration around 3.48% gave the maximal predicted lipase production (Figure 3b–f). Yeast extract was an important nitrogen source for yeast growth and any concentration of yeast extract of less than 1.32% could not fully support the cell growth [28]. However, it was reported that yeast extract rich in amino acids and peptides displayed a repressive effect on enzyme production when used at a higher concentration (>1.5%, m/v) [29]. A high glucose concentration (>10%, m/v) might be unfavorable for enzyme production due to the osmotic pressure or the carbon catabolite repression [30,31]. No significant repression effect of glucose on CaLIP10 production was found at a concentration of less than 5.5% in our study.

The above observations indicated that the maximum production of CaLIP10 (7.89 U/mL) was obtained when the initial pH of the culture, the temperature, the yeast extract concentration and the glucose concentration were at about 6.86, 25.53 °C, 1.32% and 3.48% respectively. In order to verify the predicted results, the experiment was performed under the optimized culture condition and the experimental value was 8.06 U/mL, indicating that experimental and predicted values of CaLIP10 yield were in a good agreement.

The recombinant CaLIP10 was efficiently purified by cation-exchange chromatography, and its molecular mass was determined to be about 50 kDa by SDS-PAGE analysis (Figure 4). After purification, the specific activity of CaLIP10 increased 5.68 fold with a recovery of 86.01% (Table 5). This pure CaLIP10 was used for further analysis of biochemical properties.

To investigate the hydrolysis ability toward oil, the coconut oil was used as a substrate. As shown in Figure 6, 38.64% and 43.56% of Triglyceride (TAG) were converted after 6 h and 24 h of reaction. It was found that the hydrolysis reaction almost did not progress from 6 to 24 h. The pH of the reaction solution might decrease due to accumulation of the released free fat acid, which led to decrease the activity of CaLIP10 since it was low activity and stability at low pH value. A buffering system will be used to investigate the hydrolysis reaction of the coconut oil in a subsequent study. Diacylglycerol (DAG) products were accumulated with the reaction proceeded, and 1,3-DAG was the predominant component in the DAG mixture. We are not sure whether CaLIP10 really is specific for position 2 based on the existent results. Lipase with sn-2 preference has great potential applications in industries. Until now, lipase A from Candida Antarctica (CALA) has been reported to be the only known lipase with a sn2-preference towards triglycerides [9]. Therefore, we will make effort to ensure this point in further studies. The composition of released fatty acids were analyzed (Table 7), and the CaLIP10 showed no obvious preference to the fatty acids with different carbon length.

All chemicals used in the study were analytical pure grade. The substrates p-nitrophenol caprylate (p-NPc) were purchased from Sigma-Aldrich (Germany). Other reagents were purchased from Takara (Japan) or Omega (USA).

Strain C. albicans ATCC 10231 was purchased from Guangdong Microbial Culture Collection Center (China). P. pastoris X33 and pGAPZαA (Invitrogen) were used as host and vector, respectively, for heterologous expression of the lipase, while E. coli DH5α and plasmid pBluescript SK vector (BSK, Stratagene) were used for vector construction. P. pastoris X33 was grown in shake-flasks at 30 °C in YPD medium (per liter: 10 g yeast extract, 20 g peptone, 20 g glucose). YPD containing 100 μg/mL Zeocin (Invitrogen) was used for screening of transformants. E. coli strains containing recombinant plasmids were cultured in Luria Bertani (LB) medium at 37 °C and supplemented with Zeocin when needed.

C. albicans genomic DNA was prepared using a fungal genome extraction kit (Omega) according to the manufacturer’s instruction. The mature protein-coding sequence without the signal peptide of C. albicans lipase 10 (namely lip10) was amplified by polymerase chain reaction (PCR) from genomic DNA. Specific primers 10F/10R (Table 1) were used. The PCR product was first purified and digested by restriction enzymes, then ligated with predigested pBluescript SK vector, and was termed pBluescript-lip10. The plasmid construct was used as the template for the mutagenesis to convert 2 non-universal CTG-serine codons (residue number 210 and 378) in CaLIP10 into universal TCT-serine codons. The simultaneous multiple mutagenesis was carried out by the overlap extension PCR [37]. The product of the mutagenesis was ligated into the pBluescript SK vector, and was termed pBluescript-lip10-dm. The sequence of mutated lip10 containing the mentioned replacements was confirmed by the full-length DNA sequencing. The scheme for mutation is shown in Figure 1 and the mutagenic primers are shown in Table 1. Subcloning of lip10 and lip10-dm into the expression vector pGAPZαA were performed to generate pGAPZαA-lip10 and pGAPZαA-lip10-dm.

The plasmid pGAPZαA-lip10 and pGAPZαA-lip10-dm were linearized with AvrII (NEB, USA) and transformed into P. pastoris X33 by electroporation using Gene Pulser (Bio-Rad) apparatus. Transformants were screened on YPD plates (containing 2% agar and 100 μg/mL Zeocin) to isolate Zeocin-resistant clones. Individual colonies were pitched and patched on 1% tributyrin-emulsion YPD plates to identify lipase-secreting transformants which would form a clear zone on the opaque tributyrin emulsion. P. pastoris transformed with pGAPZαA was used as a negative control. Strains with high yield of CaLIP10 were maintained at −80 °C in 50% glycerol (v/v).

All procedures were performed at 4 °C. The culture broth was centrifuged (12,000 × g, 4 min) to remove the cells. The crude enzyme solution was adjusted to pH 5.5 using HCl, and applied onto a CM Sepharose^TM Fast Flow column (CM FF, 1.6 cm × 28 cm, GE Healthcare) equilibrated with 20 mM sodium acetate buffer (pH 5.5). The column was washed with 20 mM sodium acetate buffer (pH 5.5) and then bound protein was eluted with a gradient of the same buffer containing 0–1.0 M NaCl at a flow rate of 3 mL/min. The active fractions (50 mL) were pooled, concentrated and 10 mL loaded onto the Sephadex G25 column (2.5 cm × 20 cm) equilibrated with 100 mM sodium phosphate buffer (pH 7.0). The column was then washed with the same buffer and the lipase-active fractions pooled and stored at 4 °C.

The molecular mass of the purified recombinant lipase was determined in denaturing conditions by SDS-PAGE with 5% polyacrylamide-stacking gel and a 12% polyacrylamide-resolving gel. The total protein in the samples was quantified with a BCA Protein Assay Kit (Sangon, Shanghai, China) using bovine serum albumin as standard according to the manufacturer’s instruction.

The lipase activity was determined spectrophotometrically using p-nitrophenol caprylate as substrate. The reaction mixture consisted of 50 mM Tris-HCl buffer (pH 8.0), 2 mM p-NPc and an appropriate amount of the lipase and the amount of released p-nitrophenol quantified after 5 min reaction time by its absorbance at 405 nm. 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.