Strains, vectors, media, and chemicals
E. coli DH5α (dlacZ Delta M15 Delta(lacZYA-argF) U169 recA1 endA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1) was continually preserved in our laboratory. P. pastoris KM71 (his4 arg4 aox1∆::ARG4) was purchased from Invitrogen (USA). pPICZαA and pPIC9K vectors were purchased from Invitrogen (USA). Restriction enzymes (EcoRI, KpnI, PmeI, SacI, and XbaI) and T4 DNA ligase were from NEB (USA).
LB (1% peptone, 0.5% yeast extract, 1% NaCl, 100 μg/mL ampicillin) and LSLB (1% peptone, 0.5% yeast extract, 0.5% NaCl, and 25 μl/mL zeocin) were used for E. coli growth. YPDS Agar (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol, 1.5% agar) was used for the P. pastoris screening. YPD Broth (1% yeast extract, 2% peptone, 2% dextrose, and 2% agar) was used for the P. pastoris growth. BMGY (1% yeast extract, 2% peptone, 0.34% YNB, 4 × 10−5% biotin, 1% glycerol, and 100 mM potassium phosphate buffer pH 6.0) was used for the P. pastoris pre-induction growth medium. BMMY (1% yeast extract, 2% peptone, 0.34% YNB, 4 × 10−5% biotin, and 100 mM potassium phosphate buffer pH 6.0) was used for the P. pastoris induction medium.
Isolation of the LipEMP48-D gene and signal sequence elimination
The source of gene encoding lipase from LipEMP48-D had previously been cloned into pGEM-T Easy vector and maintained in E. coli DH5α [28]. LipEMP48-D had been isolated originally from M. luteus [34]. LipEMP48-D gene was released from pGEM-T Easy vector using EcoRI and verified by using specific primers Lip013f (5′-CCC CGA CGC TAG CCG AG-3′) and Lip013r (5′-CAT CTG CAT CCG AGA GAC CG-3′), with procedure reaction as a denaturation at 95 °C for 5 min, 30 cycles (95 °C for 45 s, 56 °C for 30 s, 72 °C for 90 s), finally extension at 72 °C for 5 min. The PCR products were purified by using QIAquick Gel Extraction Kit Protocol from Qiagen (USA) and used for the next procedure.
The native signal sequence of LipEMP48-D was eliminated from the ORF of lipase gene (GenBank: MK618664.1) by using primers Lipml_KpnI-F (5′-TTA ATG GTA CCG CCC AGG AGT CGG CCC-3′) and Lipml_XbaI-R (5′-TTA ATT CTA GAG AAC CAC CCG CAC GAG TCG-3′). Primers were designed to carry the KpnI restriction site at the 5′-end and XbaI at the 3′-end. The PCR reaction was performed for pre-denaturation at 94 °C for 5 min, followed by 30 cycles of 95 °C for 45 s, 67 °C for 60 s, and 72 °C for 90 s, and extension at 72 °C for 5 min. The lipase gene was digested by KpnI and XbaI, and used as template sources (EMP-N) and control.
Codon optimization and synthesis of the LipEMP48-D gene
The LipEMP48-D gene without native signal sequence (EMP-N) was optimized according to the codon usage preference of P. pastoris (http://www.kazusa.or.jp/codon/) by using the OPTIMIZER web-based server (http://genomes.urv.es/OPTIMIZER/) [35]. The optimized DNA sequence was synthesized by Gene Synthesis (Macrogen, South Korea). The DNA-synthesized product (EMP-S) was ligated into pTOP Blunt V2 vector. The specific primers, Emp2_EcoRI-F (5′-AAG CTG AAT TCC AAG AAT CTG CTC CAG CTC CAG ATG C-3′) and Emp2_EcoRI-R (5′-TAT ATG AAT TCA AAC CAA CCA CAA GAA TCA GCA CAC C-3′) were also designed with EcoRI restriction site for cloning preparation. The recombinant plasmid, pTOP+EMP-S, was used as the template for PCR amplification. The PCR reaction procedure was set as pre-denaturation step at 94 °C for 5 min, 30 cycles of 95 °C for 45 s, 67 °C for 60 s, 72 °C for 90 s, and followed by an extension step at 72 °C for 5 min. The purified PCR product was digested with EcoRI and used as template for the next methods.
Construction of recombinant strains
The EMP-N was inserted into the multiple cloning sites of pPICZαA vector between KpnI and XbaI restriction sites. The recombinant plasmid was verified by enzyme digestion and sequence analysis. The EMP-S was ligated into pPICZαA and pPIC9K vectors to construct recombinant plasmids. The recombinant plasmids were verified by enzyme digestion and sequence analysis.
Transformation and production of protein
The ligated constructs were transformed by heat shock into E. coli DH5α cells. Then, the recombinant plasmids pPICZαA+EMP-N and pPICZαA+EMP-S were linearized by PmeI, and pPIC9K+EMP-S was linearized with SacI. All of the recombinant plasmids were transformed into P. pastoris KM71 competent cells by electroporation (2 kV 400 Ω). The transformants were spread on selective YPDS plates containing zeocin (100 μl/mL) for pPICZαA recombinants and geneticin (0.25 mg/mL) for pPIC9K recombinants. Recombinant strains were picked up and kept on new YPDS plates that have been supplemented with antibiotics.
The seed culture and enzyme production were carried out based on Pichia Expression Kit protocol from Invitrogen (USA). The positive recombinant strains were grown on the YPD plates for 2–3 days. Then, 5 mL of the YPD medium was inoculated with a single colony and incubated at 30 °C and 225 rpm in 50 mL shake flasks for 18 h or until the OD600 reached 2–6. Seed culture (10%) was inoculated onto 50 mL BMGY medium (pH 6.0) at 30 °C and 225 rpm for 18 h. The cells were centrifugated at 4000×g at room temperature for 10 min. The cell pellet was resuspended in BMMY medium (pH 6.0). The culture was maintained at 30 °C, 225 rpm for 48 h, and every 24 h, methanol (1% v/v) was added to the culture to induce protein expression. The culture was harvested by centrifugation at 5000×g at 4 °C for 15 min. The supernatant formed was analyzed as crude extracellular enzyme extract. The recombinant protein was detected by 12% (v/v) SDS-PAGE.
Assay of enzyme activity
The lipase activity was measured by alkali titration using olive oil as the substrate. Olive oil was emulsified with 4% w/v PVA at a ratio of 1:3 (v/v) for 2 × 3 min. The reaction mixture was composed of 5 ml of substrate, 0.5 ml of 150 mM citrate-phosphate (pH 5.0), and 0.75 ml of ddH2O, pre-incubated at 40 °C and 175 rpm for 5 min. Then, 0.25 ml of enzyme was added to the mixture and incubated at 175 rpm for 15 min under the optimum temperature of LipEMP48-D (40 °C) [28]. These conditions (40 °C, pH 5.0, 15 min) will be referred to as standard conditions. The reaction was stopped by the addition of 5 ml 95% ethanol solution. The reaction solution was titrated with 0.025 M NaOH standard solution. One unit of enzyme activity is defined as the amount of enzyme that released 1 μmol of fatty acids per minute. Specific enzyme activity was expressed as units per mg of protein.
Characterization of the LipEMP48-D
The optimum pH of LipEMP48-D was investigated with pH ranging from pH 3.0–4.0 (citrate buffer 0.1 M), pH 5.0 (citrate-phosphate buffer 0.15 M), pH 6.0–7.0 (phosphate buffer 0.1 M), pH 8.0–9.0 (Tris-HCl buffer 0.1 M), and pH 10.0 (glycine-NaOH buffer 0.08 M) at 40 °C. The pH stability was evaluated by incubating the LipEMP48-D lipase in 3 various buffers (pH 3.0, pH 5.0, and pH 8.0) for 120 min at 40 °C. The samples were collected at different time intervals (30 min, 60 min, 90 min, and 120 min). The effect of organic solvents on LipEMP48-D stability was checked after pre-incubation of enzyme with organic solvents at a ratio 1:1 (v/v) for 15 min at 40 °C. Types of organic solvents used include methanol, ethanol, butanol, isopropanol, acetonitrile, n-hexane, and n-heptane. The effect of metal ions on LipEMP48-D lipase activity was determined by incubating the enzyme in 0.01 M solutions of NaCl, KCl, CaCl2, MgCl2, MnCl2, FeCl2, and FeSO4 for 15 min. The hydrolysis activity on various substrates was evaluated towards olive oil, corn oil, sunflower oil, canola oil, rice bran oil, palm oil, and soya oil. All triglycerides were adjusted to the emulsions. The activity was measured under the assay conditions as described above.
Each experiment was independently repeated three times. Student’s t test was used for the analysis of statistical significance (P value) in this study, and Minitab v.19.1.1 (USA) was used for these analyses. A P value of less than 0.05 was considered significant. Control was defined as enzyme activity without the treatment of each variable. Blanks were made according to the measurement conditions of each variable without the addition of the enzymes. The measurement results are displayed in the form of relative activity values for data on the effect of variables on enzyme activity and residual activity value for enzyme stability data.
Transesterification activity of the LipEMP48-D
The transesterification activity of crude enzyme extract was measured based on the principle of the enzymatic synthesis of biodiesel from free fatty acids (FFAs). The types of FFAs used are palm fatty acid distillate (PFAD) and fatty matter (FM) which are regularly used in our laboratory. The fatty acid composition of PFAD was 45.9% of palmitic acid, 36.5% of oleic acid, and 10.3% of linoleic acid. The fatty acid composition of FM was 44.7% of palmitic acid, 37.9% of oleic acid, and 10.2% of linoleic acid.
Before use, the FFA was heated until it melts. Transesterification reaction for LipEMP48-D lipase was carried out in a 50-mL shake flask using a rotary orbital shaker. The reaction mixture is composed of 8 ml of FFA and 110 U/mg enzyme. Some stainless-steel beads were added to aid the stirring process during the reaction. The reaction mixture was incubated at 50 °C [36]; 1 mL of methanol was added every 1 h until the third hour and left for 5 h. FFA which has been successfully converted into fatty acid methyl ester (FAME) (biodiesel) was indicated by the formation of two layers in the shake flask.