Strains, plasmids, and reagents
Escherichia coli DH5α (Biomed, Beijing, China) was used for propagation of plasmids, and P. pastoris GS115 (his4) was used for protein expression. Trizol reagent (Invitrogen, Carlsbad, USA) and Oligotex mRNA Midi Kit (Qiagen, Dusseldorf, Germany) were used for total RNA extraction and mRNA purification. BD SMART™ RACE cDNA Amplification Kit was purchased from Clontech (Palo Alto, CA, USA). Ex Taq DNA polymerase, PrimeSTAR HS DNA polymerase, restriction endonucleases, and pMD18-T were purchased from TaKaRa (Tokyo, Japan). T4 DNA ligase was purchased from New England Biolabs (Ipswich, MA, USA). The Pichia pastoris expression kit was obtained from Invitrogen (Carlsbad, USA). p-Nitrophenol (pNP), pNP acetate (pNPA), pNP butyrate (pNPB), pNP hexanoate (pNPH), pNP caprylate (pNPC), pNP decanoate (pNPD), pNP laurate (pNPL), pNP myristate (pNPM), and pNP palmitate (pNPP) were purchased from Sigma Chemical Co. (St. Lous, MO, USA). pNP hexanoate (pNPH) was obtained from HEOWNS Company (Tianjin, China). Olive oil, soybean oil, and peanut oil were purchased from a local market. All other chemicals used were of analytical grade unless otherwise stated.
Microorganism and RNA extraction
Rhizomucor endophyticus deposited in the China General Microbiological Culture Collection Center (CGMCC) under accession number 3.4684 was used in this study. For isolation of genomic DNA, R. endophyticus was inoculated in the medium and cultured at 25 °C for 4 days with a rotation speed of 200 rpm. The medium contained (g L−1): soybean 20, yeast extract 10, tryptone 10, MgSO4·7H2O 0.3, KH2PO4 5, and CaCl2 0.3. Fungal mycelia were collected by centrifugation (5000×g, 10 min), washed twice with sterilized water at 4 °C and then ground to fine power in liquid nitrogen. The total RNA was extracted with the Trizol reagent, and mRNAs were purified using the Oligotex mRNA Midi Kit.
Cloning of a lipase gene from R. endophyticus and its sequence analysis
The degenerate primers: LipDF (5′-CGGCCACTCCCTGggnggngcnca-3′; n = A/T/C/G) and LipDR (5′-TGAGGAGGGACGTGGggnacdatrtc-3′; n = A/T/C/G, d = A/G/T, r = A/G) were designed on the basis of two conserved sequences (TGHSLGGAQ and RDIVPHVPPQ) of known fungal lipases using the CODEHOP algorithm . PCR was performed using R. endophyticus genomic DNA as the template. PCR conditions were as follows: a hot start at 94 °C for 5 min, ten cycles of 94 °C for 30 s, 60–55 °C for 30 s, and 72 °C for 1 min, followed by 20 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The PCR products were gel-purified, ligated to pMD18-T vector, and sequenced.
The full length cDNA sequence of the lipase was obtained by 5′ and 3′ RACE (rapid amplification of cDNA ends) using a BD SMART™ RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). 5′ end of the cDNA was amplified using the primer Lip5′GSP (5′-AGGAGGAACATGGGGAACAATATC-3′) and adapter primer UPM, followed by a nested PCR using nested gene-specific primer Lip5′NGSP (5′-GAACAATATCCCTATCATTAACAGAAC-3′) and adapter primer NUP. For the 3′ end of the cDNA, the primary PCR was performed with two primers: Lip3′GSP (5′-CACTCCCTTGGTGGTGCACAAGC-3′) and UPM, followed by a nested PCR using Lip3′NGSP (5′-CAAGCTTTGCTCGCTGGTATG-3′) and NUP. The PCR conditions for RACE were: 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 1 min at 72 °C, and finally 10 min at 72 °C. The PCR products were gel-purified, ligated to pMD18-T vector, transferred into E. coli DH5α for sequencing, and subjected to BLAST analysis.
Sequence assembly and analysis were performed with DNAMAN software (LynnonBiosoft, USA). Database homology searches of nucleotide sequences were carried out using BLAST in GenBank at the NCBI (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple alignment analysis was performed by Clustal W program (ftp-igbmc.u-strasbg.fr/pub/ClustalW/). Signal peptide was analyzed at SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/). N-Glycosylation sites were predicted using NetNGlyc1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/).
Expression of the lipase gene in P. pastoris
The lipase gene (ReLipA) without signal sequence was amplified from the genome of R. endophyticus using the primers: ReLipAF (5′-ATTCCGGAATTCCCTGCTGCTGGCACCAA-3′) and ReLipAR (5′-AAATATGCGGCCGCTTAAAGACAAAGTCCTTCATT-3′). Two restriction sites, EcoRI and NotI were inserted at both ends of the gene. PCR conditions were as follows: a hot start at 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 80 s, and a final extension cycle at 72 °C for 10 min. The amplified PCR product was cloned in-frame at the downstream site of the α-factor (signal peptide) in pPIC9K vector, yielding the recombinant plasmid pPIC9K-ReLipA. The recombinant plasmid was linearized by SalI and transformed into P. pastoris GS115.
The colonies with multiple copies of the integrated plasmid were screened on MD (minimal dextrose) plates at different geneticin 418 (Life Technologies, Gaithersburg, MD, US) concentrations (0.5–4.0 mg mL−1). Positive colonies were selected and inoculated in 5 mL of BMGY medium (buffered minimal glycerol complex medium) at 30 °C with a rotation speed of 200 rpm till the optical density (OD600) reached to 2.0–6.0. The P. pastoris cells were then harvested by centrifugation and re-suspended in 10 mL of BMMY medium (buffered minimal methanol complex medium) to obtain a final OD600 of 1.0. After the medium was continuously cultured in shake flasks for 5 days, methanol was supplemented every 24 h to ensure a final concentration of 0.5 % (v/v) to induce the expression. The culture was centrifuged at 12000×g for 20 min, and the supernatant was collected and checked for lipase activity.
The selected strain with the highest lipase activity in shake-flask culture was subjected to high cell-density fermentation in a 5-L fermentor at 30 °C according the method described in the Pichia Fermentation Guidelines (Version B, 053,002, Invitrogen Inc.). Samples withdrawn at different time intervals during the methanol induction phase were assayed for OD600, wet weight of the cells, lipase activity, and protein concentration.
Enzyme assay and protein determination
Lipase activity was determined by a spectrophotometric assay using p-nitrophenyl laurate (pNPL) as the substrate. Namely, 50 μL of substrate solution (dissolved in isopropanol with a final concentration of 10 mM) was mixed with 400 μL of 50 mM citrate buffer (pH 6.0), and pre-incubation at 40 °C for 2 min, and then the reaction was initiated by the addition of 50 μL of appropriately diluted enzyme. After incubation at 40 °C for 10 min, the reaction was terminated by adding 500 μL of a moving alkaline copper phosphate suspension. The mixture was centrifuged at 10000×g for 3 min, and the absorbance of the supernatant at 410 nm was measured immediately. One unit of enzyme activity was defined as the amount of enzyme liberating 1 μmol of pNP per minute under the above conditions. Protein concentration was measured by the method of Lowry et al.  using bovine serum albumin (BSA) as the standard.
Purification of the recombinant lipase
The cell-free crude supernatant was harvested by centrifugation (10000×g) at 4 °C for 20 min, and dialyzed against buffer A (20 mM citrate buffer pH 5.0) for 16 h. The dialysate was then loaded onto a SP Sepharose column pre-equilibrated with buffer A. After washing with buffer A till the OD280 reached to baseline, the bound proteins were eluted with 0–200 mM NaCl gradient at a flow rate of 1 mL min−1. The fractions showing high lipase activity were collected and checked for purity by SDS-PAGE.
SDS-PAGE and molecular mass determination
The homogeneity and subunit molecular mass of ReLipA were determined by SDS-PAGE as described by Laemmli  using 12.5 % separation gel. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250. The low molecular mass calibration kit (Amersham) contained phosphorylase b (97.0 kDa), albumin (66.0 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (30.0 kDa), trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). The native molecular mass of ReLipA was determined by size-exclusion chromatography on a Superdex-75 gel filtration column (1 × 40 cm) which was pre-equilibrated with 20 mM phosphate buffer (pH 7.0) containing 150 mM NaCl at a flow rate of 0.3 mL min−1. Standard proteins used were phophorylase b (97.2 kDa), albumin (66.2 kDa), albumin (45.0 kDa), α-chymotrypsinogen A (25.7 kDa), and α-lactalbumin (14.4 kDa).
Characterization of the recombinant lipase
The optimal pH of the lipase was determined by measuring enzyme’s activity at 40 °C using the following buffers (50 mM): citrate (pH 3.0–6.0), 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5–6.5), phosphate (pH 6.0–8.0), Tris–HCl (pH 7.5–9.0). To determine pH stability, the enzyme was incubated in different buffers mentioned above at 30 °C for 30 min, and the residual lipase activity was then assayed in 50 mM citrate buffer (pH 6.0). The optimal temperature was determined by measuring the lipase’s activity in 50 mM citrate buffer (pH 6.0) at different temperatures (0–60 °C). Thermal inactivation studies were performed by measuring the residual activity after pre-incubation of the enzyme in 50 mM citrate buffer (pH 6.0) at a temperature range of 30–60 °C for 30 min.
The effect of some surfactants on the enzyme stability was investigated after incubation of the lipase in 50 mM citrate buffer (pH 6.0) at 30 °C for 1 h in the presence of 5 % (w/v) of the following detergents: Tween 20, Tween 60, Tween 80, Triton X-100 and SDS. The stability of the lipase against 30 % (v/v) of various organic reagents was evaluated by the similar method. The organic solvents included methanol, ethanol, butanol, acetone, acetonitrile, isopropanol, cyclohexane, and heptane.
Substrate specificity, kinetic parameters, and positional specificity of ReLipA
Substrate specificity of the purified lipase was determined by measuring the enzyme’s activity according to the standard assay using different pNP esters (pNPA, pNPB, pNPH, pNPC, pNPD, pNPL, pNPM, and pNPP), and triglycerides (triacetin, tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, trimyristin, and tripalmitin) as the substrates. The kinetic parameters of ReLipA toward pNPC (C8), pNPL (C12), and pNPM (C14) were determined by measuring the enzyme’s activity in 50 mM citrate buffer (pH 6.0) at 40 °C for 5 min using different substrate concentrations. The K
m and V
max values were calculated by “GraFit” software.
The positional specificity of ReLipA was examined by analyzing the hydrolysis products of triolein using thin layer chromatography (TLC). Briefly, 1 mL of reaction mixture containing 50 mM citrate buffer (pH 6.0), 100 mM triolein, and 50 U of lipase was incubated at 40 °C for 8 h with a shaking speed of 180 rpm. The reaction products were extracted with the same volume of n-hexane, and then analyzed by TLC as the method described by Lu et al. .
Synthesis of biodiesel by ReLipA
The reaction mixture including 5 mL of isooctane solvent containing 1.68 g oleic acid, 1098 μL of alcohols (methanol, ethanol, or butanol), and 100 U of the lipase in a 25-mL triangular flask was incubated at 35 °C for 24 h with continuously shaking (150 rpm). The samples were withdrawn at different time intervals, and analyzed by TLC according to the method of Guan et al. . The products were quantified by gas chromatography (Agilent 6890 N) which was equipped with a DB-wax capillary column (30 m × 0. 25 mm × 0. 25 μm) and a flame ionization detector (FID). Nitrogen was used as a carrier gas. Both the injector and detector temperatures were held at 250 °C. The initial column temperature at 180 °C for 3 min was increased to 200 °C by 10 °C min−1 and held steady for 3 min, and then increased to a final temperature of 230 °C at a rate of 3 °C min−1.