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Gene Cloning, High-Level Expression, and Characterization of an Alkaline and Thermostable Lipase from Trichosporon coremiiforme V3
Gene Cloning, High-Level Expression, and Characterization of an Alkaline and Thermostable Lipase from Trichosporon coremiiforme V3
Journal of Microbiology and Biotechnology. 2015. Jun, 25(6): 845-855
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : August 18, 2014
  • Accepted : January 15, 2015
  • Published : June 28, 2015
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About the Authors
Jian-Rong Wang
Guangdong Feed Additive Research and Biotechnology Center, Zhuhai 519060, Guangdong, P.R. China
Yang-Yuan Li
Guangdong Feed Additive Research and Biotechnology Center, Zhuhai 519060, Guangdong, P.R. China
Danni Liu
Guangdong Feed Additive Research and Biotechnology Center, Zhuhai 519060, Guangdong, P.R. China
danni_vtr@163.com

Abstract
The present study describes the gene cloning and high-level expression of an alkaline and thermostable lipase gene from Trichosporon coremiiforme V3. Nucleotide analysis revealed that this lipase gene has an open reading frame of 1,692 bp without any introns, encoding a protein of 563 amino acid residues. The lipase gene without its signal sequence was cloned into plasmid pPICZ α A and overexpressed in Pichia pastoris X33. The maximum lipase activity of recombinant lipase was 5,000 U/ml, which was obtained in fed-batch cultivation after 168 h induction with methanol in a 50 L bioreactor. The purified lipase showed high temperature tolerance, and being stable at 60℃ and kept 45% enzyme activity after 1 h incubation at 70℃. The stability, effects of metal ions and other reagents were also determined. The chain length specificity of the recombinant lipase showed high activity toward triolein (C18:1) and tripalmitin (C16:0).
Keywords
Introduction
Lipases (E.C. 3.1.1.3) are a class of hydrolases that catalyze a variety of reactions, such as the hydrolysis of fatty acid ester, trans-esterification, and ester synthesis at the interface between the insoluble substrate and water [20] . Therefore, they are of great importance in various facets of biotechnology such as in organic synthesis, biodiesel production, food, and pharmaceutical and detergent industries [13 , 17 , 21] .
Lipases are ubiquitous in nature, including plants, animals, and microorganisms. However, microbial lipases have gained special industrial attention owing to their stability, selectivity, and broad substrate specificity [14 , 30] . Lipases produced by various yeasts have been known for many years and the majority of yeast lipases are extracelluar. Candida rugosa is the most frequently used organism for lipase synthesis, and the lipase from Candida rugosa is becoming one of the most industrially used enzymes [6] . Lipase B from Candida antarctica (CalB) is a highly versatile biocatalyst mainly used for organic synthesis at the laboratory and commercial scales [1] . The deuteromycotinous yeast Trichosporon also produces a large amount of extracellular lipase. In a previous study, a Trichosporon fermentans named WU-C12 was isolated from soil and showed maximum lipase activity of 128 U/ml after 4 days of growth at 30℃ [3] . A lipase from Trichosporon asteroides could increase the concentration of eicosapentaenoic acid and docosahexaenoic acid in tuna oil, which could potentially be used for the concentration step in the production process of such polyunsaturated fatty acids [9] . The production of extracellular lipase from Trichosporon asahii MSR54 was improved by medium optimization, and this lipase showed high activity to hydrolysis of phenylethyl acetate, which could be used in the pharmaceutical sector for chiral selective reactions [23] . Meanwhile, a thiolactivated lipase from T. asahii MSR54 could be a good candidate for developing a presoak formulation that can be used along with many available commercial detergents at ambient washing temperatures [24] . A thermoactive and thermostable lipase was isolated from Trichosporon coremiiforme and showed to be a good candidature for industrial and biotechnological applications [25] .
Although there are many lipases with potential commercial application isolated from Trichosporon , the production of lipase from Trichosporon is too low for economic use. For commercial exploitation of Trichosporon lipase, it is essential to achieve high yield and activity. The characteristics of high yield and high activity mean that the lipase product has greater market competitiveness. Heterologous expression of the lipase gene is an effective method to improve lipase yield. The Pichia pastoris ( P. pastoris ) expression system is most suitable for the production of extracellular lipase because of its powerful secretion ability and the mature fermentation technology [7] . The expression level of lipase genes in this host can be hundreds of times higher than that in the native host [27] .
Until now, there are few reports about the gene cloning and high-level expression of lipase from Trichosporon . In this study, we report the gene cloning and high-level expression of a lipase gene from Trichosporon coremiiforme in P. pastoris , with the aim of allowing the production of a high amount of the interesting enzyme. Furthermore, we report the biochemical characterization of the recombinant lipase for evaluation of its potential applications.
Materials and Methods
- Strains, Plasmids, and Materials
T. coremiiforme V3 was purchased from the China Center of Industrial Culture Collection (Beijing, China). The E. coli strain Top 10 is routinely conserved in our laboratory. P. pastoris X-33, the expression vector pPICZ α A, and zeocin were purchased from Invitrogen (Carlsbad, CA, USA). Restriction enzymes, T 4 -DNA ligase, and Pfu DNA polymerase were purchased from Sangon Biotech (Shanghai, China). The oligonucleotides were synthesized by the Shanghai Generay Company (Shanghai, China). The different lipase substrates (tributyrin, tricaprylin, trilaurin, tripalmitin, tristearin, triolein, and fatty acid methyl esters, including methyl butyrate, methyl caprylin, methyl laurate, methyl palmitate, and methyl stearate) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
- Gene Cloning and Bioinformatics Analysis
Genomic DNA and total RNA from T. coremiiforme V3 were extracted by using the Rapid Yeast Genomic DNA Isolation Kit (Sangon, China) and E.Z.N.A. Fungal RNA Kit (Omega, USA), respectively. Based on the complete nucleotide sequences of lipase genes from Trichosporon fermentans WU-C12 (GenBank No. AB000260), Geotrichum candidum (GenBank No. AB000260), and Galactomyces geotrichum (GenBank No. ACX69980), two degenerated primers TcF (5’-ATGGTWTCCAAAAVCTTKTTYTTRGC-3’) and TcR (5’-TTAACCGTAGAGATTAACGTCAGWCTC-3’) were designed for PCR and RT-PCR amplification. The cloned fragments were then ligated into the pMD18-T vector and sequenced.
The cloned sequences were first analyzed via online BLASTn provided by the National Center for Biotechnology Information, USA. The sequences were analyzed for identity using DNAman ver. 6. The signal peptide was analyzed by SignalP 4.0 server ( http://www.cbs.dtu.dk/ services/ SignalP/ ).
- Vector Construction and Transformation and Selection ofP. pastorisClones
The tcl ( T . coremiiforme V3 lipase gene without the signal sequence) was amplified by using genomic DNA of T . coremiiforme V3 as the template. The amplification primers containing the restriction sites for Eco RI and Xba I were designed as follows: TF, 5’-CAGCGAATTCCAGGCCCCCACGGCCGTTCTC-3’ and TR, 5’-GACGTCTAGATTAACCGTAGAGATTAACGTC-3’. The PCR products were double digested with Eco RI and Xba I, and then ligated into pPICZ α A, forming pPICZ α A- tcl . Finally, the recombinant expressing vector pPICZ α A- tcl was used to transform E. coli TOP 10. Through DNA sequencing, pPICZ α A- tcl was confirmed to contain the tcl gene.
P. pastoris X-33 was transformed with 10 µg of Sac I-linearized pPICZ α A- tcl vector by electrotransformation, according to Invitrogen’s recommendations. Transformants were plated on YPDS plates (10g/l yeast extract, 20g/l peptone, 20g/l dextrose, 20g/l agar, and 1 M sorbitol) containing 100 µg/ml Zeocin to isolate resistant clones. Transformed colonies were confirmed by both PCR and sequencing.
- Shaking Flask Cultures
Fifty clones from the YPDS plates were selected for shaking flask cultures. The seeds were incubated in 10ml of BMGY (yeast extract 1% (w/v), peptone 2% (w/v), 100 mM potassium phosphate buffer at pH 6.0, yeast nitrogen base with no amino acids 1.34% (w/v), glycerol 1% (w/v), and biotin 0.04% (w/v)) in a 100 ml shake flask and incubated at 30℃ and 200 rpm until the culture reached OD 600 = 2.0–6.0. The cells were harvested by centrifugation and resuspended in 50ml of BMMY (containing 0.5% methanol instead of glycerol as the sole carbon source) and incubated at 30℃ and 200 rpm. The methanol induction temperature was set at 30℃, and 0.7% (v/v) methanol was fed at 24-h intervals for 7 days. The activities of the lipase were checked at 24, 48, 72, 96, 120, 144, and 168 h. The colony with the highest activity was selected as the strain to ferment in the 5 and 50 L bioreactors.
- High Cell Density Fermentation
The transformed strain showing the highest lipase activity in shake-flask culture was cultivated in a high cell density fermentor. High cell density fermentation was carried out in 5 and 50L bioreactors (Baoxing Co., Shanghai, China). The cultivation conditions and medium composition were the same as a previously described method [31] . The detailed protocol of high cell density fermentation is provided in Supplemental materials. The enzyme activity of the supernatant and the cell density were monitored throughout the cultivation. Cell density was expressed as grams of dry cell weigh (DCW) per liter of broth, and was obtained by centrifuging 10ml samples in a pre-weighted centrifuge tube at 8,000 × g for 10min and washing twice with deionized water, and then allowing the pellet to dry at 100℃ to constant weight.
- Assay of Lipase Activity and Total Protein Concentration
The lipase activity in the culture filtrate was determined by a titrimetry method [35] . The lipase activity in the culture filtrate was measured using olive oil as substrate. Ten percent (v/v) olive oil was emulsified in distilled water containing 2% (w/v) gum arabic as stabilizer using a homogenizer (UltraTurrax T25, Janke and Kunkel) for 10 min at maximum speed. Twenty milliliters of substrate solution was heated to 50℃ and adjusted to pH 10.0. After addition of 5–20 µl of the enzyme solution, the activity was measured with a pH stat (Metrohm, Switzerland). Liberated fatty acids were titrated automatically with 0.01 N NaOH to maintain the pH constant at 10.0. One unit (U) of lipase activity was defined as the amount of enzyme that liberates 1 µmol fatty acid per minute under assay conditions. The protein content was determined according to the Bradford method using BSA as standard.
- Purification, Deglycosylation, and SDS-PAGE Analysis of RecombinantT. coremiiformeLipase
After fermentation, cells from the cultures were removed by centrifuging at 8,000 × g for 10min. The supernatant was concentrated by ultrafiltration using a Millipore set-up according to the manufacturer’s instructions with a membrane of 10 kDa cut off, and subsequently dialyzed against 20mM Tris-HCl buffer (pH 7.0) at 4℃ overnight. For further purification, the enzyme solution was loaded onto a DEAE-Sepharose Fast Flow column (2.0cm × 20cm) equilibrated with 20mM Tris-HCl (pH 7.0). Elution was performed with a linear gradient of 0–1 M NaCl at a rate of 2 ml/min. The active fractions were pooled, and the purity of lipase was analyzed by SDS-PAGE. Purified recombinant T. coremiiforme lipase (rTCL) was deglycosylated using 300 U of Endo H f for 3 h at 37℃ according to the manufacturer’s instructions (NEB, USA). The deglycosylated and untreated samples were analyzed by SDS-PAGE.
- Effects of pH and Temperature on Enzyme Activity and Stability
The activity of the lipase at different temperature and pH values was measured by pH-stat assay using olive oil as the substrate. The optimum temperature of purified recombinant rTCL was measured at different temperatures ranging from 30℃ to 80℃. The relative activity at different temperatures was calculated by setting 50 ℃ as 100 %. The thermal stability of rTCL and deglycosylated rTCL was studied by incubating lipase at various temperatures (30–80℃) in Tris-HCl buffer (pH 9.0) up to 30 min. The residual enzyme activity was measured at 50℃ with olive oil as substrate and the residual activity was calculated by taking the non-heated lipase activity as 100%. Optimal pH was determined by assessing the activity of the purified rTCL at pH 6.0–12.0. The relative activity at different pH values was calculated by setting pH 10.0 as 100%. The pH stability was determined by measuring the residual enzyme activities after incubating purified rTCL at various pH (50mM acetic acid-sodium acetic acid buffer (pH 3.0– 5.0), 50 mM Na 2 HPO 4 -NaH 2 PO 4 buffer (pH 6.0–7.0), 50 mM TrisHCl buffer (pH 8.0–9.0), and KCl-NaOH (10.0-12.0)) for 24 h at 30℃. The residual activity was calculated by taking the activity of purified rTCL without buffer treatment as 100%. All measurements were carried out in triplicate.
- Effect of Metal Ions on Lipase Activity
The effect of metal ions on lipase activity was analyzed by incubating enzyme samples for 6 h at room temperature in 50mM Tris-HCl buffer (pH 9.0 ), containing 1 mM of Ca 2+ , Mg 2+ , Cu 2+ , Na + , K + , Li + , Zn 2+ , Mn 2+ , Co 2+ , and Fe 2+ . The residual activity was determined as described previously. The activity of rTCL was determined in the buffer with no addition of metal ions and set as 100%. All measurements were carried out in triplicate.
- Effect of Surfactants and Oxidizing Agents on Lipase Activity
The effects of some surfactants (Triton X-100, Tween-80, Tween-20 , and SDS) and oxidizing agents (sodium perborate and H 2 O 2 ) on enzyme stability were studied by pre-incubating the enzyme for 1 h at room temperature. The residual activity was determined as described previously. The enzyme activity of a control, without surfactants and oxidizing agents, incubated under similar conditions, was taken as 100%.
- Effect of Organic Solvents on Lipase
The effect of organic solvents on enzyme activity was determined by measuring residual activity after pre-incubation of 2 ml of enzyme solution for 6 h at room temperature, in 2 ml of methanol, ethanol, acetone, isopropanol, glycerol, butanol, chloroform, diethyl ether, isooctane, and hexane, respectively. Besides this, enzyme stability in various concentrations of methanol and ethanol was evaluated since enzyme stability in these solvents is highly desirable for the enzymatic biodiesel production.
- Substrate Specificity
For the determination of substrate specificity, 10% (v/v) triglycerides, including tributyrin, tricaprylin, trilaurin, tripalmitin, tristearin, triolein, olive oil, and fatty acid methyl esters, including methyl butyrate, methyl caprylin, methyl laurate, methyl palmitate, and methyl stearate were used as substrates with 2% (w/v) gum arabic as stabilizer in the pH-stat assay. Meanwhile, the activity of rTCL against a series of vegetable oils was determined in a similar manner. Activities on each substrate are expressed as the percentage of that on olive oil. All measurements were carried out in triplicate.
- Nucleotide Sequence Accession Number
The nucleotide sequence accession number of the Trichosporon coremiiforme V3 lipase gene has been deposited into the GenBank database under the accession number KM099068.
Results and Discussion
- Cloning and Sequence Analysis of the Gene Encoding Lipase
Based on the consensus sequences of lipases from T. fermentans WU-C12, G. candidum , and G. geotrichum , about 1.6 kb fragments were amplified from genomic DNA and cDNA sources by both PCR and RT-PCR. Nucleotide sequencing of the fragments revealed that tcl has an ORF of 1,692 bp without any introns, encoding a protein of 563 amino acid residues, including a potential signal sequence of 19 amino acid residues. The NCBI protein–protein BLAST showed that the deduced TCL amino acid sequence has 85% identity to lipases I from T. fermentans WU-C12 (Accession No. AB000260), followed by G. candidum strain lip42 (83% identity, Accession No. AB000260). Results of a multiple alignment analysis of these lipase amino acid sequences are shown in Fig. 1 . As previously reported, a characteristic flap covering the active site of the lipase was found at position 81-123 [28] . A conservative motif (-Gly 234 -Glu-Ser-Ala-Gly 238 -) was found at position 234-238, which is a characteristic of triacylglycerol hydrolases [33] . In all these alignment lipases, the catalytic triad (-Ser 236 -Glu 373 -His 482 -) was found at perfectly conserved positions. The protein contained four Cys residues involving two disulfide bridges (-Cys 80 ~Cys 124 - and –Cys 295 ~Cys 307 -) and two potential glycosylation sites (-Ala 383 -Asn-Thr 385 and -Ala 456 -Asn-Thr 458 -).
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Multiple sequence alignment of the complete lipase of T. coremiiforme V3 against other related complete lipases. Sequences of other species were obtained from the GenBank (Tc: T. coremiiforme V3, GenBank No. KM099068; Tf: T. fermentans WU-C12, GenBank No. AB000260; Gc: G. candidum, GenBank No. AB000260; Gg: G. geotrichum, GenBank No. ACX69980). The bold line indicates the flap domain. The catalytic triad (Ser236-Glu373-His482) is marked by triangles, and rectangles show the disulfide bonds. The conservative motif (Gly234-Glu-Ser-Ala-Gly238) is boxed.
- Vector Construction and Scre
A 1,635 bp tcl (without the signal sequence) was integrated in frame with the Saccharomyces cerevisiae α -factor secretion signal sequence under the control of the AOX1 promoter in plasmid pPICZ α A to obtain the expression vector pPICZ α A- tcl . Plasmid pPICZ α A- tcl was transformed into P. pastoris X33. To select the lipase-producing clones, we randomly picked up 50 positive colonies and inoculated them into 500 ml shaking flasks containing 50 ml of BMGY medium to check the lipase production. In shaking flasks, the lipase activity increased gradually and reached the highest activity after 144 h cultivation. A positive clone that exhibited a maximum lipase activity of 150 U/ml (by pHstat assay using olive oil as substrate) was enhanced 7.5-fold compared with the activity of native T. coremiiforme V3 cultured at flask level (20 U/ml, by pH-stat assay using olive oil as substrate). The positive clone with the highest activity was chosen for all further experiments.
- High Cell Density Fermentation
To obtain a large amount of active protein, fed-batch studies were carried out in 5 and 50 L fermentors. The fed-batch fermentation continued for a period of 8 days. Upon methanol induction, the maximum lipase activity and cell density reached 4,100 U/ml (by pH-stat assay using olive oil as substrate) and 170 g DCW/L, respectively, in the 5 L fed-batch bioreactor ( Fig. 2 A). The maximum lipase activity and cell density obtained in the 50 L fed-batch bioreactor were 5,000 U/ml (by pH-stat assay using olive oil as substrate) and 198 g DCW/L, respectively ( Fig. 2 B). Compared with the shaking flasks procedure, the high cell density fed batch of P. pastoris produced a significantly higher level of the recombinant enzyme. Under these conditions, a 33-fold increase in the volumetric lipase activity was obtained when scaling from shaking flasks to the 50 L bioreactor (150 and 5,000 U/ml, respectively). In this study, the total protein concentrations in the supernatant achieved in 5 and 50 L fermentors were 4.5 and 5.4 g/l, respectively. To the best of our knowledge, it is significantly higher than most other reported results. Yu et al. [36] expressed Rhizopus chinensis lipase in P. pastoris GS115 and the maximum total protein of the recombinant was 1.6 g/l after 61 h of methanol induction in a 7 L bioreactor. Lipase B from C. antarctica was expressed in P. pastoris X33, which reached a maximum total protein of 1.18 g/l after 105 h of cultivation in a 5 L bioreactor [10] . We deduced one of the reasons for the high total protein concentrations of rTCL was the high dry cell weight (DCW). In this study, the maximum DCW of recombinant in the 50 L fed-batch bioreactor was 198 g/l, which was significantly higher than lipase B from C. antarctica (maximum DCW of 135.7 g/l after 105 h of cultivation in a 5 L bioreactor), lipase 2 from Y. lipolytica (maximum DCW of 68 g/l after 192 h of cultivation in a 7.5 L bioreactor), and lipase from R. chinensis (maximum DCW of 98 g/l after 105 h of cultivation in a 7 L bioreactor) in P. pastoris [10 , 35 , 36] . The methylotrophic yeast P. pastoris is an excellent host for the production of proteins from different sources [15 , 32] . Compared with E. coli and S. cerevisiae , the methylotrophic yeast P. pastoris has many advantages as a host for the production of recombinant heterologous proteins, such as high cell density, high levels of productivity, ease of genetic manipulation, the ability to perform complex posttranslational modifications, and very low secretion levels of endogenous proteins [7 , 27] .
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Lipase production and cell growth during fed-batch fermentation in 5 L (A) and 50 L (B) bioreactors. The lipase activity was measured with a pH-stat (Metrohm) and using olive oil as substrate at pH 10.0, 50℃. Dry cell weight was obtained by centrifuging 10 ml samples in a pre-weight centrifuge tube at 8,000 ×g for 10 min and washing twice with deionized water, and then allowing the pellet to dry at 105℃ to constant weight.
- Purification of Recombinant Lipase
Cells were separated from the fermentation broth by centrifuging at 8,000 × g for 10 min.
The recombinant enzymes from the culture supernatant were purified by a two-step purification protocol. A summary of the purification process is given in Table 1 . Since P. pastoris secreted few native proteins at low levels to the medium, the rTCL was approximately free from contaminating proteins. As shown in Fig. 3 , the purified rTCL showed two forms of rTCL with molecular masses close to 70 kDa on SDS-PAGE, which is about 10 kDa larger than 60 kDa, the calculated molecular mass of the nonglycosylated TCL. The discrepancy between the actual molecular mass and that estimated based on amino acid sequence was probably due to glycosylation of the enzyme. As shown in Fig. 3 , Endo H f treatment of recombinant rTCL resulted in a shift in the protein band on SDS-PAGE and yielded a single band of 60 kDa, suggesting that rTCL is a glycoprotein.
Purification of rTCL from culture supernatant.
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Purification of rTCL from culture supernatant.
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SDS-PAGE of rTCL produced by P. pastoris. Lane M shows the molecular weight standards; Lane 1 shows lipase from ultrafiltration; Lane 2 shows the purified lipase from DEAESepharose FF chromatography; Lane 3 shows the deglycosylated recombinant rTCL and Endo Hf.
- Effect of pH on rTCL Activity and Stability
The influence of pH on rTCL activity and stability is presented in Fig. 4 . The activity of rTCL was measured over a pH range of 6.0–12.0. As shown in Fig. 4 A, rTCL remained active at a pH range of 9.0-11.0 and showed maximum activity at pH 10. The optimum pH for rTCL is different from recombinant lipases of G. candidum and G. geotrichum whose optimum pH was at a range of pH 7.0-7.5 [11 , 18] . In the pH stability study, the lipase was stable over a broad range of pH values of 5.0-11.0 after 24 h of incubating at 30℃. Almost 72% of the maximum activity remained stable at pH 11.0 ( Fig. 4 B), indicating that rTCL is an alkaline lipase. The pH stability of rTCL is comparable to the lipase from Ralstonia sp. CS274 [37] . Lipases active and stable in alkaline media are very attractive and a great deal of research is currently going into developing lipases that will work under alkaline conditions. For example, lipase produced by Acinetobacter radioresistens has an optimum pH of 10.0 and it was stable over a pH range of 6.0-10.0, which has a great potential for application in the detergent industry [4] .
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Influence of pH on rTCL activity (A) and stability (B). The lipase activity was measured with a pH-stat (Metrohm), using olive oil as substrate. The optimal pH was determined by assessing the activity of the purified rTCL at pH 6.0–12.0. The relative activity at different pH values was calculated by setting pH 10.0 as 100%. The pH stability was determined by measuring the residual enzyme activities after incubating purified rTCL at various pH values for 24 h at 30℃. The residual activity was calculated by taking the activity of purified rTCL without buffer treatment as 100%.
- Effect of Temperature on rTCL Activity and Stability
The influence of temperature on rTCL activity and stability is presented in Fig. 5 . As shown in Fig. 5 A, the recombinant lipase showed optimum activity at 50℃ and activity dropped above 60℃. The optimum temperature of rTCL is higher than previously reported for lipase of G. candidum and G. geotrichum (25-40℃) [11 , 28] . Thermostability was examined by incubating rTCL at different temperatures for 1 h, and the residual activity was measured at 50℃ under the conditions mentioned above. The activity of rTCL was stable at temperatures below 60℃, but it decreased dramatically when the temperature was above 70°C. rTCL showed only 26% residual activity after 1 h incubation at 80°C. Thermostable enzymes have long been of interest to biochemists. In this study, rTCL exhibited better thermal stability than most reported lipases from other fungi. The extracellular lipase 2 from Yarrowia lipolytica lost activity completely at 50℃ after 30 min incubation [35] . The recombinant lipase from C. rugosa suffered inactivation when heated to 50℃ [8] . A lipase from Penicillium expansum showed no residual activity after 15 min at 50℃ [2] . Our results showed that rTCL was one of the thermostable enzymes among the published lipases in eukaryotic organisms. The good temperature stability of rTCL may make it useful in industry applications.
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Influence of temperature on rTCL activity (A) and stability (B). The optimum temperature of purified rTCL was measured at different temperatures ranging from 30℃ to 80℃. The relative activity at different temperatures was calculated by setting 50℃ as 100%. The thermal stability of rTCL and deglycosylated rTCL was studied by incubating lipase at various temperatures (30–80℃) up to 30 min. The residual enzyme activity was measured at 50℃ and the residual activity was calculated by taking the non-heated lipase activity as 100%.
Mechanisms for the thermostability of proteins have been studied. In previous studies, glycosylation helped to improve the thermostability of recombinant protein. The possible explanation is that glycosylation is involved in protein rigid structure formation, which increases the thermostability of protein [38] . In this study, TCL contained two potential glycosylation sites (-Ala 383 -Asn-Thr 385 and – Ala 456 -Asn-Thr 458 -) that help to improve the thermostability of rTCL. The thermostability of deglycosylated rTCL is shown in Fig. 5 B. The activity of deglycosylated rTCL was stable at temperatures below 50°C, but it decreased dramatically when the temperature was above 60°C. The thermostability of rTCL was higher than deglycosylated rTCL. rTCL showed 45% and 26% residual activity after 1 h incubation at 70℃and 80°C, whereas deglycosylated rTCL showed only 10% and 5%.
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Substrate specificity of rTCL on various oils. Activities on each substrate are expressed as the percentage of that on olive oil. All measurements were carried out in triplicate.
- Effect of Metal Ions on rTCL Activity
The effect of metal ions was tested at 1 mM in 0.1 M Tris-HCl buffer at pH 9.0 ( Table 2 ). Metal ions, particularly Ca 2+ , play important roles in influencing the structure and function of enzyme, and calcium-stimulated lipases have been reported [19] . In this study, maximum enhancement (by 18%) was found in the presence of 1 mM Ca 2+ . Zn 2+ and Cu 2+ inhibited the activity by 38% and 25%. The activity enhancement caused by Ca 2+ and inhibition caused by Cu 2+ and Zn 2+ of rTCL were almost similar to Pseudomonas aeruginosa LX1 lipase [22] . Na + , K + , Mg 2+ , Li + , Mn 2+ , Fe 2+ , and Co 2+ had no significant effect on enzyme activity in our study (80%-102%).
Effect of metal ions on rTCL activity.
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The effect of metal ions on lipase activity was analyzed by incubating enzyme samples for 6 h at room temperature in 50 mM Tris-HCl buffer (pH 9.0). The activity was determined under the standard assay conditions. The activity of rTCL was determined in the buffer with no addition of metal ions and set as 100%.
- Effect of Surfactants and Oxidizing Agents on rTCL Stablity
For use in laundry detergents, a good detergent enzyme must be compatible and stable with all commonly used detergent compounds such as surfactants and oxidizing agents [29] . The effects of various detergents in rTCL activity are presented in Table 3 . Non-ionic detergent Triton X-100 (1% (v/v)) and Tween 20 (1% (v/v)) increased the enzyme activity to 110% and 115%, respectively. rTCL activity was also highly stable in the presence of 5% Triton X-100, 5% Tween 20, and 5% Tween 80, retaining 100% of the original activity after 1 h incubation at 40°C. Furthermore, rTCL was less stable against the anionic detergent SDS and retained only 40% of its activity in the presence of 0.1% (w/v) SDS. In addition, we investigated the effects of oxidizing agents (H 2 O 2 and sodium perborate) on rTCL stability. As shown in Table 3 , rTCL activity was little influenced by oxidizing agents and retained 80% and 75% of its activity after incubation for 1 h at 40℃ in the presence of 0.1% (v/v) sodium perborate and H 2 O 2 , respectively.
Stability of rTCL in the presence of various surfactants and oxidizing agents.
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rTCL was incubated with different surfactants and oxidizing agents for 1 h at 40℃ and the remaining activity was measured under standard conditions. The activity is expressed as percentage of the activity level in the absence of additives.
- Effect of Organic Solvents on rTCL Stablity
The stability of rTCL in the presence of various organic solvents is presented in Table 4 . In this study, glycerol slightly activated the enzyme activity with relative activity of 105%. Above 82% of the original activity remained stable in isooctane, diethyl ether, and hexane. Methanol, ethanol, acetone, isopropanol, butanol, and chloroform had negative effect on enzyme activity in our study, with relative activity from 10% to 65%, probably because they provoked a rapid protein denaturation. The enzyme stability was also evaluated in the presence of various concentrations of methanol and ethanol ( Table 5 ). In 6 h incubation, above 85% of the original activity remained stable when the ethanol concentration was below 20% and this tendency was found to be even better in the presence of methanol (above 93% of the original activity). However, the lipase stability decreased when the ethanol and methanol concentrations were increased up to 30%. Yang et al. [34] reported that lipases from Pseudomonas cepacia, Candida rugosa , and Candida antarctica were inactivated by 10% methanol in 30 min incubation; therefore, rTCL has better methanol tolerance than those lipases. The stability of this lipase in methanol suggests it could be used as a biocatalyst in biodiesel production, since biodiesel production is generally carried out by methylation of various oils. Hence, the result evidenced that rTCL is suitable for this purpose.
Influence of various organic solvents on the activity of rTCL.
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Enzyme samples were mixed with solvents (50% (v/v)) and incubated for 6 h in a rotary shaker set at 160 rpm and 25℃ prior to determining the residual activity. All measurements were carried out in triplicate.
Influence of methanol and ethanol concentrations on the activity of rTCL.
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Enzyme samples were incubated with various concentrations of methanol and ethanol for 6 h in a shaker set at 160 rpm and 25℃. All measurements were carried out in triplicate.
- Substrate Specificity
The substrate preferences of rTCL were characterized with various triglycerides and fatty acid methyl esters. As shown in Table 6 , the purified rTCL showed preferential specificity for long-chain substrates. For triglycerides, the lipase showed high activity towards triolein (C18:1) and tripalmitin (C16:0). For fatty acid methyl ester, the lipase also preferred long-chain fatty acid esters (C12, C16, C18). Compared with rTCL, C. rugosa lipase 1 and C. antarctica lipase B showed different substrate specificities. Among the different triglycerides, the lipolytic activity of C. rugosa lipase 1 displayed the highest activity for hydrolysis of the medium-chain triglyceride (trilaurin; C12) [5] . For triglycerides, the lipase B from C. antarctica showed high activity towards short-chain triglyceride (tributyrin; C4) [26] . The substrate specificity of different lipases may be attributed to differences in the geometry and size of their active sites [16] . As shown in Fig. 5 , rTCL showed relatively high activity on various emulsified vegetable oils (from 73% to 116%), especially on cotton seed oil. An explanation for this can be vegetable oils containing a large number of long fatty acyl chains, such as oleic acid, linoleic acid, and so on. The reason for the different relative activity towards different vegetable oils may be the different composition of fatty acyl chains.
Substrate specificity of rTCL on various triglycerides and fatty acid methyl esters.
PPT Slide
Lager Image
Activities on each substrate are expressed as the percentage of that on olive oil. All measurements were carried out in triplicate.
In conclusion, in this study, an alkaline and thermostable lipase gene was cloned from T. coremiiforme V3 and functionally expressed in P. pastoris . To the best of our knowledge, this is the first report of the gene cloning, highlevel expression, and characterization of TCL. The production of rTCL was improved greatly in a 50 L bioreactor, being 33 times higher than that in shake flask, and was about 247 times higher than that of native Trichosporon coremiiforme V3 cultured at flask level. Furthermore, rTCL exhibited several properties of significant industrial importance, such as high temperature and pH stability, wide organic solvent tolerance, and broad hydrolysis range on vegetable oils. Such a combination of properties makes TCL a highly interesting candidate for future practical applications.
Acknowledgements
This work was supported by the National HighTechnology Project of P.R. China (No. 2014AA093514), and the Key Science and Technology Program of Zhuhai (No. 2012D0201990042).
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