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High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase
Protein Expression and Purification 28 (2003) 102–110 www.elsevier.com/locate/yprep
High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase Dinh Thi Quyen,a,* Claudia Schmidt-Dannert,b and Rolf D. Schmidc a
b
National Center of Natural Science and Technology, Institute of Biotechnology, 18 Hoang Quoc Viet Road, Nghiado, Distr. Caugiay, 10600 Hanoi, Viet Nam California Institute of Technology, Chemical Engineering 210-41, 120 E. California Blvd., Pasadena, CA 91125, USA c Institut f€ur Technische Biochemie, Universit€at Stuttgart, Allmandring 31, Stuttgart, Germany Received 5 August 2002, and in revised form 6 November 2002
Abstract The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZaA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPa-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4 L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000 U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000 U/mg protein towards tributyrin. The pure enzyme was characterized and its physicochemical properties were compared to those of the BTL2 lipase, which had previously been expressed in Escherichia coli under the control of its native promoter on pUC18 or under the control of the strong temperature inducible promoter kPL , yielding 600 U/g or 54,000 U/ g wet cells, respectively. The three proteins showed the same N-terminal sequence and had very similar pH optimum, pH stability, temperature optimum, thermostability, and substrate specificity profiles. Three enzymes were extremely stable in the presence of several organic solvents and detergents. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Lipase; Expression; Bacillus thermocatenulatus; Pichia pastoris; Properties
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis of triglycerides at the interface between the insoluble substrate and water [1] and have been utilized widely in hydrolysis and transesterification of triglycerides and enantioselective synthesis and hydrolysis of a variety of esters [2–4]. Lipases from thermophiles often show their extreme stability at elevated temperatures and in organic solvents [5]. Thus, they have become objects of special interest for structural investigations and also for industrial applications [6].
* Corresponding author. Fax: +84-4-8363144. E-mail address: [email protected] (D.T. Quyen).
The thermophile Bacillus thermocatenulatus produces two lipases, named BTL1 and BTL2 [7]. The gene of the BTL2 lipase has been cloned and expressed in Escherichia coli under the control of the native promoter [8] or under the control of the strong temperature inducible kPL promoter [9]. The lipase was overexpressed in the E. coli vector pCYTEXP1. However, the BTL2 lipase was expressed in E. coli as intracellular protein which demands a lot of purification steps. Recently, many lipases have been overexpressed as extracellular protein in Pichia pastoris [10–14]. In this paper, we report cloning of the BTL2 lipase gene in the yeast P. pastoris GS115, high-level expression of the lipase as extracellular protein in large quantities, characterization, and comparison of the physicochemical properties of the BTL2 lipases expressed both in E. coli as well as in P. pastoris.
1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-5928(02)00679-4
D.T. Quyen et al. / Protein Expression and Purification 28 (2003) 102–110
Materials and methods Materials The BTL2 lipase gene from B. thermocatenulatus (GenBank X95309) was isolated from the plasmid pT1BTL2 that was described by Rua et al. [9]. The plasmid pPICZaA (Fig. 1a) and yeast strain P. pastoris GS115 were purchased from Invitrogen. E. coli DH5a (supE44 DlacU169 [U80lacZDM15] hsdR17 recA1 endA1 gyrA 96 thi-1 relA1) was used for subcloning. The triacylglycerols tributyrin, tricaprin, trilaurin, trimyristin, tripalmitin, and triolein were purchased from Fluka (Diesenhofen, Germany). Triacetin, tricaproin, tricaprylin, and nitrophenyl palmitate were supplied by Sigma (Deisenhofen, Germany). The detergents Triton X-100 and sodium dodecyl sulfate (SDS) were from Fluka (Deisenhofen, Germany), sodium cholate was from Sigma (Deisenhofen, Germany), and Tween 20 and Tween 80 were from Riedel-de Haen (Hannover, Germany). Bacto-tryptone, yeast extract, and yeast nitrogen bases were from Difco (Ausburg, Germany). a-Naphthyl acetate, glucose, and gum arabic were from Fluka (Deisenhofen, Germany), and Fast Red TR-salt was from Serva (Heidelberg, Germany). All other reagents were of analytical grade unless otherwise stated. Plasmid construction, subcloning, and transformation The BTL2 lipase operon of 1.2 kb from B. thermocatenulatus was amplified from the expression vector in E. coli pT1-BTL2 [9] by PCR with two primers 50 -GAG ACT GAA TTC GCC GAC AAC TAC GCG-30 and 50 GGC GGA TCC TAA GTA AGT AGA ATT CTG AGT AGG-30 introducing EcoRI sites lying immedi-
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ately upstream of the start codon ATG and downstream of the stop codon. It was followed by the ligation of the EcoRI digested PCR products with pPICZaA linearized by the same enzyme, resulting in pPa-BTL2 (Fig. 1b) under the control of the alcohol oxidase promoter AOX1 induced by methanol and possessing the Zeocin marker. The plasmid pPa-BTL2 was subcloned in E. coli DH5a and the recombinant gene was confirmed by the fluorescence-based dideoxy DNA cycle sequencing method. DNA sequencing was performed with the Taq Dye Deoxy Cycle Sequencing Kit (Applied Biosystems) and with a 373A DNA Sequencing System (Applied Biosystems) according to manufacturerÕs instructions. Other standard recombinant DNA techniques were carried out as described by Sambrook et al. [15] and Ausubel et al. [16]. The PmeI linearized plasmid pPa-BTL2 was introduced into the yeast P. pastoris GS115 by electroporation according to manufacturerÕs recommendations. Gene expression and fermentation The P. pastoris GS115 pPa-BTL2 was cultivated in 50 mL BMGY medium (1% (w/v) of yeast extract, 2% (w/v) of bacto-peptone, 100 mM potassium phosphate, pH 7.0, 4 105 % (w/v) of biotin, and 1% (v/v) of glycerol) for 24 h, at 30 °C and agitated at 220 rpm. After 24 h of cultivation, the culture was centrifuged and the cell pellet was transferred into the BMMY medium, which contains the same components as the BMGY medium except for 0.5% (v/v) of methanol instead of 1% (v/v) of glycerol. The culture was cultivated at 30 °C, 220 rpm. As much as 0.5% (v/v) of methanol was added to the culture every 24 h and 2 mL of the culture was
Fig. 1. Expression vectors pPICZaA (a) and pPa-BTL2 (b) in P. pastoris.
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taken to determine the cell growth and the lipase activity. To produce large quantities of lipase, the P. pastoris GS115 harboring the plasmid pPa-BTL2 was grown in a 5-L bioreactor (Infors, Switzerland). Forty mL culture (in the BMGY medium at 30 °C, 220 rpm for 24 h) was inoculated in 400 mL BMGY medium and grown under the same cultivation conditions. After 24 h of growth, cells were harvested by centrifugation, resuspended in 100 mL BMMY medium, and inoculated in 4 L BMMY medium in 5-L bioreactor. The culture was grown at 30 °C, aerated at the rate of 40% pO2 and agitated at 500 200 rpm. Methanol was added continuously to the culture at the rate of 917 lL of 100% methanol per hour ( 22 mL/day) to induce lipase production. Culture samples were collected at different times to estimate the lipase activity and cell growth. After 120 h of cultivation, the crude lipase solution was harvested by centrifugation and concentrated to a volume of 350 mL by cross-flow filtration with the membrane of 10 kDa nominal MW cutoff.
maximum speed, as described by Schmidt-Dannert et al. [7]. A 20 mL volume of the triglyceride solution was heated to 65 °C and adjusted to pH 8.5. Autohydrolysis was measured in 10 min without addition of enzyme. After addition of 5–50 lL of the lipase solution, the activity was measured with a pH-stat (Metrohm) for 10 min. One unit was defined as the amount of enzyme, which released 1 lmol of fatty acid per minute. Gel electrophoresis SDS–PAGE was carried out, as described by Laemmli [17] with Biometra and Bio-Rad equipment. SDS– PAGE was usually performed with gels of 12.5% (w/v) of acrylamide according to manufacturerÕs recommendations. Gels were stained for protein detection by a silver stain procedure described by Butcher and Tomkins [18]. Detection of hydrolytic activity with a-naphthyl acetate as the substrate was performed by coupling with Fast Red TR salt according to Schmidt-Dannert et al. [8] after renaturation of the protein in 0.1 mM Tris buffer, pH 7.5.
Enzyme purification N-terminal sequence analysis Twenty mL concentrate of the crude enzyme containing a total activity of 51,900 U was loaded on a butyl-Sepharose (Sigma) column (2.6 cm 32 cm with 100 mL packed resin) equilibrated with 50 mM Tris buffer (pH 8.0) containing 0.1 M NaCl at a flow rate of 1 mL/min. Afterwards, the column was washed with the equilibration buffer until no further protein was eluted. The BTL2 lipase bound on the butyl-Sepharose column was then eluted with 1% (w/v) cholic acid in 50 mM Tris buffer (pH 8.0). Fractions with activity were pooled and concentrated by ultrafiltration through a 10 kDa membrane (Filtron) by a factor of up to 10. Enzyme assays Spectrometric assay The spectrophotometric assay was carried out using p-nitrophenyl palmitate (pNPP) as the substrate in a Ultrospec 3000 spectrophotometer (Pharmacia). Cleavage of pNPP was measured at 60 °C using 0.1 M Tris buffer (pH 7.5) according to Schmidt-Dannert et al. [7]. One unit was defined as the amount of enzyme, which cleaved 1 lmol pNPP to p-nitrophenol and palmitate per minute under the assay conditions. pH-stat assay Lipase activity was estimated using triglycerides (C4– C18) as substrate in a pH-stat (Metrohm). Ten mM triglycerides were emulsified in distilled water containing 2% (w/v) of gum arabic as stabilizer using a homogenizer (Ultraturrax T25, Janke and Kunkel) for 5 min at
Purified proteins were applied to a SDS gel. After blotting, the polyvinylidene difluoride membranes were stained with Coomassie brilliant blue R-250, the protein bands of interest were cut out and used for aminoterminal sequence determination. Amino-terminal sequence analysis was performed in a gas-phase sequencer 470A (Applied Biosystems) following manufacturerÕs recommendations. Effect of pH, temperature, organic solvents, and detergents on lipase activity and stability The optimum pH for the enzyme activity was measured at 60 °C by pH-stat assay using tributyrin and triolein as substrates. The assays were carried out by incubation of the reaction mixtures at 60 °C at various pH values from 5.0 to 10.0. The effect of pH on the lipase stability was determined by incubating aliquots of pure lipase solutions for 14 h at 30 °C in 0.1 M (glycine/ HCl, Tris/HCl or glycine/NaOH) buffers at different pH values. Residual activity was measured by photometric assay using pNPP as substrate. The optimum temperature for the enzyme activity was measured at pH 8.0 with tributyrin and triolein as substrates. The assay was carried out by incubating the reaction mixture at various temperatures from 35 to 80 °C. The effect of temperature on the lipase stability was determined by incubating aliquots of pure lipase solution for 30 min in 0.1 M Tris buffer, pH 8.0, at various temperatures from 30 to 80 °C. Residual activity was determined by photometric assay using pNPP as the substrate.
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The effect of detergents on the lipase activity was determined by incubating the enzyme for 1.5 h at 30 °C in 0.1 M Tris buffer (pH 8.0) containing 1% (w/v) of detergents. The lipase activity was measured only at the end of the incubation time by photometric assay with pNPP as substrate. The effect of 30% (v/v) of organic solvent on the lipase activity was determined in a similar way as that of detergents. The reaction mixtures were incubated for 1.5 h at 30 °C; the control contained no organic solvent. All measurements were carried out three times and from these values the average value was taken. Determination of substrate specificity To determine the substrate specificity of the BTL2 lipase, the triglycerides triacetin (C2), tributyrin (C4), tricaproin (C6), tricaprylin (C8), tricaprin (C10), trilaurin (C12), trimyristin (C14), tripalmitin (C16), and tristearin (C18) at a final concentration of 10 mM were each emulsified in distilled water containing 2% (w/v) of gum arabic and used as substrate solution. The lipase activity of the purified enzyme towards triacylglycerides of fatty acids of different chain lengths was determined by pH-stat assay at 65 °C and two different pHs (7.5 and 8.5).
Results Plasmid construction The BTL2 lipase gene was removed from the plasmid pT1-BTL2 by PCR with two flanking primers complementary to the 50 -end and reverse complementary to the 30 -end of the lipase gene and containing EcoRI restriction site. The PCR product was digested with EcoRI and inserted into the vector pPICZaA that was also digested with this enzyme, resulting in the plasmid pPa-BTL2 (Fig. 1b). After subcloning in E. coli DH5a and confirming by DNA sequencing, the plasmid pPa-BTL2 linearized with PmeI was transformed in P. pastoris GS115. On the plate containing 1% (w/v) of tributyrin and 50 lg/mL Zeocin, many colonies showed a halo ring. Transformed colonies were confirmed by the PCR. Lipase expression P. pastoris GS115 harboring the PmeI linearized plasmid pPa-BTL2 as integrating in its genomic DNA was cultivated in 2-L shaking flask containing 400 mL BMMY medium. At different times, samples were taken from the culture and assayed for cell growth (OD600 ) and for lipase activity by using p-nitrophenyl palmitate as substrate (data not shown). The activity reached a maximum of 406 U/mL supernatant after 120 h of
Fig. 2. Cell growth ðjÞ and lipase activity ðdÞ of the BTL2 expressed in P. pastoris. Cultivation was performed, as described in the text. Lipase activity was measured with p-nitrophenyl palmitate (pNPP) as substrate at 60 °C, pH 7.5.
cultivation. From this result, the strain was cultivated further in a 5-L bioreactor filled with 4 L of the BMMY medium. A profile of cell growth and lipase activity was studied at different times, as shown in Fig. 2. P. pastoris GS115 cells were grown at 30 °C for 120 h. The cell growth OD600 increased gradually from 25 at 24 h to 42 at 120 h whereas the lipase activity increased steeply from 56 U/mL supernatant at 24 h to 309 U/mL supernatant as the maximum at 96 h of cultivation and decreased to 209 U/mL supernatant at 120 h of cultivation. This activity profile was a little different from that of shaking flask culture (data not shown). Protein purification and sequencing Twenty mL concentrate of the crude enzyme of total activity of 51,800 U with a specific activity of 35 U/mg was loaded on a butyl-Sepharose column and 91% of the loaded lipase activity was adsorbed to the matrix. By using 1% (w/v) cholic acid in 50 mM Tris buffer, pH 8.0, only 9500 U (18%) of the bound lipase activity was eluted from the column. The enzyme was purified 660fold with a specific activity of 23,000 U/mg (Table 1). This fraction was studied for the protein sequencing and characterization of the BTL2 lipase expressed in P. pastoris. The crude enzyme solution and the purified lipase on SDS–PAGE showed a protein band, corresponding to the lipase molecular weight (43 kDa) (Fig. 3). Purified protein of 1 mL lipase solution was precipitated using trichloroacetic acid and subjected to SDS– PAGE. After blotting, the protein band was used for amino-terminal sequence determination. The N-terminal sequence was E–F–A–S–P–R–A–N–D. The N-terminal fragment of the deduced BTL2 lipase sequence was M–A– S–P–R–A–N–D. The extra amino acids E and F were the result of introduction of EcoRI site (GAATTC).
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Table 1 Summary of the purification of recombinant lipase BTL2 expressed with Pichia pastoris pPa-BTL2 Step Crude enzyme Butyl-Sepharose a b
Total units (U)a 51,800 9500
Specific activity (U/mg)b 35 23,000
Purification factor
Yield (%)
1 660
100 18
From 180 mL of culture supernatant. Specific activity is given as units of lipase towards tributyrin per milligram of lipase.
Fig. 3. SDS–PAGE of the BTL2 lipase before and after purification. Lane M, molecular mass standards indicated in kDa; lane 1, sample of the supernatant after 120 h of cultivation; lane 2, sample of the purified lipase after butyl-Sepharose.
Effect of pH and temperature on lipase activity and stability The optimum activity of the BTL2 lipase was investigated at 60 °C using tributyrin and triolein as substrates (Fig. 4a) with a pH range from 5.0 to 9.0 and from 7.5 to 10.0, respectively. The lipase showed maximum activity at pH 7.5 and pH 9.0 with tributyrin and triolein, respectively. With tributyrin, the relative lipase
activity increased from 34% at pH 5.0 steeply to 71% at pH 6.0, and then slowly to 80% at pH 7.0 and the maximum (100%) at pH 7.5. It decreased slowly to 96% at pH 8.0, 86% at pH 8.5, and remained 80% at pH 9.0. With triolein the relative activity increased as well as decreased steeply around pH 9.0. It increased from 20% at pH 7.5 to 65% at pH 8.0, to 86% at pH 8.5, and to the maximum (100%) at pH 9.0 and then decreased to 80% at pH 9.5 and 58% at pH 10.0. At pH 7.0, no lipase activity with triolein was observed whereas 80% residual activity was still detected with tributyrin. Different pH values of incubation buffers of 0.1 M (glycine/HCl, pH 4.0–6.0, Tris/HCl, pH 7.0–9.0 or glycine/NaOH, pH 9.0–12.0) showed an obvious effect on the lipase stability with p-nitrophenyl palmitate as substrate after 14 h of incubation at 30 °C. The BTL2 lipase stability was low at the pH range from 4.0 to 6.0 (30– 80%) or from pH 12.0 (55%). However, the BTL2 lipase showed a high relatively constant stability within the broad pH range from 7.0 to 11.0 after 14 h incubation at 30 °C (Fig. 4b). Within this pH range from 7.0 to 11.0, the relative residual activity remained at least 90% (pH 10.0) and showed the maximum of 100% at pH 8.0. The optimum temperature of the BTL2 lipase was also investigated using tributyrin and triolein as substrates at a constant pH of 8.0. The maximum temperature was 65 and 75 °C for hydrolysis of tributyrin and triolein, respectively (Fig. 5a). With tributyrin, the rel-
Fig. 4. Effect of pH on lipase activity (a) and stability (b). (a) Effect of pH on lipase activity was determined by the pH-stat assay with tributyrin ðjÞ and triolein ðdÞ. (b) Purified lipase was incubated for 14 h at 30 °C in various 0.1 M buffers: ðjÞ, and glycine/HCl buffer; ðdÞ, Tris/HCl buffer; ðNÞ, glycine/NaOH buffer. The residual activity was measured at 65 °C with the photometric assay using pNPP as substrate.
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Fig. 5. Effect of temperature on lipase activity (a) and stability (b). (a) Effect of temperature on lipase activity was determined by the pH-stat assay with tributyrin ðjÞ and triolein ðdÞ as substrate. (b) Purified lipase was incubated for 30 min at pH 9.0. The residual activity was measured with the photometric assay using pNPP as substrate ðjÞ.
ative lipase activity increased very gradually from 34% at 35 °C to the maximum of 100% at 65 °C and then steeply decreased to 48% at 75 °C. With triolein, the profile of the relative lipase activity was similar to that with tributyrin. The lipase activity increased very gradually from 12% at 35 °C to the maximum of 100% at 75 °C and then decreased to 65% at 80 °C. Below the optimum temperature, the lipase activity decreased very steeply. The various incubation temperatures from 35 to 80 °C showed an obvious temperature effect on the lipase stability with pNPP as the substrate incubated in 0.1 M Tris buffer, pH 8.0, for 30 min. The relative residual lipase activity is disproportional to the incubation temperature. The relative BTL2 lipase activity was fixed as 100% at the temperature of 40 °C and increased to 120% when incubated at the lower temperature of 30 °C. The BTL2 lipase was stable up to 50 °C when incubated for 30 min at pH 8.0 in 0.1 M Tris buffer. Above this temperature, the residual activity decreased steeply to 18% at 70 °C. At 80 °C most of the lipase was inactivated and the lipase activity remained only 5% (Fig. 5b). Substrate specificity Among triglycerides, the BTL2 lipase showed the highest activity towards tributyrin (C4 acyl group) at both pH 7.5 and 8.5. The highest activity towards tributyrin at pH 8.5 was fixed as 100%. The typical profile of chain length specificity of this lipase is shown in Fig. 6. The substrate specificity profiles at pH 7.5 and 8.5 were very similar; however, there was a little difference between these profiles at pH 7.5 and 8.5. For most triglycerides, the lipase activity at pH 8.5 was higher (1– 3%) than that at pH 7.5, except for three substrates triacetin, tricaproin, and trimyristin (Fig. 6). The activity towards tricaprylin (C8 acyl group) relative to
Fig. 6. Substrate specificity at pH 7.5 and 8.5. Activity of the purified lipase towards triacylglycerols with various chain lengths of the acyl group was determined by the pH-stat assay at 65 °C and pH 7.5 (filled column) or pH 8.5 (slightly filled column).
tributyrin was around 40%. The activity of the BTL2 lipase towards tricaproin (C6 acyl group), tricaprin (C10 acyl group), and tripalmitin (C16 acyl group) remained around 20% of that towards tributyrin. The relative activity of trilaurin (C12 acyl group), trimyristin (C14 acyl group), and tristearin (C18 acyl group) in comparison to tributyrin was around 10%. Triacetin (C2 acyl group) was the poorest substrate (3–4%) at both pH 7.5 and 8.5. Effect of organic solvents and detergents on lipase activity The effect of 30% (v/v) of organic solvents including methanol, 2-propanol, and acetone on lipase activity was investigated by incubating the BTL2 lipase for 1 h at 30 °C in 0.1 M Tris buffer, pH 8.0. Methanol and acetone showed a slight inhibitory effect on the BTL2 lipase activity. The residual lipase activity towards p-nitro-
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Fig. 7. Influence of organic solvents (30% v/v) and detergents (1% w/v) on the lipase activity. Purified lipase was incubated for 30 min at 30 °C in 0.1 M Tris buffer, pH 8.0. 1, 30% (v/v) 2-propanol; 2, 30% (v/v) acetone; 3, 30% (v/v) methanol; 4, control sample; 5, 1% (w/v) Triton X-100; 6, 1% (w/v) sodium cholate; 7, 1% (w/v) Tween 80; 8, 1% (w/v) Tween 20; and 9, 1% (w/v) SDS.
phenyl palmitate in the presence of 30% (v/v) methanol or acetone decreased by 16%, whereas the addition of 30% (v/v) of 2-propanol reduced the BTL2 lipase activity by around 47% (Fig. 7). The effect of 1% (w/v) of detergents including Tween 80, Tween 20, Triton X-100, cholate, and SDS was measured by incubating the BTL2 lipase for 1.5 h at 30 °C in 0.1 M Tris buffer, pH 8.0. The addition of 1% (w/v) of SDS decreased the BTL2 lipase activity drastically, while the residual activity was only 5% of that of the control sample. The addition of 1% (w/v) of Tween 20 or 1% (w/v) of cholate decreased the lipase activity slightly by 7% and 20%, respectively. However, the addition of 1% (w/v) of Triton X-100 and Tween 80 increased the BTL2 lipase activity by around 30% and 6%, respectively.
Discussion Recently, the BTL2 lipase has been expressed in E. coli under the control of the native promoter [8,19] and under the control of the strong temperature inducible promoter kPL . Schmidt-Dannert reported that the BTL2 lipase was expressed at a low level in E. coli with a lipase yield of only 600 U/g wet cells. Probably, the native promoter may be responsible for the relatively low levels of expression of this lipase in E. coli. Rua et al. [20,21] has improved the expression levels of this lipase in E. coli using the strong temperature inducible promoter kPL with a lipase yield of 54,000 U/g wet cells. In both cases, the BTL2 was produced as intracellular protein. Thus, a lot of purification steps have to be done to obtain the purified enzyme. Using P. pastoris to produce the BTL2 lipase, the protein was secreted into
the culture medium. The protein was relatively pure in the culture medium without any purification steps and displayed a molecular weight of around 43 kDa on SDS gel, corresponding to the molecular weight for the mature lipase deduced from sequence calculation (43 kDa). The pure enzyme overexpressed in P. pastoris was characterized and its physicochemical properties were compared to those of the BTL2 lipase previously expressed at low and high levels in E. coli (Table 2). The BTL2 lipase expressed in P. pastoris showed the same amino-terminal sequence as the two lipases expressed in E. coli, except for E and F because of the introduction of EcoRI site. The molecular weight of the expressed in P. pastoris BTL2 was around 43 kDa, a little higher than that overexpressed in E. coli (40 kDa) but much more different from that expressed in E. coli at low level (32 kDa) (Table 2). The difference of 8–11 kDa between the molecular mass of the weakly expressed lipase in E. coli (32 kDa) and those of the overexpressed in E. coli (40 kDa) and P. pastoris (43 kDa) might be attributed to an abnormal migration of the weakly expressed BTL2 lipase during SDS–PAGE due to the presence of 2% (w/ v) octyl glucoside in the samples of pure lipase [8]. The BTL2 lipase expressed at low or high level in E. coli as well as overexpressed in P. pastoris showed no significant difference in the pH stability and pH optimum. The pH stability spectrum of the BTL2 overexpressed in P. pastoris was broader (7.0–11.0) than both the BTL2 lipases expressed in E. coli (9.0–11.0). Most other Bacillus lipases are stable at pH 9.0–11.0 [22–25]. The pH optimum of the overexpressed in P. pastoris BTL2 lipase was slightly lower (pH 7.5) than both BTL2
Table 2 Comparison of the properties of the weakly expressed Bacillus thermocatenulatus BTL2 lipase in E. coli [8], the overexpressed BTL2 lipase in E. coli [21], and the overexpressed BTL2 lipase in Pichia pastoris GS115 Properties
Weakly expressed BTL2 in E. coli
Overexpressed in BTL2 in E. coli
Overexpressed in P. pastoris GS115
Molecular mass (kDa) pI Topt (°C) pHopt Tstab (°C) pHstab Substrate stability Specific activity (U/mg) Detergent stability Organic solvent stability
32
40
43
7.2 60–70 8–9 40 9–11 C4 10,225
7.2 60 8 50 9–11 C4 55,000
n.d. 65 7.5 50 7–11 C4 23,000
High High
High High
High High
BTL2: Bacillus thermocatenulatus lipase 2; Topt , temperature optimum of activity; Tstab , temperature stability after 30 min of incubation; pHopt , pH optimum of activity; pHstab , pH stability; C4, tributyrin.
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lipases expressed in E. coli (pH 8.0–9.0) (Table 2). Most other Bacillus lipases [22,25] and BTL1 lipase from this strain [7] show a maximum activity in a lower pH range of 5.6–8.0 like the BTL2 lipase expressed in P. pastoris. The lipases from B. subtilis [23] and B. pumilus [24] have maximum activity at pH 8.0–10.0 as both the BTL2 lipases expressed in E. coli. Also, there were no significant differences in the thermostability between the BTL2 lipases expressed at low and high levels in E. coli as well as overexpressed in P. pastoris. The temperature stability of the overexpressed BTL2 lipase in P. pastoris and in E. coli was the same (50 °C), a little higher than that of the BTL2 expressed at low level (40 °C). The temperature optimum of the overexpressed in P. pastoris lipase towards tributyrin (65 °C) and triolein (75 °C) was slightly higher than those expressed in E. coli (60 and 70 °C, respectively) (Table 2). High thermostability was one characteristic property of most Bacillus and Pseudomonas lipases [7,22,23,25–29]. The specific activity of three BTL2 lipases towards tributyrin was very different. That of the overexpressed lipase in E. coli (54,887 U/mg) was approximately 5- and 2-fold higher than that of the previously expressed BTL2 (10,225 U/mg) and that of the overexpressed in P. pastoris (23,000 U/mg), respectively. Rua et al. [20,21] assumed that the discrepancy between the two lipases expressed in E. coli might be due to a conformational difference. Another explanation might be that the enzyme expressed in E. coli at low levels was not fully active. Why is there a difference in the specific activity between the BTL2 lipase expressed in E. coli and P. pastoris. Due to postmodification of proteins expressed in P. pastoris, the BTL2 lipase expressed in P. pastoris might be glycosylated; hence, the specific activity might be affected negatively. Another reason might be due to the degradation of the BTL2 lipase expressed in P. pastoris. All three lipases expressed in E. coli as well as in P. pastoris were specific for tributyrin. However, the relative activities of other triglycerides were more or less different. The effect of detergents on the lipase activity was somehow different. While the overexpressed BTL2 lipase in P. pastoris as well as in E. coli was activated by Triton X-100, the low-level expressed BTL2 in E. coli was inhibited. After incubating the lipase mixture for 1.5 h and at 30 °C with 1% (w/v) of Tween 80, both the BTL2 lipases expressed in E. coli were completely inactivated whereas the activity of the overexpressed in P. pastoris BTL2 lipase increased slightly by 6%. The addition of Tween 20 to the reaction mixture inactivated the overexpressed BTL2 lipase in E. coli but activated the lowlevel expressed lipase in E. coli. For the BTL2 lipase overexpressed in P. pastoris, the activity remained at 93% after treatment with 1% (w/v) of Tween 20 at 30 °C
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for 30 min. The behavior of all three BTL2 lipases expressed in E. coli as well as in P. pastoris to SDS was the same. They were most completely inhibited by 1% (w/v) of SDS. There was a significant difference of the effect of cholate on the activity of all three lipases. The activity after treatment with 1% (w/v) of cholate was increased to 80% for lipases expressed in E. coli whereas it slightly decreased for the lipase expressed in P. pastoris. All three lipases exhibited the same behavior to organic solvents in the reaction mixture. The addition of 30% (v/v) methanol, 30% (v/v) of acetone decreased the activity by around 20% for all three lipases expressed in E. coli as well as in P. pastoris. The addition of 30% (v/v) 2-propanol decreased the activity of the low-level expressed in E. coli, the overexpressed in E. coli and the overexpressed in P. pastoris slightly (5%), 20% and 47%, respectively.
Acknowledgments Dinh Thi Quyen gratefully acknowledges a scholarship from the Konrad Adenauer Foundation, Germany.
References [1] H.L. Brockman, General features of lipolysis: reaction scheme, interfacial structure and experimental approaches, in: B. Borgstr€ om, H.L. Brockman (Eds.), Lipases, Elsevier, Amsterdam, 1984, pp. 3–46. [2] J. K€ otting, H. Eibl, Lipases and phospholipases in organic synthesis, in: P. Woolley, S.B. Petersen (Eds.), Lipases, Their Structure, Biochemistry and Application, Cambridge University Press, Cambridge, 1994, pp. 289–314. [3] E. Santaniello, P. Ferraboschi, P. Grisenti, A. Manzocchi, The biocatalytic approach to the preparation of enantiometrically pure chiral building blocks, Chem. Rev. 92 (1992) 1071–1140. [4] E.N. Vulfson, Industrial applications of lipases, in: P. Woolley, S.B. Petersen (Eds.), Lipases, Their Structure, Biochemistry and Application, Cambridge University Press, Cambridge, 1994, pp. 271–288. [5] U. Bornscheuer, O.-W. Reif, R. Lausch, R. Freitag, T. Scheper, F.N. Kolisis, U. Menge, Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization, Biochim. Biophys. Acta 1201 (1994) 55–60. [6] K.-E. Jaeger, S. Ransac, B.W. Dijkstra, C. Colson, M.v. Heuvel, O. Misset, Bacterial lipases, FEMS Microbiol. Rev. 15 (1994) 29– 63. [7] C. Schmidt-Dannert, H. Sztajer, W. St€ ocklein, U. Menge, R.D. Schmid, Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus, Biochim. Biophys. Acta 1214 (1994) 43–53. [8] C. Schmidt-Dannert, M.L. Rua, H. Atomi, R.D. Schmid, Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. Molecular, nucleotide sequence, purification and some properties, Biochim. Biophys. Acta 1301 (1996) 105–114. [9] M.L. Rua, H. Atomi, C. Schmidt-Dannert, R.D. Schmid, Highlevel expression of the thermoalkalophilic lipase from Bacillus thermocatenulatus in Escherichia coli, Appl. Microbiol. Biotechnol 56 (1998) 405–410.
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[10] S. Brocca, C. Schmidt-Dannert, M. Lotti, L. Alberghina, R.D. Schmid, Functional overexpression of a 1647 synthetic gene coding for Candida rugosa LIP1, a major industrial lipase, Protein Sci. 7 (1998) 1–8. [11] E. Catoni, C. Schmidt-Dannert, R.D. Schmid, Overexpression of lipase A and B of Geotrichum candidum in Pichia pastoris: highlevel production and some properties of functional expressed lipase B, Biotechnol. Techniques 9 (1997) 689–695. [12] M. Holmquist, D.C. Tessier, M. Cycler, High-level production of recombinant Geotrichum candidum lipases in yeast Pichia pastoris, Protein Expr. Purif. 11 (1997) 35–40. [13] S. Minning, C. Schmidt-Dannert, R.D. Schmid, Functional expression of Rhizopus oryzae lipase in Pichia pastoris: high-level production and some properties, J. Biotech. 66 (1998) 147–156. [14] S. Minning, A. Serrano, P. Ferrer, C. Sola, R.D. Schmid, F. Valero, Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris, J. Biotech. 86 (2001) 59–70. [15] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. [16] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New York, 1993. [17] U.K. Laemmli, Cleavage of structure proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [18] L.A. Butcher, J.K. Tomkins, A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing, Anal. Biochem. 148 (1986) 384–388. [19] C. Schmidt-Dannert, M.L. Rua, R.D. Schmid, Two novel lipase from thermophile Bacillus thermocatenulatus: screening, purification, cloning, overexpression, and properties, Methods Enzymol. 1396 (1997) 194–220.
[20] M.L. Rua, C. Schmidt-Dannert, S. Wahl, A. Sprauer, R.D. Schmid, Thermoalkalophilic lipase of Bacillus thermocatenulatus. Large-scale production, purification and properties: aggregation behaviour and its effect on activity, J. Biotech. 56 (1997) 89–102. [21] M.L. Rua, C. Schmidt-Dannert, S. Wahl, A. Sprauer, R.D. Schmid, Bacillus thermocatenulatus lipase: a thermoalkalophilic lipase with interesting properties, Biochem. Soc. Trans. 56 (1997) 178–182. [22] H.-K. Kim, M.-H. Sung, H.-M. Kim, T.-K. Oh, Occurence of thermostable lipase in thermophilic Bacillus sp. strain 398, Biosci. Biotech. Biochem. 58 (1994) 961–962. [23] E. Lesuisse, K. Schanck, C. Colson, Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme, Eur. J. Biochem. 216 (1993) 155–160. [24] B. Moeller, R. Vetter, D. Wilke, B. Foullois, Alkaline lipases from Bacillus and the cloning of their genes, Patent: Ger. Offen. DE 4,111,321, 1991. [25] A. Sugihara, T. Tani, Y. Tominaga, Purification and characterisation of a novel thermostable lipase from Bacillus sp, J. Biochem. 109 (1991) 211–216. [26] E.J. Gilbert, A. Cornish, C.W. Jones, Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF 2, J. Gen. Mirob. 137 (1991) 2223–2229. [27] A. Makhzoum, R.K. Owusu, J.S. Knapp, The conformational stability of a lipase from a psychrotrophic Pseudomonas fluorescens, Food Chem. 46 (1993) 355–359. [28] Y. Shabati, N. Daya-Mishne, Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments, Appl. Environ. Microb. 58 (1992) 174–180. [29] A. Sugihara, M. Ueshima, Y. Shimada, S. Tsunasawa, Y. Tominaga, Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia, J. Biochem. 112 (1992) 598–603.
High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase Dinh Thi Quyen,a,* Claudia Schmidt-Dannert,b and Rolf D. Schmidc a
b
National Center of Natural Science and Technology, Institute of Biotechnology, 18 Hoang Quoc Viet Road, Nghiado, Distr. Caugiay, 10600 Hanoi, Viet Nam California Institute of Technology, Chemical Engineering 210-41, 120 E. California Blvd., Pasadena, CA 91125, USA c Institut f€ur Technische Biochemie, Universit€at Stuttgart, Allmandring 31, Stuttgart, Germany Received 5 August 2002, and in revised form 6 November 2002
Abstract The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZaA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPa-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4 L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000 U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000 U/mg protein towards tributyrin. The pure enzyme was characterized and its physicochemical properties were compared to those of the BTL2 lipase, which had previously been expressed in Escherichia coli under the control of its native promoter on pUC18 or under the control of the strong temperature inducible promoter kPL , yielding 600 U/g or 54,000 U/ g wet cells, respectively. The three proteins showed the same N-terminal sequence and had very similar pH optimum, pH stability, temperature optimum, thermostability, and substrate specificity profiles. Three enzymes were extremely stable in the presence of several organic solvents and detergents. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Lipase; Expression; Bacillus thermocatenulatus; Pichia pastoris; Properties
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis of triglycerides at the interface between the insoluble substrate and water [1] and have been utilized widely in hydrolysis and transesterification of triglycerides and enantioselective synthesis and hydrolysis of a variety of esters [2–4]. Lipases from thermophiles often show their extreme stability at elevated temperatures and in organic solvents [5]. Thus, they have become objects of special interest for structural investigations and also for industrial applications [6].
* Corresponding author. Fax: +84-4-8363144. E-mail address: [email protected] (D.T. Quyen).
The thermophile Bacillus thermocatenulatus produces two lipases, named BTL1 and BTL2 [7]. The gene of the BTL2 lipase has been cloned and expressed in Escherichia coli under the control of the native promoter [8] or under the control of the strong temperature inducible kPL promoter [9]. The lipase was overexpressed in the E. coli vector pCYTEXP1. However, the BTL2 lipase was expressed in E. coli as intracellular protein which demands a lot of purification steps. Recently, many lipases have been overexpressed as extracellular protein in Pichia pastoris [10–14]. In this paper, we report cloning of the BTL2 lipase gene in the yeast P. pastoris GS115, high-level expression of the lipase as extracellular protein in large quantities, characterization, and comparison of the physicochemical properties of the BTL2 lipases expressed both in E. coli as well as in P. pastoris.
1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-5928(02)00679-4
D.T. Quyen et al. / Protein Expression and Purification 28 (2003) 102–110
Materials and methods Materials The BTL2 lipase gene from B. thermocatenulatus (GenBank X95309) was isolated from the plasmid pT1BTL2 that was described by Rua et al. [9]. The plasmid pPICZaA (Fig. 1a) and yeast strain P. pastoris GS115 were purchased from Invitrogen. E. coli DH5a (supE44 DlacU169 [U80lacZDM15] hsdR17 recA1 endA1 gyrA 96 thi-1 relA1) was used for subcloning. The triacylglycerols tributyrin, tricaprin, trilaurin, trimyristin, tripalmitin, and triolein were purchased from Fluka (Diesenhofen, Germany). Triacetin, tricaproin, tricaprylin, and nitrophenyl palmitate were supplied by Sigma (Deisenhofen, Germany). The detergents Triton X-100 and sodium dodecyl sulfate (SDS) were from Fluka (Deisenhofen, Germany), sodium cholate was from Sigma (Deisenhofen, Germany), and Tween 20 and Tween 80 were from Riedel-de Haen (Hannover, Germany). Bacto-tryptone, yeast extract, and yeast nitrogen bases were from Difco (Ausburg, Germany). a-Naphthyl acetate, glucose, and gum arabic were from Fluka (Deisenhofen, Germany), and Fast Red TR-salt was from Serva (Heidelberg, Germany). All other reagents were of analytical grade unless otherwise stated. Plasmid construction, subcloning, and transformation The BTL2 lipase operon of 1.2 kb from B. thermocatenulatus was amplified from the expression vector in E. coli pT1-BTL2 [9] by PCR with two primers 50 -GAG ACT GAA TTC GCC GAC AAC TAC GCG-30 and 50 GGC GGA TCC TAA GTA AGT AGA ATT CTG AGT AGG-30 introducing EcoRI sites lying immedi-
103
ately upstream of the start codon ATG and downstream of the stop codon. It was followed by the ligation of the EcoRI digested PCR products with pPICZaA linearized by the same enzyme, resulting in pPa-BTL2 (Fig. 1b) under the control of the alcohol oxidase promoter AOX1 induced by methanol and possessing the Zeocin marker. The plasmid pPa-BTL2 was subcloned in E. coli DH5a and the recombinant gene was confirmed by the fluorescence-based dideoxy DNA cycle sequencing method. DNA sequencing was performed with the Taq Dye Deoxy Cycle Sequencing Kit (Applied Biosystems) and with a 373A DNA Sequencing System (Applied Biosystems) according to manufacturerÕs instructions. Other standard recombinant DNA techniques were carried out as described by Sambrook et al. [15] and Ausubel et al. [16]. The PmeI linearized plasmid pPa-BTL2 was introduced into the yeast P. pastoris GS115 by electroporation according to manufacturerÕs recommendations. Gene expression and fermentation The P. pastoris GS115 pPa-BTL2 was cultivated in 50 mL BMGY medium (1% (w/v) of yeast extract, 2% (w/v) of bacto-peptone, 100 mM potassium phosphate, pH 7.0, 4 105 % (w/v) of biotin, and 1% (v/v) of glycerol) for 24 h, at 30 °C and agitated at 220 rpm. After 24 h of cultivation, the culture was centrifuged and the cell pellet was transferred into the BMMY medium, which contains the same components as the BMGY medium except for 0.5% (v/v) of methanol instead of 1% (v/v) of glycerol. The culture was cultivated at 30 °C, 220 rpm. As much as 0.5% (v/v) of methanol was added to the culture every 24 h and 2 mL of the culture was
Fig. 1. Expression vectors pPICZaA (a) and pPa-BTL2 (b) in P. pastoris.
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taken to determine the cell growth and the lipase activity. To produce large quantities of lipase, the P. pastoris GS115 harboring the plasmid pPa-BTL2 was grown in a 5-L bioreactor (Infors, Switzerland). Forty mL culture (in the BMGY medium at 30 °C, 220 rpm for 24 h) was inoculated in 400 mL BMGY medium and grown under the same cultivation conditions. After 24 h of growth, cells were harvested by centrifugation, resuspended in 100 mL BMMY medium, and inoculated in 4 L BMMY medium in 5-L bioreactor. The culture was grown at 30 °C, aerated at the rate of 40% pO2 and agitated at 500 200 rpm. Methanol was added continuously to the culture at the rate of 917 lL of 100% methanol per hour ( 22 mL/day) to induce lipase production. Culture samples were collected at different times to estimate the lipase activity and cell growth. After 120 h of cultivation, the crude lipase solution was harvested by centrifugation and concentrated to a volume of 350 mL by cross-flow filtration with the membrane of 10 kDa nominal MW cutoff.
maximum speed, as described by Schmidt-Dannert et al. [7]. A 20 mL volume of the triglyceride solution was heated to 65 °C and adjusted to pH 8.5. Autohydrolysis was measured in 10 min without addition of enzyme. After addition of 5–50 lL of the lipase solution, the activity was measured with a pH-stat (Metrohm) for 10 min. One unit was defined as the amount of enzyme, which released 1 lmol of fatty acid per minute. Gel electrophoresis SDS–PAGE was carried out, as described by Laemmli [17] with Biometra and Bio-Rad equipment. SDS– PAGE was usually performed with gels of 12.5% (w/v) of acrylamide according to manufacturerÕs recommendations. Gels were stained for protein detection by a silver stain procedure described by Butcher and Tomkins [18]. Detection of hydrolytic activity with a-naphthyl acetate as the substrate was performed by coupling with Fast Red TR salt according to Schmidt-Dannert et al. [8] after renaturation of the protein in 0.1 mM Tris buffer, pH 7.5.
Enzyme purification N-terminal sequence analysis Twenty mL concentrate of the crude enzyme containing a total activity of 51,900 U was loaded on a butyl-Sepharose (Sigma) column (2.6 cm 32 cm with 100 mL packed resin) equilibrated with 50 mM Tris buffer (pH 8.0) containing 0.1 M NaCl at a flow rate of 1 mL/min. Afterwards, the column was washed with the equilibration buffer until no further protein was eluted. The BTL2 lipase bound on the butyl-Sepharose column was then eluted with 1% (w/v) cholic acid in 50 mM Tris buffer (pH 8.0). Fractions with activity were pooled and concentrated by ultrafiltration through a 10 kDa membrane (Filtron) by a factor of up to 10. Enzyme assays Spectrometric assay The spectrophotometric assay was carried out using p-nitrophenyl palmitate (pNPP) as the substrate in a Ultrospec 3000 spectrophotometer (Pharmacia). Cleavage of pNPP was measured at 60 °C using 0.1 M Tris buffer (pH 7.5) according to Schmidt-Dannert et al. [7]. One unit was defined as the amount of enzyme, which cleaved 1 lmol pNPP to p-nitrophenol and palmitate per minute under the assay conditions. pH-stat assay Lipase activity was estimated using triglycerides (C4– C18) as substrate in a pH-stat (Metrohm). Ten mM triglycerides were emulsified in distilled water containing 2% (w/v) of gum arabic as stabilizer using a homogenizer (Ultraturrax T25, Janke and Kunkel) for 5 min at
Purified proteins were applied to a SDS gel. After blotting, the polyvinylidene difluoride membranes were stained with Coomassie brilliant blue R-250, the protein bands of interest were cut out and used for aminoterminal sequence determination. Amino-terminal sequence analysis was performed in a gas-phase sequencer 470A (Applied Biosystems) following manufacturerÕs recommendations. Effect of pH, temperature, organic solvents, and detergents on lipase activity and stability The optimum pH for the enzyme activity was measured at 60 °C by pH-stat assay using tributyrin and triolein as substrates. The assays were carried out by incubation of the reaction mixtures at 60 °C at various pH values from 5.0 to 10.0. The effect of pH on the lipase stability was determined by incubating aliquots of pure lipase solutions for 14 h at 30 °C in 0.1 M (glycine/ HCl, Tris/HCl or glycine/NaOH) buffers at different pH values. Residual activity was measured by photometric assay using pNPP as substrate. The optimum temperature for the enzyme activity was measured at pH 8.0 with tributyrin and triolein as substrates. The assay was carried out by incubating the reaction mixture at various temperatures from 35 to 80 °C. The effect of temperature on the lipase stability was determined by incubating aliquots of pure lipase solution for 30 min in 0.1 M Tris buffer, pH 8.0, at various temperatures from 30 to 80 °C. Residual activity was determined by photometric assay using pNPP as the substrate.
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The effect of detergents on the lipase activity was determined by incubating the enzyme for 1.5 h at 30 °C in 0.1 M Tris buffer (pH 8.0) containing 1% (w/v) of detergents. The lipase activity was measured only at the end of the incubation time by photometric assay with pNPP as substrate. The effect of 30% (v/v) of organic solvent on the lipase activity was determined in a similar way as that of detergents. The reaction mixtures were incubated for 1.5 h at 30 °C; the control contained no organic solvent. All measurements were carried out three times and from these values the average value was taken. Determination of substrate specificity To determine the substrate specificity of the BTL2 lipase, the triglycerides triacetin (C2), tributyrin (C4), tricaproin (C6), tricaprylin (C8), tricaprin (C10), trilaurin (C12), trimyristin (C14), tripalmitin (C16), and tristearin (C18) at a final concentration of 10 mM were each emulsified in distilled water containing 2% (w/v) of gum arabic and used as substrate solution. The lipase activity of the purified enzyme towards triacylglycerides of fatty acids of different chain lengths was determined by pH-stat assay at 65 °C and two different pHs (7.5 and 8.5).
Results Plasmid construction The BTL2 lipase gene was removed from the plasmid pT1-BTL2 by PCR with two flanking primers complementary to the 50 -end and reverse complementary to the 30 -end of the lipase gene and containing EcoRI restriction site. The PCR product was digested with EcoRI and inserted into the vector pPICZaA that was also digested with this enzyme, resulting in the plasmid pPa-BTL2 (Fig. 1b). After subcloning in E. coli DH5a and confirming by DNA sequencing, the plasmid pPa-BTL2 linearized with PmeI was transformed in P. pastoris GS115. On the plate containing 1% (w/v) of tributyrin and 50 lg/mL Zeocin, many colonies showed a halo ring. Transformed colonies were confirmed by the PCR. Lipase expression P. pastoris GS115 harboring the PmeI linearized plasmid pPa-BTL2 as integrating in its genomic DNA was cultivated in 2-L shaking flask containing 400 mL BMMY medium. At different times, samples were taken from the culture and assayed for cell growth (OD600 ) and for lipase activity by using p-nitrophenyl palmitate as substrate (data not shown). The activity reached a maximum of 406 U/mL supernatant after 120 h of
Fig. 2. Cell growth ðjÞ and lipase activity ðdÞ of the BTL2 expressed in P. pastoris. Cultivation was performed, as described in the text. Lipase activity was measured with p-nitrophenyl palmitate (pNPP) as substrate at 60 °C, pH 7.5.
cultivation. From this result, the strain was cultivated further in a 5-L bioreactor filled with 4 L of the BMMY medium. A profile of cell growth and lipase activity was studied at different times, as shown in Fig. 2. P. pastoris GS115 cells were grown at 30 °C for 120 h. The cell growth OD600 increased gradually from 25 at 24 h to 42 at 120 h whereas the lipase activity increased steeply from 56 U/mL supernatant at 24 h to 309 U/mL supernatant as the maximum at 96 h of cultivation and decreased to 209 U/mL supernatant at 120 h of cultivation. This activity profile was a little different from that of shaking flask culture (data not shown). Protein purification and sequencing Twenty mL concentrate of the crude enzyme of total activity of 51,800 U with a specific activity of 35 U/mg was loaded on a butyl-Sepharose column and 91% of the loaded lipase activity was adsorbed to the matrix. By using 1% (w/v) cholic acid in 50 mM Tris buffer, pH 8.0, only 9500 U (18%) of the bound lipase activity was eluted from the column. The enzyme was purified 660fold with a specific activity of 23,000 U/mg (Table 1). This fraction was studied for the protein sequencing and characterization of the BTL2 lipase expressed in P. pastoris. The crude enzyme solution and the purified lipase on SDS–PAGE showed a protein band, corresponding to the lipase molecular weight (43 kDa) (Fig. 3). Purified protein of 1 mL lipase solution was precipitated using trichloroacetic acid and subjected to SDS– PAGE. After blotting, the protein band was used for amino-terminal sequence determination. The N-terminal sequence was E–F–A–S–P–R–A–N–D. The N-terminal fragment of the deduced BTL2 lipase sequence was M–A– S–P–R–A–N–D. The extra amino acids E and F were the result of introduction of EcoRI site (GAATTC).
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Table 1 Summary of the purification of recombinant lipase BTL2 expressed with Pichia pastoris pPa-BTL2 Step Crude enzyme Butyl-Sepharose a b
Total units (U)a 51,800 9500
Specific activity (U/mg)b 35 23,000
Purification factor
Yield (%)
1 660
100 18
From 180 mL of culture supernatant. Specific activity is given as units of lipase towards tributyrin per milligram of lipase.
Fig. 3. SDS–PAGE of the BTL2 lipase before and after purification. Lane M, molecular mass standards indicated in kDa; lane 1, sample of the supernatant after 120 h of cultivation; lane 2, sample of the purified lipase after butyl-Sepharose.
Effect of pH and temperature on lipase activity and stability The optimum activity of the BTL2 lipase was investigated at 60 °C using tributyrin and triolein as substrates (Fig. 4a) with a pH range from 5.0 to 9.0 and from 7.5 to 10.0, respectively. The lipase showed maximum activity at pH 7.5 and pH 9.0 with tributyrin and triolein, respectively. With tributyrin, the relative lipase
activity increased from 34% at pH 5.0 steeply to 71% at pH 6.0, and then slowly to 80% at pH 7.0 and the maximum (100%) at pH 7.5. It decreased slowly to 96% at pH 8.0, 86% at pH 8.5, and remained 80% at pH 9.0. With triolein the relative activity increased as well as decreased steeply around pH 9.0. It increased from 20% at pH 7.5 to 65% at pH 8.0, to 86% at pH 8.5, and to the maximum (100%) at pH 9.0 and then decreased to 80% at pH 9.5 and 58% at pH 10.0. At pH 7.0, no lipase activity with triolein was observed whereas 80% residual activity was still detected with tributyrin. Different pH values of incubation buffers of 0.1 M (glycine/HCl, pH 4.0–6.0, Tris/HCl, pH 7.0–9.0 or glycine/NaOH, pH 9.0–12.0) showed an obvious effect on the lipase stability with p-nitrophenyl palmitate as substrate after 14 h of incubation at 30 °C. The BTL2 lipase stability was low at the pH range from 4.0 to 6.0 (30– 80%) or from pH 12.0 (55%). However, the BTL2 lipase showed a high relatively constant stability within the broad pH range from 7.0 to 11.0 after 14 h incubation at 30 °C (Fig. 4b). Within this pH range from 7.0 to 11.0, the relative residual activity remained at least 90% (pH 10.0) and showed the maximum of 100% at pH 8.0. The optimum temperature of the BTL2 lipase was also investigated using tributyrin and triolein as substrates at a constant pH of 8.0. The maximum temperature was 65 and 75 °C for hydrolysis of tributyrin and triolein, respectively (Fig. 5a). With tributyrin, the rel-
Fig. 4. Effect of pH on lipase activity (a) and stability (b). (a) Effect of pH on lipase activity was determined by the pH-stat assay with tributyrin ðjÞ and triolein ðdÞ. (b) Purified lipase was incubated for 14 h at 30 °C in various 0.1 M buffers: ðjÞ, and glycine/HCl buffer; ðdÞ, Tris/HCl buffer; ðNÞ, glycine/NaOH buffer. The residual activity was measured at 65 °C with the photometric assay using pNPP as substrate.
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Fig. 5. Effect of temperature on lipase activity (a) and stability (b). (a) Effect of temperature on lipase activity was determined by the pH-stat assay with tributyrin ðjÞ and triolein ðdÞ as substrate. (b) Purified lipase was incubated for 30 min at pH 9.0. The residual activity was measured with the photometric assay using pNPP as substrate ðjÞ.
ative lipase activity increased very gradually from 34% at 35 °C to the maximum of 100% at 65 °C and then steeply decreased to 48% at 75 °C. With triolein, the profile of the relative lipase activity was similar to that with tributyrin. The lipase activity increased very gradually from 12% at 35 °C to the maximum of 100% at 75 °C and then decreased to 65% at 80 °C. Below the optimum temperature, the lipase activity decreased very steeply. The various incubation temperatures from 35 to 80 °C showed an obvious temperature effect on the lipase stability with pNPP as the substrate incubated in 0.1 M Tris buffer, pH 8.0, for 30 min. The relative residual lipase activity is disproportional to the incubation temperature. The relative BTL2 lipase activity was fixed as 100% at the temperature of 40 °C and increased to 120% when incubated at the lower temperature of 30 °C. The BTL2 lipase was stable up to 50 °C when incubated for 30 min at pH 8.0 in 0.1 M Tris buffer. Above this temperature, the residual activity decreased steeply to 18% at 70 °C. At 80 °C most of the lipase was inactivated and the lipase activity remained only 5% (Fig. 5b). Substrate specificity Among triglycerides, the BTL2 lipase showed the highest activity towards tributyrin (C4 acyl group) at both pH 7.5 and 8.5. The highest activity towards tributyrin at pH 8.5 was fixed as 100%. The typical profile of chain length specificity of this lipase is shown in Fig. 6. The substrate specificity profiles at pH 7.5 and 8.5 were very similar; however, there was a little difference between these profiles at pH 7.5 and 8.5. For most triglycerides, the lipase activity at pH 8.5 was higher (1– 3%) than that at pH 7.5, except for three substrates triacetin, tricaproin, and trimyristin (Fig. 6). The activity towards tricaprylin (C8 acyl group) relative to
Fig. 6. Substrate specificity at pH 7.5 and 8.5. Activity of the purified lipase towards triacylglycerols with various chain lengths of the acyl group was determined by the pH-stat assay at 65 °C and pH 7.5 (filled column) or pH 8.5 (slightly filled column).
tributyrin was around 40%. The activity of the BTL2 lipase towards tricaproin (C6 acyl group), tricaprin (C10 acyl group), and tripalmitin (C16 acyl group) remained around 20% of that towards tributyrin. The relative activity of trilaurin (C12 acyl group), trimyristin (C14 acyl group), and tristearin (C18 acyl group) in comparison to tributyrin was around 10%. Triacetin (C2 acyl group) was the poorest substrate (3–4%) at both pH 7.5 and 8.5. Effect of organic solvents and detergents on lipase activity The effect of 30% (v/v) of organic solvents including methanol, 2-propanol, and acetone on lipase activity was investigated by incubating the BTL2 lipase for 1 h at 30 °C in 0.1 M Tris buffer, pH 8.0. Methanol and acetone showed a slight inhibitory effect on the BTL2 lipase activity. The residual lipase activity towards p-nitro-
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Fig. 7. Influence of organic solvents (30% v/v) and detergents (1% w/v) on the lipase activity. Purified lipase was incubated for 30 min at 30 °C in 0.1 M Tris buffer, pH 8.0. 1, 30% (v/v) 2-propanol; 2, 30% (v/v) acetone; 3, 30% (v/v) methanol; 4, control sample; 5, 1% (w/v) Triton X-100; 6, 1% (w/v) sodium cholate; 7, 1% (w/v) Tween 80; 8, 1% (w/v) Tween 20; and 9, 1% (w/v) SDS.
phenyl palmitate in the presence of 30% (v/v) methanol or acetone decreased by 16%, whereas the addition of 30% (v/v) of 2-propanol reduced the BTL2 lipase activity by around 47% (Fig. 7). The effect of 1% (w/v) of detergents including Tween 80, Tween 20, Triton X-100, cholate, and SDS was measured by incubating the BTL2 lipase for 1.5 h at 30 °C in 0.1 M Tris buffer, pH 8.0. The addition of 1% (w/v) of SDS decreased the BTL2 lipase activity drastically, while the residual activity was only 5% of that of the control sample. The addition of 1% (w/v) of Tween 20 or 1% (w/v) of cholate decreased the lipase activity slightly by 7% and 20%, respectively. However, the addition of 1% (w/v) of Triton X-100 and Tween 80 increased the BTL2 lipase activity by around 30% and 6%, respectively.
Discussion Recently, the BTL2 lipase has been expressed in E. coli under the control of the native promoter [8,19] and under the control of the strong temperature inducible promoter kPL . Schmidt-Dannert reported that the BTL2 lipase was expressed at a low level in E. coli with a lipase yield of only 600 U/g wet cells. Probably, the native promoter may be responsible for the relatively low levels of expression of this lipase in E. coli. Rua et al. [20,21] has improved the expression levels of this lipase in E. coli using the strong temperature inducible promoter kPL with a lipase yield of 54,000 U/g wet cells. In both cases, the BTL2 was produced as intracellular protein. Thus, a lot of purification steps have to be done to obtain the purified enzyme. Using P. pastoris to produce the BTL2 lipase, the protein was secreted into
the culture medium. The protein was relatively pure in the culture medium without any purification steps and displayed a molecular weight of around 43 kDa on SDS gel, corresponding to the molecular weight for the mature lipase deduced from sequence calculation (43 kDa). The pure enzyme overexpressed in P. pastoris was characterized and its physicochemical properties were compared to those of the BTL2 lipase previously expressed at low and high levels in E. coli (Table 2). The BTL2 lipase expressed in P. pastoris showed the same amino-terminal sequence as the two lipases expressed in E. coli, except for E and F because of the introduction of EcoRI site. The molecular weight of the expressed in P. pastoris BTL2 was around 43 kDa, a little higher than that overexpressed in E. coli (40 kDa) but much more different from that expressed in E. coli at low level (32 kDa) (Table 2). The difference of 8–11 kDa between the molecular mass of the weakly expressed lipase in E. coli (32 kDa) and those of the overexpressed in E. coli (40 kDa) and P. pastoris (43 kDa) might be attributed to an abnormal migration of the weakly expressed BTL2 lipase during SDS–PAGE due to the presence of 2% (w/ v) octyl glucoside in the samples of pure lipase [8]. The BTL2 lipase expressed at low or high level in E. coli as well as overexpressed in P. pastoris showed no significant difference in the pH stability and pH optimum. The pH stability spectrum of the BTL2 overexpressed in P. pastoris was broader (7.0–11.0) than both the BTL2 lipases expressed in E. coli (9.0–11.0). Most other Bacillus lipases are stable at pH 9.0–11.0 [22–25]. The pH optimum of the overexpressed in P. pastoris BTL2 lipase was slightly lower (pH 7.5) than both BTL2
Table 2 Comparison of the properties of the weakly expressed Bacillus thermocatenulatus BTL2 lipase in E. coli [8], the overexpressed BTL2 lipase in E. coli [21], and the overexpressed BTL2 lipase in Pichia pastoris GS115 Properties
Weakly expressed BTL2 in E. coli
Overexpressed in BTL2 in E. coli
Overexpressed in P. pastoris GS115
Molecular mass (kDa) pI Topt (°C) pHopt Tstab (°C) pHstab Substrate stability Specific activity (U/mg) Detergent stability Organic solvent stability
32
40
43
7.2 60–70 8–9 40 9–11 C4 10,225
7.2 60 8 50 9–11 C4 55,000
n.d. 65 7.5 50 7–11 C4 23,000
High High
High High
High High
BTL2: Bacillus thermocatenulatus lipase 2; Topt , temperature optimum of activity; Tstab , temperature stability after 30 min of incubation; pHopt , pH optimum of activity; pHstab , pH stability; C4, tributyrin.
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lipases expressed in E. coli (pH 8.0–9.0) (Table 2). Most other Bacillus lipases [22,25] and BTL1 lipase from this strain [7] show a maximum activity in a lower pH range of 5.6–8.0 like the BTL2 lipase expressed in P. pastoris. The lipases from B. subtilis [23] and B. pumilus [24] have maximum activity at pH 8.0–10.0 as both the BTL2 lipases expressed in E. coli. Also, there were no significant differences in the thermostability between the BTL2 lipases expressed at low and high levels in E. coli as well as overexpressed in P. pastoris. The temperature stability of the overexpressed BTL2 lipase in P. pastoris and in E. coli was the same (50 °C), a little higher than that of the BTL2 expressed at low level (40 °C). The temperature optimum of the overexpressed in P. pastoris lipase towards tributyrin (65 °C) and triolein (75 °C) was slightly higher than those expressed in E. coli (60 and 70 °C, respectively) (Table 2). High thermostability was one characteristic property of most Bacillus and Pseudomonas lipases [7,22,23,25–29]. The specific activity of three BTL2 lipases towards tributyrin was very different. That of the overexpressed lipase in E. coli (54,887 U/mg) was approximately 5- and 2-fold higher than that of the previously expressed BTL2 (10,225 U/mg) and that of the overexpressed in P. pastoris (23,000 U/mg), respectively. Rua et al. [20,21] assumed that the discrepancy between the two lipases expressed in E. coli might be due to a conformational difference. Another explanation might be that the enzyme expressed in E. coli at low levels was not fully active. Why is there a difference in the specific activity between the BTL2 lipase expressed in E. coli and P. pastoris. Due to postmodification of proteins expressed in P. pastoris, the BTL2 lipase expressed in P. pastoris might be glycosylated; hence, the specific activity might be affected negatively. Another reason might be due to the degradation of the BTL2 lipase expressed in P. pastoris. All three lipases expressed in E. coli as well as in P. pastoris were specific for tributyrin. However, the relative activities of other triglycerides were more or less different. The effect of detergents on the lipase activity was somehow different. While the overexpressed BTL2 lipase in P. pastoris as well as in E. coli was activated by Triton X-100, the low-level expressed BTL2 in E. coli was inhibited. After incubating the lipase mixture for 1.5 h and at 30 °C with 1% (w/v) of Tween 80, both the BTL2 lipases expressed in E. coli were completely inactivated whereas the activity of the overexpressed in P. pastoris BTL2 lipase increased slightly by 6%. The addition of Tween 20 to the reaction mixture inactivated the overexpressed BTL2 lipase in E. coli but activated the lowlevel expressed lipase in E. coli. For the BTL2 lipase overexpressed in P. pastoris, the activity remained at 93% after treatment with 1% (w/v) of Tween 20 at 30 °C
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for 30 min. The behavior of all three BTL2 lipases expressed in E. coli as well as in P. pastoris to SDS was the same. They were most completely inhibited by 1% (w/v) of SDS. There was a significant difference of the effect of cholate on the activity of all three lipases. The activity after treatment with 1% (w/v) of cholate was increased to 80% for lipases expressed in E. coli whereas it slightly decreased for the lipase expressed in P. pastoris. All three lipases exhibited the same behavior to organic solvents in the reaction mixture. The addition of 30% (v/v) methanol, 30% (v/v) of acetone decreased the activity by around 20% for all three lipases expressed in E. coli as well as in P. pastoris. The addition of 30% (v/v) 2-propanol decreased the activity of the low-level expressed in E. coli, the overexpressed in E. coli and the overexpressed in P. pastoris slightly (5%), 20% and 47%, respectively.
Acknowledgments Dinh Thi Quyen gratefully acknowledges a scholarship from the Konrad Adenauer Foundation, Germany.
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