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Characteristics and vegetable oils degumming of recombinant phospholipase B
Chemical Engineering Journal 237 (2014) 23–28
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Characteristics and vegetable oils degumming of recombinant phospholipase B Shen Huang, Meili Liang 1, Yinghua Xu, Aamir Rasool, Chun Li ⇑ School of Life Science, Beijing Institute of Technology, 100081 Beijing, PR China
h i g h l i g h t s The recombinant phospholipase B displayed 2-fold higher activity compared to native strain. The recombinant enzyme could degum the phosphorous content of vegetable oils <5 mg/kg. Phospholipase B from Pseudomonas fluorescens BIT-18 was overexpressed in Pichia pastoris.
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Article history: Received 22 July 2013 Received in revised form 22 September 2013 Accepted 30 September 2013 Available online 12 October 2013 Keywords: Phospholipase B Vegetable oil degumming Pseudomonas fluorescens BIT-18 Expression
a b s t r a c t Phospholipase B from Pseudomonas fluorescens BIT-18 can cleave acyl chains at the sn-1 and sn-2 positions of a phospholipid and has been successfully used to degum vegetable oils in our previous work. This study focused on the heterologous overexpression of phospholipase B (Pf-PLB-P) in Pichia pastoris to investigate its characteristics and application in degumming vegetable oils. After optimizing the fermentation conditions, the maximum achieved enzyme activity was 65 U/ml, which was twice the enzyme activity of wild-strain P. fluorescens BIT-18. Purified Pf-PLB-P was obtained by ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The kinetic constants Km and Vmax were determined to be 4.75 mM and 98.67 mmol/(L min), respectively. Pf-PLB-P enzyme activity was detected at 25–55 °C and pH 4.5–9.5, and the temperature range was observed to be slightly broadened than that of the wild type. Based on these characteristics, Pf-PLB-P was also successfully used to degum soybean and peanut oils, whose phosphorus contents decreased from 125.1 mg/kg to 4.96 mg/kg and 96 mg/kg to 3.54 mg/kg, respectively. These results indicate that Pf-PLB-P produced by P. pastoris has potential industrial use. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Phospholipase B (PLB) is a heterogeneous group of phospholipases that harbor three distinct activities: a phospholipase A1 or phospholipase A2, a lysophospholipase, and a lysophospholipid– transacylase [1,2]. PLBs are universally found in bacteria [3], fungi [4], plants [5], rat [6], guinea pig [7], and human epidermis [8]. PLB has applications in the food and pharmaceutical industries to produce phospholipid derivatives where phospholipid hydrolysis is needed. Our group has previously reported that PLB from Pseudomonas fluorescens BIT-18 (Pf-PLB) isolated from soil can be used for vegetable oil degumming in which phospholipids are easily hydrolyzed and removed. The phosphorous content is <5 mg/kg after digestion
⇑ Corresponding author. Tel./fax: +86 010 68913171. 1
E-mail address: [email protected] (C. Li). Meili Liang contributed equally to this work.
1385-8947/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cej.2013.09.109
[9,10]. Enzymatic degumming of vegetable oils can replace the conventional method that requires acid, alkali, and soft water [11]. However, the industrial application of Pf-PLB in wild-type P. fluorescens BIT-18 induced by soybean phospholipid is limited by low yields [9]. Thus, the gene of Pf-PLB has been isolated and expressed in Escherichica coli to improve Pf-PLB productivity. Although recombinant PLB has been successfully expressed in E. coli [12], most recombinant enzymes only exist in the form of inclusion bodies. The methylotrophic Pichia pastoris expression system has several advantages over the E. coli expression system, including the use of alcohol oxidase 1 gene (AOX1) promoter, the ability to culture cells at high density, a more simplified purification procedure for secreted heterologous proteins, and the ability to express even highly toxic antimicrobial proteins at a large scale [13]. More importantly, the recombinant proteins expressed by this system are safe for human use. Numerous enzymes in the food industry have already been expressed in P. pastoris, such as a-amylase [14,15], protease [16], glucoamylase [17], chymosin [18], and glucose oxidase [19].
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S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28
In this work, PLB (Pf-PLB-P) was heterologous overexpressed in P. pastoris. After optimizing the fermentation conditions, Pf-PLB-P was then purified, characterized, and successfully used to degum soybean and peanut oils. The results can serve as a foundation for further large-scale production and application of PLB in vegetable oil degumming. 2. Materials and methods 2.1. Strains, vectors, and reagents The P. pastoris host strain GS115 and secretion expression vector pPIC9K were purchased from Invitrogen (CA, USA). E. coli DH5a was used for routine plasmid amplification. All primers were synthesized by BGI (Shenzhen, China). All restriction enzymes, DNA markers, and protein markers were purchased from Takara (Dalian, China). Yeast nitrogen base, yeast extract, and tryptone were obtained from OXOID Ltd. (Basingstoke, England). PCR purification, gel extraction, and miniprep kits for plasmid extraction were obtained from Biomed Corporation (Beijing, China). 2.2. pfplb/pPIC9K construction and transformation The strategy for pfplb/pPIC9K construction and transformation into P. pastoris are depicted in Fig. 1. pfplb DNA was amplified by PCR from a prokaryotic plasmid constructed in our laboratory [12] encoding full-length PLB. The forward and reverse primers were 50 -CCGGAATTCATGAAAAAAGTCATGCTCAA-30 and 50 -ATTTGC GGCCGCTCAGAAGCGGTAGGTCGCGC-30 , with EcoR I and Not I sites underlined, respectively. The PCR products were digested with the restriction enzymes and ligated into EcoR I/Not I digested pPIC9K, which inserted the fragment in-frame to the a-factor secretion
signal downstream of alcohol oxidase I promoter. The resulting plasmid (pfplb/pPIC9K) was transformed into E. coli DH5a. Then, the positive colonies were selected by colony-PCR and sequence analysis. pfplb/pPIC9K purified from the positive colony was linearized with Bgl II and then transformed into P. pastoris GS115 by the PEG method according to the manufacturer’s protocol (Multi-Copy Pichia Expression Kit, Invitrogen). Muts transformants were selected by MD plates. The multi-copy transformants were obtained on YPD plates at different G418 concentrations (0.5, 1.0, 1.5, and 2.0 mg/ml). Integration of pfplb in the recombinant P. pastoris genome was confirmed by genomic PCR. 2.3. Expression and PLB in P. pastoris Protein expression trials were used to identify Pf-PLB-P expression conditions, and analyzed the secretion levels among recombinant P. pastoris clones. The selected strains were grown in 30 ml of YPD medium for approximately 24 h at 28 °C with constant shaking at 170 rpm. These cells were cultured further in 70 ml of BMGY medium (1% yeast extract, 2% tryptone, 1.34% YNB, 4 105% biotin, 1% glycerol, and 100 mM potassium phosphate, pH 6.1) until the culture reached OD600 4–6, whereupon the cells were harvested by centrifugation for 5 min at 5000 g and 4 °C. The supernatant was carefully decanted, and the cell pellet was resuspended in 70 ml of BMMY (1% yeast extract, 2% tryptone, 1.34% YNB, 4 105% biotin, 1% methanol, and 100 mM potassium phosphate, pH 7.0) for methanol induction of protein expression. Samples (3 ml) of the expression medium were collected for expression analysis by SDS–PAGE [20] and enzyme assay. The effects of initial pH (5.5–7.5, 100 mM Na2HPO4–citric acid buffer) in BMMY medium, the inoculum amount of recombinant P. pastoris (1–5%, v/v), and the methanol dosage (0.5–2%, v/v) were determined at 28 °C. The optimum temperature for Pf-PLB-P expression was determined at 20–30 °C and pH 6.0 after methanol addition, and an enzyme producing curve was drawn under the optimum condition. 2.4. Purification of recombinant PLB A maximum PLB activity of 65 U/ml was observed after 36 h of induction. The extracellular enzyme was isolated by centrifuging the fermentation broth at 15,000g and 4 °C for 10 min. The supernatant was brought to 60% saturation with ammonium sulfate, left undisturbed for 2 h, and centrifuged. The precipitate was dissolved in a small volume of 20 mM Tris–HCl buffer (pH 7.3) and dialyzed overnight against the buffer. The crude enzyme solution was passed through a anion-exchange chromatography column (HiPrep EDAE FF 16/10 (USA GE)) with 1 ml/min, and bound protein was eluted with a gradient NaCl (0–0.4 M) with the same flow rate. The fractions containing the highest activities were pooled, concentrated, and passed through a gel filtration column (Superdex™ 75 10/300 GL (USA GE)) equilibrated and eluted with 0.02 M Tris–HCl (pH 7.3) and 0.1 M NaCl buffer with 1 ml/min flow rate. The chromatographic processes were done using the ÄKTA purifier 10 (USA GE). The active fractions were flash frozen by liquid nitrogen and stored at 80 °C. 2.5. Characterization of recombinant phospholipase B
Fig. 1. Schematic of pfplb/pPIC9K construction and transformation into Pichia pastoris GS115.
PLB activity was performed as described by [9]. For kinetic studies of Pf-PLB-P, the initial velocities of the enzymatic reaction were examined by varying the concentration of soybean phospholipids (from 1 g/l to 8 g/l). Values of the Michaelis constants (Km) and maximal velocity (Vmax) were obtained by the Lineweaver–Burk plot. The parameters were determined by three separate
S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28
Fig. 2. Gel electrophoretogram of single and double digested pfplb/pPIC9K (Lane M1: 1 kb DNA ladder; Lane M2:DL2000 marker; Lane 1–2: double digested pfplb/ pPIC9K by Not I and EcoR I; Lane 3–4: single digested pfplb/pPIC9K by EcoR I).
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experiments. Protein concentration was measured using the Bradford assay and bovine serum albumin as a standard [21]. The optimum pH of Pf-PLB-P was investigated from pH 4.0 to 9.0 (100 mM Na2HPO4–citric acid buffer, pH 4.0–7.0; or 100 mM Tris–HCl buffer, pH 7.5–9.0) at 30 °C. The effect of pH on the Pf-PLB-P stability was estimated by measuring the residual enzyme activity after enzyme incubation in the absence of substrate at 4 °C for 12 h. The optimum temperature of Pf-PLB was determined at 25–55 °C and pH 6.5. The effect of temperature on enzyme stability was estimated by measuring the residual enzyme activity after incubation of Pf-PLB-P in the absence of substrate at 25–55 °C for 20 min. 2.6. Enzymatic degumming and phosphorus content analysis Degumming of crude soybean and peanut oil by Pf-PLB-P and Pf-PLB were carried out according to [9]. Phosphorus content was determined by the molybdenum blue method in accordance with GB/T 5537 (National Standard of the People’s Republic China, 2008). 3. Results and discussion 3.1. Expression vector construction and transformation
Fig. 3. SDS–PAGE analysis. Lane 1, concentrated supernatant from the negative stain (Pichia pastoris pPIC9K) after induction by methanol at 48 h. Lanes 2, concentrated supernatant from the positive stain (P. pastoris pfplb/pPIC9K) after induction by methanol at 48 h. Lane M, protein size markers.
A gene (pfplb) (GenBank accession No. CP000094) from P. fluorescens BIT-18 encoding the enzyme with PLB activity reportedly consists of 1272 bp with an open-reading frame encoding a peptide of 423 amino acids [12]. In this work, pfplb was amplified by PCR from pET-28a-pfplb constructed by our laboratory and ligated into pPIC9K downstream of AOX1 promoter (Fig. 1).
Fig. 4. Optimum conditions for the expression of recombinant PLB (Pf-PLB-P). (a) Effects of different level of pH on Pf-PLB-P activity, (b) effects of different temperature on Pf-PLB-P activity, (c) eof different methanol concentration on Pf-PLB-P activity and (d) effects of different inoculum volume on Pf-PLB-P activity.
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reached to maximum was advanced 24 h than that of the wild stain (see Fig. 5). 3.3. Purification of Pf-PLB-P Crude extract was precipitated by 60% saturated ammonium sulfate for Pf-PLB-P purification by anion-exchange chromatography and gel filtration. After the component with PLB activity was applied to a DEAE FF column, the targeted enzyme (Fig. 6c, lane 2) was obtained after elution with 200 mM NaCl (Fig. 6a). The concentrated enzyme solution was then fractionated by gel filtration
Fig. 5. Relationship curve of cell growth and enzyme production (Note: (–j–) curve of cell growth; (–.–) curve of enzyme production).
The resultant construct harbored a single open-reading frame consisting of the a-factor secretion signal peptide and Pf-PLB protein. The integrity of the recombinant was confirmed by direct sequencing. Two additional restriction endonuclease sites Not I and EcoR I were introduced at the 50 and 30 ends of pfplb, respectively. The recombinant expression vectors (pfplb/pPIC9K), which had been experimentally proved to be correct as show in Fig. 2, was linearized with Bgl II and transformed into P. pastoris GS115 competent cells. Many colonies were selected on the MD plates, in which multi-copy transformants were selected with G418 (2 mg/ml). Eventually, one transformant with the highest enzyme activity was selected. The molecular mass of recombinant enzyme expressed in P. pastoris as determined by SDS–PAGE was about 46 kDa (Fig. 3, lane 2), which was similar to native Pf-PLB secreted by P. fluorescens BIT-18 [9]. 3.2. Optimizing of the productivity PLB in P. pastoris A transformant with high PLB-secreting ability was selected to optimize the heterologous expression of Pf-PLB-P under various cultivation conditions, including different pH values, temperatures, inoculation volumes, and methanol concentrations. The highest enzyme activity was observed at pH 6.5, as shown in Fig. 4a, which increased 29% and 31% compared with pH 5.5 and pH 7.5, respectively. Therefore, controlling the medium pH during the fermentation process is necessary, the optimum pH may depends on individual properties of the protein [22]. The highest enzyme activity of 40 U/ml was detected at 26 °C, as shown in Fig. 4b. The temperatures for enzyme induction with methanol in the P. pastoris expression system were different while most other protein expressed at about 30 °C [23–28]. A benefit of lowering the temperature is to reduce the proteolytic degradation of the recombinant protein in the culture medium [22]. This phenomenon may result from the poor stability of the recombinant protein and folding problems at high temperatures [25]. The optimum methanol concentration of 1% (v/v) (Fig. 4c) and optimum inoculum volume of 3% (v/v) (Fig. 4d) were determined by varying the methanol concentration from 0.5% to 2% and inoculum volume from 1% to 5%. Methanol (1%) was added to the culture for inducing enzyme expression when P. pastoris had OD600 4 under the optimum conditions (pH 6.5, 26 °C, 3% inoculation volume). Pf-PLB-P activity was detected 12 h after adding the inducer. Results showed that enzyme activity sharply increased in the logarithmic phase of cell growth with the total enzyme activity reaching the maximum value of 65 U/ml at 36 h, which was twice the value expressed by P. fluorescens BIT-18. In addition, the time that the PLB activity
Fig. 6. Purification of the recombinant PLB (Pf-PLB-P). (a) Purification chromatograph of the Pf-PLB-P by DEAE anion exchange-chromatography. The bound material was eluted with a gradient of sodium chloride (dashed line), (b) purification chromatograph of the Pf-PLB-P by Superdex 75 gel filtration and (c) SDS–PAGE chromatograph (M: protein marker; Lane 1: the recombinant PLB by Superdex 75; Lane 2: the recombinant PLB by DEAE: Lane: the fermentation).
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S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28 Table 1 Purification of recombinant phospholipase B from Pichia pastoris. Purification steps
Total protein (mg)
Total activity (U)
Specific activity (U tmg1)
Recovery (%)
Purification (fold)
The fermentation (NH4)2SO4 precipitation Anion-exchange chromatography Gel filtration
52.4 15.8 9.1 6.3
3461 2815 2448 1855
66 178 268 294
100.00 81.35 70.74 53.60
1.0 2.7 4.1 4.5
chromatography using a Superdex 75 column (Fig. 6b). Highly purified Pf-PLB-P was obtained after these steps as proved by the single band corresponding to a molecular mass around 46 kDa in the SDS–PAGE gel (Fig. 6c, lane 1). The recovery and purification factors of Pf-PLB-P at different purification steps were summarized in Table 1. 3.4. Enzymatic properties The enzymatic properties of Pf-PLB-P are shown in Fig. 7. The optimum reaction temperature was determined to be 30 °C (Fig. 7a), which was similar to that of PLB from native P. fluorescens BIT-18 [9] and the one expressed by E. coli [12]. The optimum reaction pH was 6.5, similar to that of native PLB (pH 6.5) [9,10] but 0.5 higher than that of PLB expressed by E. coli (pH 6.0) [12]. Additionally, >70% of enzyme activity remained with increased pH from 5.0 to 7.5, which was almost the same as that of PLB expressed by E. coli. In conclusion, Pf-PLB-P displayed a slightly broader temperature range for soybean phospholipids hydrolysis than the wild type. Table 2 shows that Km and Vmax of Pf-PLB-P for soybean phospholipids hydrolysis were 4.75 mM and 98.67 mmol/(L min), respectively. kcat was 36.54 s1 and kcat/Km was 7.69 s1/mM. Understanding the enzyme kinetic properties may help understand the catalytic mechanism. 3.5. Application of Pf-PLB-P in degumming vegetable oil
Fig. 7. (a) Effect of pH on the activity (–j–) and stability (–d–) of Pf-PLB-P and (b) effect of temperature on the activity (–N–) and stability (–.–) of Pf-PLB-P.
The phosphorus contents of soybean and peanut oils decreased from 125.1 mg/kg to 4.96 mg/kg and 96.86 mg/kg to 3.54 mg/kg, respectively, within 5 h after applying Pf-PLB-P, as shown in Table 3. This phosphorous level after degumming was acceptable for industrial applications. These results exhibited that Pf-PLB-P had similar degumming characteristics of soybean oil but showed a much higher enzyme productivity of 65 U/ml, which was twice higher than native Pf-PLB [9]. In traditional chemical vegetable oil degumming process; during the non-hydratable phosphatides degumming acid is added at temperatures between 85 and 90 °C and neutralized with base [29,30]. While enzymatic degumming requires enzymes (PLA1,
Table 2 Kinetic parameters of recombinant phospholipase B from Pseudomonas fluorescens BIT-18 expressed in P. pastoris reacting with soybean phospholipids. Substrate Soybean phospholipids
Km (mM) 4.75
Vmax (mmol L1 min1) 98.67
kcat (s1) 36.54
kcat/Km (s1 mM1) 7.69
Each value represents the mean of triplicate experiments.
Table 3 Application of recombinant phospholipase B in both soybean and peanut oil degumming. Enzyme
Phosphorus content before degumming
Phosphorus content after degumming
Pf-PLB
Soybean oil Peanut oil
121.23 ± 11.47 96.86 ± 10.5
4.86 ± 0.62 3.73 ± 0.42
Pf-PLB-P
Soybean oil Peanut oil
121.23 ± 11.47 96.86 ± 10.5
4.96 ± 0.59 3.54 ± 0.41
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PLA2, PLB, etc.) at 40–50 °C to degum the non-hydratable phosphatides of the vegetable oil [31,32]. If we draw a comparison between these two degumming methods the cost of the enzymatic degumming is the cost of the enzyme. Therefore, the cost of the enzymatic degumming could be significantly lowered by constructing a strong PLB expression system and it will provide a cheaper enzyme for industrial applications. The requirement of the small quantity of water, acid and base for enzymatic degumming also make it cost effective and industrially applicable.
4. Conclusions PLB expressed by P. pastoris was successfully produced. After optimization of the fermentation conditions, the enzyme activity was twice that of native PLB from P. fluorescens BIT-18. High-purity PLB was obtained by ammonium sulfate fractionation, anionexchange chromatography and gel filtration successively. Km of the enzyme for soybean phospholipid was 4.75 mM, and Vmax was 98.67 mmol/(L min). The purified enzyme was used to degum soybean and peanut oils, whose phosphorus contents decreased to <5 mg/kg. The development of suitable fermentation strategies, some of which are underway, may enable the large-scale production of Pf-PLB for industrial application. Acknowledgements This work was financially supported by National Science Foundation of China (Nos. 21176028, 21276025, 21376028), Doctoral Fund of Ministry of Education of China (Nos. 20091101110036, 20121101110050), the Major State Basic Research Development Program of China (973 Program) (No. 2013CB733900), and the National High Technology Research and Development Program of China (863 Program) (No. 2012AA02A704). References [1] G.A. Köhler, A. Brenot, E. Haas-Stapleton, N. Agabian, R. Deva, S. Nigam, Phospholipase A2 and phospholipase B activities in fungi, Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1761 (2006) 1391– 1399. [2] M.A. Ghannoum, Potential role of phospholipases in virulence and fungal pathogenesis, Clinical Microbiology Reviews 13 (2000) 122–143. [3] J.L. Farn, R.A. Strugnell, P.A. Hoyne, W.P. Michalski, J.M. Tennent, Molecular characterization of a secreted enzyme with phospholipase B activity from Moraxella bovis, Journal of Bacteriology 183 (2001) 6717–6720. [4] S.C. Chen, L.C. Wright, J.C. Golding, T.C. Sorrell, Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans, Biochemical Journal 347 (2000) 431. [5] D.K. Kim, H.J. Lee, Y. Lee, Detection of two phospholipase A2 (PLA2) activities in leaves of higher plant Vicia faba and comparison with mammalian PLA2’s, FEBS Letters 343 (1994) 213. [6] T. Lu, M. Ito, U. Tchoua, H. Takemori, M. Okamoto, H. Tojo, Identification of essential residues for catalysis of rat intestinal phospholipase B/lipase, Biochemistry 40 (2001) 7133–7139. [7] M. Nauze, L. Gonin, B. Chaminade, C. Perès, F. Hullin Matsuda, B. Perret, H. Chap, A. Gassama Diagne, Guinea pig phospholipase B, identification of the catalytic serine and the proregion involved in its processing and enzymatic activity, Journal of Biological Chemistry 277 (2002) 44093–44099. [8] S. Xu, L. Zhao, A. Larsson, P. Venge, The identification of a phospholipase B precursor in human neutrophils, FEBS Journal 276 (2009) 175–186.
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Characteristics and vegetable oils degumming of recombinant phospholipase B Shen Huang, Meili Liang 1, Yinghua Xu, Aamir Rasool, Chun Li ⇑ School of Life Science, Beijing Institute of Technology, 100081 Beijing, PR China
h i g h l i g h t s The recombinant phospholipase B displayed 2-fold higher activity compared to native strain. The recombinant enzyme could degum the phosphorous content of vegetable oils <5 mg/kg. Phospholipase B from Pseudomonas fluorescens BIT-18 was overexpressed in Pichia pastoris.
a r t i c l e
i n f o
Article history: Received 22 July 2013 Received in revised form 22 September 2013 Accepted 30 September 2013 Available online 12 October 2013 Keywords: Phospholipase B Vegetable oil degumming Pseudomonas fluorescens BIT-18 Expression
a b s t r a c t Phospholipase B from Pseudomonas fluorescens BIT-18 can cleave acyl chains at the sn-1 and sn-2 positions of a phospholipid and has been successfully used to degum vegetable oils in our previous work. This study focused on the heterologous overexpression of phospholipase B (Pf-PLB-P) in Pichia pastoris to investigate its characteristics and application in degumming vegetable oils. After optimizing the fermentation conditions, the maximum achieved enzyme activity was 65 U/ml, which was twice the enzyme activity of wild-strain P. fluorescens BIT-18. Purified Pf-PLB-P was obtained by ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The kinetic constants Km and Vmax were determined to be 4.75 mM and 98.67 mmol/(L min), respectively. Pf-PLB-P enzyme activity was detected at 25–55 °C and pH 4.5–9.5, and the temperature range was observed to be slightly broadened than that of the wild type. Based on these characteristics, Pf-PLB-P was also successfully used to degum soybean and peanut oils, whose phosphorus contents decreased from 125.1 mg/kg to 4.96 mg/kg and 96 mg/kg to 3.54 mg/kg, respectively. These results indicate that Pf-PLB-P produced by P. pastoris has potential industrial use. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Phospholipase B (PLB) is a heterogeneous group of phospholipases that harbor three distinct activities: a phospholipase A1 or phospholipase A2, a lysophospholipase, and a lysophospholipid– transacylase [1,2]. PLBs are universally found in bacteria [3], fungi [4], plants [5], rat [6], guinea pig [7], and human epidermis [8]. PLB has applications in the food and pharmaceutical industries to produce phospholipid derivatives where phospholipid hydrolysis is needed. Our group has previously reported that PLB from Pseudomonas fluorescens BIT-18 (Pf-PLB) isolated from soil can be used for vegetable oil degumming in which phospholipids are easily hydrolyzed and removed. The phosphorous content is <5 mg/kg after digestion
⇑ Corresponding author. Tel./fax: +86 010 68913171. 1
E-mail address: [email protected] (C. Li). Meili Liang contributed equally to this work.
1385-8947/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cej.2013.09.109
[9,10]. Enzymatic degumming of vegetable oils can replace the conventional method that requires acid, alkali, and soft water [11]. However, the industrial application of Pf-PLB in wild-type P. fluorescens BIT-18 induced by soybean phospholipid is limited by low yields [9]. Thus, the gene of Pf-PLB has been isolated and expressed in Escherichica coli to improve Pf-PLB productivity. Although recombinant PLB has been successfully expressed in E. coli [12], most recombinant enzymes only exist in the form of inclusion bodies. The methylotrophic Pichia pastoris expression system has several advantages over the E. coli expression system, including the use of alcohol oxidase 1 gene (AOX1) promoter, the ability to culture cells at high density, a more simplified purification procedure for secreted heterologous proteins, and the ability to express even highly toxic antimicrobial proteins at a large scale [13]. More importantly, the recombinant proteins expressed by this system are safe for human use. Numerous enzymes in the food industry have already been expressed in P. pastoris, such as a-amylase [14,15], protease [16], glucoamylase [17], chymosin [18], and glucose oxidase [19].
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In this work, PLB (Pf-PLB-P) was heterologous overexpressed in P. pastoris. After optimizing the fermentation conditions, Pf-PLB-P was then purified, characterized, and successfully used to degum soybean and peanut oils. The results can serve as a foundation for further large-scale production and application of PLB in vegetable oil degumming. 2. Materials and methods 2.1. Strains, vectors, and reagents The P. pastoris host strain GS115 and secretion expression vector pPIC9K were purchased from Invitrogen (CA, USA). E. coli DH5a was used for routine plasmid amplification. All primers were synthesized by BGI (Shenzhen, China). All restriction enzymes, DNA markers, and protein markers were purchased from Takara (Dalian, China). Yeast nitrogen base, yeast extract, and tryptone were obtained from OXOID Ltd. (Basingstoke, England). PCR purification, gel extraction, and miniprep kits for plasmid extraction were obtained from Biomed Corporation (Beijing, China). 2.2. pfplb/pPIC9K construction and transformation The strategy for pfplb/pPIC9K construction and transformation into P. pastoris are depicted in Fig. 1. pfplb DNA was amplified by PCR from a prokaryotic plasmid constructed in our laboratory [12] encoding full-length PLB. The forward and reverse primers were 50 -CCGGAATTCATGAAAAAAGTCATGCTCAA-30 and 50 -ATTTGC GGCCGCTCAGAAGCGGTAGGTCGCGC-30 , with EcoR I and Not I sites underlined, respectively. The PCR products were digested with the restriction enzymes and ligated into EcoR I/Not I digested pPIC9K, which inserted the fragment in-frame to the a-factor secretion
signal downstream of alcohol oxidase I promoter. The resulting plasmid (pfplb/pPIC9K) was transformed into E. coli DH5a. Then, the positive colonies were selected by colony-PCR and sequence analysis. pfplb/pPIC9K purified from the positive colony was linearized with Bgl II and then transformed into P. pastoris GS115 by the PEG method according to the manufacturer’s protocol (Multi-Copy Pichia Expression Kit, Invitrogen). Muts transformants were selected by MD plates. The multi-copy transformants were obtained on YPD plates at different G418 concentrations (0.5, 1.0, 1.5, and 2.0 mg/ml). Integration of pfplb in the recombinant P. pastoris genome was confirmed by genomic PCR. 2.3. Expression and PLB in P. pastoris Protein expression trials were used to identify Pf-PLB-P expression conditions, and analyzed the secretion levels among recombinant P. pastoris clones. The selected strains were grown in 30 ml of YPD medium for approximately 24 h at 28 °C with constant shaking at 170 rpm. These cells were cultured further in 70 ml of BMGY medium (1% yeast extract, 2% tryptone, 1.34% YNB, 4 105% biotin, 1% glycerol, and 100 mM potassium phosphate, pH 6.1) until the culture reached OD600 4–6, whereupon the cells were harvested by centrifugation for 5 min at 5000 g and 4 °C. The supernatant was carefully decanted, and the cell pellet was resuspended in 70 ml of BMMY (1% yeast extract, 2% tryptone, 1.34% YNB, 4 105% biotin, 1% methanol, and 100 mM potassium phosphate, pH 7.0) for methanol induction of protein expression. Samples (3 ml) of the expression medium were collected for expression analysis by SDS–PAGE [20] and enzyme assay. The effects of initial pH (5.5–7.5, 100 mM Na2HPO4–citric acid buffer) in BMMY medium, the inoculum amount of recombinant P. pastoris (1–5%, v/v), and the methanol dosage (0.5–2%, v/v) were determined at 28 °C. The optimum temperature for Pf-PLB-P expression was determined at 20–30 °C and pH 6.0 after methanol addition, and an enzyme producing curve was drawn under the optimum condition. 2.4. Purification of recombinant PLB A maximum PLB activity of 65 U/ml was observed after 36 h of induction. The extracellular enzyme was isolated by centrifuging the fermentation broth at 15,000g and 4 °C for 10 min. The supernatant was brought to 60% saturation with ammonium sulfate, left undisturbed for 2 h, and centrifuged. The precipitate was dissolved in a small volume of 20 mM Tris–HCl buffer (pH 7.3) and dialyzed overnight against the buffer. The crude enzyme solution was passed through a anion-exchange chromatography column (HiPrep EDAE FF 16/10 (USA GE)) with 1 ml/min, and bound protein was eluted with a gradient NaCl (0–0.4 M) with the same flow rate. The fractions containing the highest activities were pooled, concentrated, and passed through a gel filtration column (Superdex™ 75 10/300 GL (USA GE)) equilibrated and eluted with 0.02 M Tris–HCl (pH 7.3) and 0.1 M NaCl buffer with 1 ml/min flow rate. The chromatographic processes were done using the ÄKTA purifier 10 (USA GE). The active fractions were flash frozen by liquid nitrogen and stored at 80 °C. 2.5. Characterization of recombinant phospholipase B
Fig. 1. Schematic of pfplb/pPIC9K construction and transformation into Pichia pastoris GS115.
PLB activity was performed as described by [9]. For kinetic studies of Pf-PLB-P, the initial velocities of the enzymatic reaction were examined by varying the concentration of soybean phospholipids (from 1 g/l to 8 g/l). Values of the Michaelis constants (Km) and maximal velocity (Vmax) were obtained by the Lineweaver–Burk plot. The parameters were determined by three separate
S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28
Fig. 2. Gel electrophoretogram of single and double digested pfplb/pPIC9K (Lane M1: 1 kb DNA ladder; Lane M2:DL2000 marker; Lane 1–2: double digested pfplb/ pPIC9K by Not I and EcoR I; Lane 3–4: single digested pfplb/pPIC9K by EcoR I).
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experiments. Protein concentration was measured using the Bradford assay and bovine serum albumin as a standard [21]. The optimum pH of Pf-PLB-P was investigated from pH 4.0 to 9.0 (100 mM Na2HPO4–citric acid buffer, pH 4.0–7.0; or 100 mM Tris–HCl buffer, pH 7.5–9.0) at 30 °C. The effect of pH on the Pf-PLB-P stability was estimated by measuring the residual enzyme activity after enzyme incubation in the absence of substrate at 4 °C for 12 h. The optimum temperature of Pf-PLB was determined at 25–55 °C and pH 6.5. The effect of temperature on enzyme stability was estimated by measuring the residual enzyme activity after incubation of Pf-PLB-P in the absence of substrate at 25–55 °C for 20 min. 2.6. Enzymatic degumming and phosphorus content analysis Degumming of crude soybean and peanut oil by Pf-PLB-P and Pf-PLB were carried out according to [9]. Phosphorus content was determined by the molybdenum blue method in accordance with GB/T 5537 (National Standard of the People’s Republic China, 2008). 3. Results and discussion 3.1. Expression vector construction and transformation
Fig. 3. SDS–PAGE analysis. Lane 1, concentrated supernatant from the negative stain (Pichia pastoris pPIC9K) after induction by methanol at 48 h. Lanes 2, concentrated supernatant from the positive stain (P. pastoris pfplb/pPIC9K) after induction by methanol at 48 h. Lane M, protein size markers.
A gene (pfplb) (GenBank accession No. CP000094) from P. fluorescens BIT-18 encoding the enzyme with PLB activity reportedly consists of 1272 bp with an open-reading frame encoding a peptide of 423 amino acids [12]. In this work, pfplb was amplified by PCR from pET-28a-pfplb constructed by our laboratory and ligated into pPIC9K downstream of AOX1 promoter (Fig. 1).
Fig. 4. Optimum conditions for the expression of recombinant PLB (Pf-PLB-P). (a) Effects of different level of pH on Pf-PLB-P activity, (b) effects of different temperature on Pf-PLB-P activity, (c) eof different methanol concentration on Pf-PLB-P activity and (d) effects of different inoculum volume on Pf-PLB-P activity.
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reached to maximum was advanced 24 h than that of the wild stain (see Fig. 5). 3.3. Purification of Pf-PLB-P Crude extract was precipitated by 60% saturated ammonium sulfate for Pf-PLB-P purification by anion-exchange chromatography and gel filtration. After the component with PLB activity was applied to a DEAE FF column, the targeted enzyme (Fig. 6c, lane 2) was obtained after elution with 200 mM NaCl (Fig. 6a). The concentrated enzyme solution was then fractionated by gel filtration
Fig. 5. Relationship curve of cell growth and enzyme production (Note: (–j–) curve of cell growth; (–.–) curve of enzyme production).
The resultant construct harbored a single open-reading frame consisting of the a-factor secretion signal peptide and Pf-PLB protein. The integrity of the recombinant was confirmed by direct sequencing. Two additional restriction endonuclease sites Not I and EcoR I were introduced at the 50 and 30 ends of pfplb, respectively. The recombinant expression vectors (pfplb/pPIC9K), which had been experimentally proved to be correct as show in Fig. 2, was linearized with Bgl II and transformed into P. pastoris GS115 competent cells. Many colonies were selected on the MD plates, in which multi-copy transformants were selected with G418 (2 mg/ml). Eventually, one transformant with the highest enzyme activity was selected. The molecular mass of recombinant enzyme expressed in P. pastoris as determined by SDS–PAGE was about 46 kDa (Fig. 3, lane 2), which was similar to native Pf-PLB secreted by P. fluorescens BIT-18 [9]. 3.2. Optimizing of the productivity PLB in P. pastoris A transformant with high PLB-secreting ability was selected to optimize the heterologous expression of Pf-PLB-P under various cultivation conditions, including different pH values, temperatures, inoculation volumes, and methanol concentrations. The highest enzyme activity was observed at pH 6.5, as shown in Fig. 4a, which increased 29% and 31% compared with pH 5.5 and pH 7.5, respectively. Therefore, controlling the medium pH during the fermentation process is necessary, the optimum pH may depends on individual properties of the protein [22]. The highest enzyme activity of 40 U/ml was detected at 26 °C, as shown in Fig. 4b. The temperatures for enzyme induction with methanol in the P. pastoris expression system were different while most other protein expressed at about 30 °C [23–28]. A benefit of lowering the temperature is to reduce the proteolytic degradation of the recombinant protein in the culture medium [22]. This phenomenon may result from the poor stability of the recombinant protein and folding problems at high temperatures [25]. The optimum methanol concentration of 1% (v/v) (Fig. 4c) and optimum inoculum volume of 3% (v/v) (Fig. 4d) were determined by varying the methanol concentration from 0.5% to 2% and inoculum volume from 1% to 5%. Methanol (1%) was added to the culture for inducing enzyme expression when P. pastoris had OD600 4 under the optimum conditions (pH 6.5, 26 °C, 3% inoculation volume). Pf-PLB-P activity was detected 12 h after adding the inducer. Results showed that enzyme activity sharply increased in the logarithmic phase of cell growth with the total enzyme activity reaching the maximum value of 65 U/ml at 36 h, which was twice the value expressed by P. fluorescens BIT-18. In addition, the time that the PLB activity
Fig. 6. Purification of the recombinant PLB (Pf-PLB-P). (a) Purification chromatograph of the Pf-PLB-P by DEAE anion exchange-chromatography. The bound material was eluted with a gradient of sodium chloride (dashed line), (b) purification chromatograph of the Pf-PLB-P by Superdex 75 gel filtration and (c) SDS–PAGE chromatograph (M: protein marker; Lane 1: the recombinant PLB by Superdex 75; Lane 2: the recombinant PLB by DEAE: Lane: the fermentation).
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S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28 Table 1 Purification of recombinant phospholipase B from Pichia pastoris. Purification steps
Total protein (mg)
Total activity (U)
Specific activity (U tmg1)
Recovery (%)
Purification (fold)
The fermentation (NH4)2SO4 precipitation Anion-exchange chromatography Gel filtration
52.4 15.8 9.1 6.3
3461 2815 2448 1855
66 178 268 294
100.00 81.35 70.74 53.60
1.0 2.7 4.1 4.5
chromatography using a Superdex 75 column (Fig. 6b). Highly purified Pf-PLB-P was obtained after these steps as proved by the single band corresponding to a molecular mass around 46 kDa in the SDS–PAGE gel (Fig. 6c, lane 1). The recovery and purification factors of Pf-PLB-P at different purification steps were summarized in Table 1. 3.4. Enzymatic properties The enzymatic properties of Pf-PLB-P are shown in Fig. 7. The optimum reaction temperature was determined to be 30 °C (Fig. 7a), which was similar to that of PLB from native P. fluorescens BIT-18 [9] and the one expressed by E. coli [12]. The optimum reaction pH was 6.5, similar to that of native PLB (pH 6.5) [9,10] but 0.5 higher than that of PLB expressed by E. coli (pH 6.0) [12]. Additionally, >70% of enzyme activity remained with increased pH from 5.0 to 7.5, which was almost the same as that of PLB expressed by E. coli. In conclusion, Pf-PLB-P displayed a slightly broader temperature range for soybean phospholipids hydrolysis than the wild type. Table 2 shows that Km and Vmax of Pf-PLB-P for soybean phospholipids hydrolysis were 4.75 mM and 98.67 mmol/(L min), respectively. kcat was 36.54 s1 and kcat/Km was 7.69 s1/mM. Understanding the enzyme kinetic properties may help understand the catalytic mechanism. 3.5. Application of Pf-PLB-P in degumming vegetable oil
Fig. 7. (a) Effect of pH on the activity (–j–) and stability (–d–) of Pf-PLB-P and (b) effect of temperature on the activity (–N–) and stability (–.–) of Pf-PLB-P.
The phosphorus contents of soybean and peanut oils decreased from 125.1 mg/kg to 4.96 mg/kg and 96.86 mg/kg to 3.54 mg/kg, respectively, within 5 h after applying Pf-PLB-P, as shown in Table 3. This phosphorous level after degumming was acceptable for industrial applications. These results exhibited that Pf-PLB-P had similar degumming characteristics of soybean oil but showed a much higher enzyme productivity of 65 U/ml, which was twice higher than native Pf-PLB [9]. In traditional chemical vegetable oil degumming process; during the non-hydratable phosphatides degumming acid is added at temperatures between 85 and 90 °C and neutralized with base [29,30]. While enzymatic degumming requires enzymes (PLA1,
Table 2 Kinetic parameters of recombinant phospholipase B from Pseudomonas fluorescens BIT-18 expressed in P. pastoris reacting with soybean phospholipids. Substrate Soybean phospholipids
Km (mM) 4.75
Vmax (mmol L1 min1) 98.67
kcat (s1) 36.54
kcat/Km (s1 mM1) 7.69
Each value represents the mean of triplicate experiments.
Table 3 Application of recombinant phospholipase B in both soybean and peanut oil degumming. Enzyme
Phosphorus content before degumming
Phosphorus content after degumming
Pf-PLB
Soybean oil Peanut oil
121.23 ± 11.47 96.86 ± 10.5
4.86 ± 0.62 3.73 ± 0.42
Pf-PLB-P
Soybean oil Peanut oil
121.23 ± 11.47 96.86 ± 10.5
4.96 ± 0.59 3.54 ± 0.41
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PLA2, PLB, etc.) at 40–50 °C to degum the non-hydratable phosphatides of the vegetable oil [31,32]. If we draw a comparison between these two degumming methods the cost of the enzymatic degumming is the cost of the enzyme. Therefore, the cost of the enzymatic degumming could be significantly lowered by constructing a strong PLB expression system and it will provide a cheaper enzyme for industrial applications. The requirement of the small quantity of water, acid and base for enzymatic degumming also make it cost effective and industrially applicable.
4. Conclusions PLB expressed by P. pastoris was successfully produced. After optimization of the fermentation conditions, the enzyme activity was twice that of native PLB from P. fluorescens BIT-18. High-purity PLB was obtained by ammonium sulfate fractionation, anionexchange chromatography and gel filtration successively. Km of the enzyme for soybean phospholipid was 4.75 mM, and Vmax was 98.67 mmol/(L min). The purified enzyme was used to degum soybean and peanut oils, whose phosphorus contents decreased to <5 mg/kg. The development of suitable fermentation strategies, some of which are underway, may enable the large-scale production of Pf-PLB for industrial application. Acknowledgements This work was financially supported by National Science Foundation of China (Nos. 21176028, 21276025, 21376028), Doctoral Fund of Ministry of Education of China (Nos. 20091101110036, 20121101110050), the Major State Basic Research Development Program of China (973 Program) (No. 2013CB733900), and the National High Technology Research and Development Program of China (863 Program) (No. 2012AA02A704). References [1] G.A. Köhler, A. Brenot, E. Haas-Stapleton, N. Agabian, R. Deva, S. Nigam, Phospholipase A2 and phospholipase B activities in fungi, Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1761 (2006) 1391– 1399. [2] M.A. Ghannoum, Potential role of phospholipases in virulence and fungal pathogenesis, Clinical Microbiology Reviews 13 (2000) 122–143. [3] J.L. Farn, R.A. Strugnell, P.A. Hoyne, W.P. Michalski, J.M. Tennent, Molecular characterization of a secreted enzyme with phospholipase B activity from Moraxella bovis, Journal of Bacteriology 183 (2001) 6717–6720. [4] S.C. Chen, L.C. Wright, J.C. Golding, T.C. Sorrell, Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans, Biochemical Journal 347 (2000) 431. [5] D.K. Kim, H.J. Lee, Y. Lee, Detection of two phospholipase A2 (PLA2) activities in leaves of higher plant Vicia faba and comparison with mammalian PLA2’s, FEBS Letters 343 (1994) 213. [6] T. Lu, M. Ito, U. Tchoua, H. Takemori, M. Okamoto, H. Tojo, Identification of essential residues for catalysis of rat intestinal phospholipase B/lipase, Biochemistry 40 (2001) 7133–7139. [7] M. Nauze, L. Gonin, B. Chaminade, C. Perès, F. Hullin Matsuda, B. Perret, H. Chap, A. Gassama Diagne, Guinea pig phospholipase B, identification of the catalytic serine and the proregion involved in its processing and enzymatic activity, Journal of Biological Chemistry 277 (2002) 44093–44099. [8] S. Xu, L. Zhao, A. Larsson, P. Venge, The identification of a phospholipase B precursor in human neutrophils, FEBS Journal 276 (2009) 175–186.
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