Chemical Engineering Journal 237 (2014) 23–28

Contents lists available at ScienceDirect

Chemical Engineering Journal

journal homepage: www.elsevier .com/locate /cej

Characteristics and vegetable oils degumming of recombinantphospholipase B

1385-8947/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.cej.2013.09.109

⇑ Corresponding author. Tel./fax: +86 010 68913171.E-mail address: lichun@bit.edu.cn (C. Li).

1 Meili Liang contributed equally to this work.

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 2013Received in revised form 22 September2013Accepted 30 September 2013Available online 12 October 2013

Keywords:Phospholipase BVegetable oil degummingPseudomonas fluorescens BIT-18Expression

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 posi-tions of a phospholipid and has been successfully used to degum vegetable oils in our previous work. Thisstudy focused on the heterologous overexpression of phospholipase B (Pf-PLB-P) in Pichia pastoris toinvestigate its characteristics and application in degumming vegetable oils. After optimizing the fermen-tation conditions, the maximum achieved enzyme activity was 65 U/ml, which was twice the enzymeactivity of wild-strain P. fluorescens BIT-18. Purified Pf-PLB-P was obtained by ammonium sulfate precip-itation, anion-exchange chromatography, and gel filtration. The kinetic constants Km and Vmax weredetermined to be 4.75 mM and 98.67 mmol/(L min), respectively. Pf-PLB-P enzyme activity was detectedat 25–55 �C and pH 4.5–9.5, and the temperature range was observed to be slightly broadened than thatof the wild type. Based on these characteristics, Pf-PLB-P was also successfully used to degum soybeanand peanut oils, whose phosphorus contents decreased from 125.1 mg/kg to 4.96 mg/kg and 96 mg/kgto 3.54 mg/kg, respectively. These results indicate that Pf-PLB-P produced by P. pastoris has potentialindustrial use.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Phospholipase B (PLB) is a heterogeneous group of phospholip-ases that harbor three distinct activities: a phospholipase A1 orphospholipase 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]. PLBhas applications in the food and pharmaceutical industries to pro-duce phospholipid derivatives where phospholipid hydrolysis isneeded.

Our group has previously reported that PLB from Pseudomonasfluorescens BIT-18 (Pf-PLB) isolated from soil can be used for vege-table oil degumming in which phospholipids are easily hydrolyzedand removed. The phosphorous content is <5 mg/kg after digestion

[9,10]. Enzymatic degumming of vegetable oils can replace theconventional method that requires acid, alkali, and soft water[11]. However, the industrial application of Pf-PLB in wild-typeP. fluorescens BIT-18 induced by soybean phospholipid is limitedby low yields [9]. Thus, the gene of Pf-PLB has been isolated andexpressed in Escherichica coli to improve Pf-PLB productivity.Although recombinant PLB has been successfully expressed inE. coli [12], most recombinant enzymes only exist in the form ofinclusion bodies. The methylotrophic Pichia pastoris expressionsystem has several advantages over the E. coli expression system,including the use of alcohol oxidase 1 gene (AOX1) promoter, theability to culture cells at high density, a more simplified purifica-tion procedure for secreted heterologous proteins, and the abilityto express even highly toxic antimicrobial proteins at a large scale[13]. More importantly, the recombinant proteins expressed bythis system are safe for human use. Numerous enzymes in thefood industry have already been expressed in P. pastoris, such asa-amylase [14,15], protease [16], glucoamylase [17], chymosin[18], and glucose oxidase [19].

24 S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28

In this work, PLB (Pf-PLB-P) was heterologous overexpressed inP. pastoris. After optimizing the fermentation conditions, Pf-PLB-Pwas then purified, characterized, and successfully used to degumsoybean and peanut oils. The results can serve as a foundationfor further large-scale production and application of PLB in vegeta-ble oil degumming.

2. Materials and methods

2.1. Strains, vectors, and reagents

The P. pastoris host strain GS115 and secretion expression vec-tor pPIC9K were purchased from Invitrogen (CA, USA). E. coli DH5awas used for routine plasmid amplification. All primers were syn-thesized by BGI (Shenzhen, China). All restriction enzymes, DNAmarkers, and protein markers were purchased from Takara (Dalian,China). Yeast nitrogen base, yeast extract, and tryptone wereobtained from OXOID Ltd. (Basingstoke, England). PCR purification,gel extraction, and miniprep kits for plasmid extraction wereobtained from Biomed Corporation (Beijing, China).

2.2. pfplb/pPIC9K construction and transformation

The strategy for pfplb/pPIC9K construction and transformationinto P. pastoris are depicted in Fig. 1. pfplb DNA was amplified byPCR from a prokaryotic plasmid constructed in our laboratory[12] encoding full-length PLB. The forward and reverse primerswere 50-CCGGAATTCATGAAAAAAGTCATGCTCAA-30 and 50-ATTTGCGGCCGCTCAGAAGCGGTAGGTCGCGC-30, with EcoR I and Not I sitesunderlined, respectively. The PCR products were digested with therestriction enzymes and ligated into EcoR I/Not I digested pPIC9K,which inserted the fragment in-frame to the a-factor secretion

Fig. 1. Schematic of pfplb/pPIC9K construction and transformation into Pichiapastoris GS115.

signal downstream of alcohol oxidase I promoter. The resultingplasmid (pfplb/pPIC9K) was transformed into E. coli DH5a. Then,the positive colonies were selected by colony-PCR and sequenceanalysis.

pfplb/pPIC9K purified from the positive colony was linearizedwith Bgl II and then transformed into P. pastoris GS115 by thePEG method according to the manufacturer’s protocol (Multi-CopyPichia Expression Kit, Invitrogen). Muts transformants wereselected by MD plates. The multi-copy transformants wereobtained on YPD plates at different G418 concentrations (0.5, 1.0,1.5, and 2.0 mg/ml). Integration of pfplb in the recombinantP. 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 expres-sion conditions, and analyzed the secretion levels among recombi-nant P. pastoris clones. The selected strains were grown in 30 ml ofYPD medium for approximately 24 h at 28 �C with constant shak-ing at 170 rpm. These cells were cultured further in 70 ml of BMGYmedium (1% yeast extract, 2% tryptone, 1.34% YNB, 4 � 10�5% bio-tin, 1% glycerol, and 100 mM potassium phosphate, pH 6.1) untilthe culture reached OD600 4–6, whereupon the cells were har-vested by centrifugation for 5 min at 5000 � g and 4 �C. The super-natant was carefully decanted, and the cell pellet was resuspendedin 70 ml of BMMY (1% yeast extract, 2% tryptone, 1.34% YNB,4 � 10�5% 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 expressionanalysis by SDS–PAGE [20] and enzyme assay.

The effects of initial pH (5.5–7.5, 100 mM Na2HPO4–citric acidbuffer) in BMMY medium, the inoculum amount of recombinantP. pastoris (1–5%, v/v), and the methanol dosage (0.5–2%, v/v) weredetermined at 28 �C. The optimum temperature for Pf-PLB-Pexpression was determined at 20–30 �C and pH 6.0 after methanoladdition, and an enzyme producing curve was drawn under theoptimum condition.

2.4. Purification of recombinant PLB

A maximum PLB activity of 65 U/ml was observed after 36 h ofinduction. The extracellular enzyme was isolated by centrifugingthe fermentation broth at 15,000g and 4 �C for 10 min. The super-natant was brought to 60% saturation with ammonium sulfate, leftundisturbed for 2 h, and centrifuged. The precipitate was dissolvedin a small volume of 20 mM Tris–HCl buffer (pH 7.3) and dialyzedovernight against the buffer. The crude enzyme solution waspassed through a anion-exchange chromatography column (HiPrepEDAE FF 16/10 (USA GE)) with 1 ml/min, and bound protein waseluted with a gradient NaCl (0–0.4 M) with the same flow rate.The fractions containing the highest activities were pooled, con-centrated, and passed through a gel filtration column (Superdex™75 10/300 GL (USA GE)) equilibrated and eluted with 0.02 MTris–HCl (pH 7.3) and 0.1 M NaCl buffer with 1 ml/min flowrate. The chromatographic processes were done using the ÄKTApurifier 10 (USA GE). The active fractions were flash frozen byliquid nitrogen and stored at �80 �C.

2.5. Characterization of recombinant phospholipase B

PLB activity was performed as described by [9]. For kinetic stud-ies of Pf-PLB-P, the initial velocities of the enzymatic reaction wereexamined by varying the concentration of soybean phospholipids(from 1 g/l to 8 g/l). Values of the Michaelis constants (Km) andmaximal velocity (Vmax) were obtained by the Lineweaver–Burkplot. The parameters were determined by three separate

Fig. 2. Gel electrophoretogram of single and double digested pfplb/pPIC9K (LaneM1: 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).

Fig. 3. SDS–PAGE analysis. Lane 1, concentrated supernatant from the negativestain (Pichia pastoris pPIC9K) after induction by methanol at 48 h. Lanes 2,concentrated supernatant from the positive stain (P. pastoris pfplb/pPIC9K) afterinduction by methanol at 48 h. Lane M, protein size markers.

Fig. 4. Optimum conditions for the expression of recombinant PLB (Pf-PLB-P). (a) EffectsPf-PLB-P activity, (c) eof different methanol concentration on Pf-PLB-P activity and (d) e

S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28 25

experiments. Protein concentration was measured using the Brad-ford assay and bovine serum albumin as a standard [21].

The optimum pH of Pf-PLB-P was investigated from pH 4.0 to9.0 (100 mM Na2HPO4–citric acid buffer, pH 4.0–7.0; or 100 mMTris–HCl buffer, pH 7.5–9.0) at 30 �C. The effect of pH on thePf-PLB-P stability was estimated by measuring the residual enzymeactivity after enzyme incubation in the absence of substrate at 4 �Cfor 12 h. The optimum temperature of Pf-PLB was determined at25–55 �C and pH 6.5. The effect of temperature on enzymestability was estimated by measuring the residual enzyme activityafter incubation of Pf-PLB-P in the absence of substrate at 25–55 �Cfor 20 min.

2.6. Enzymatic degumming and phosphorus content analysis

Degumming of crude soybean and peanut oil by Pf-PLB-P andPf-PLB were carried out according to [9]. Phosphorus content wasdetermined by the molybdenum blue method in accordance withGB/T 5537 (National Standard of the People’s Republic China,2008).

3. Results and discussion

3.1. Expression vector construction and transformation

A gene (pfplb) (GenBank accession No. CP000094) fromP. fluorescens BIT-18 encoding the enzyme with PLB activityreportedly consists of 1272 bp with an open-reading frameencoding a peptide of 423 amino acids [12]. In this work, pfplb wasamplified by PCR from pET-28a-pfplb constructed by our laboratoryand ligated into pPIC9K downstream of AOX1 promoter (Fig. 1).

of different level of pH on Pf-PLB-P activity, (b) effects of different temperature onffects of different inoculum volume on Pf-PLB-P activity.

Fig. 5. Relationship curve of cell growth and enzyme production (Note: (–j–) curveof cell growth; (–.–) curve of enzyme production).

26 S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28

The resultant construct harbored a single open-reading frameconsisting of the a-factor secretion signal peptide and Pf-PLB pro-tein. The integrity of the recombinant was confirmed by directsequencing. Two additional restriction endonuclease sites Not Iand EcoR I were introduced at the 50 and 30 ends of pfplb, respec-tively. The recombinant expression vectors (pfplb/pPIC9K), whichhad been experimentally proved to be correct as show in Fig. 2,was linearized with Bgl II and transformed into P. pastoris GS115competent 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 enzymeactivity was selected. The molecular mass of recombinant enzymeexpressed in P. pastoris as determined by SDS–PAGE was about46 kDa (Fig. 3, lane 2), which was similar to native Pf-PLB secretedby P. fluorescens BIT-18 [9].

Fig. 6. Purification of the recombinant PLB (Pf-PLB-P). (a) Purification chromato-graph of the Pf-PLB-P by DEAE anion exchange-chromatography. The boundmaterial 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 bySuperdex 75; Lane 2: the recombinant PLB by DEAE: Lane: the fermentation).

3.2. Optimizing of the productivity PLB in P. pastoris

A transformant with high PLB-secreting ability was selected tooptimize the heterologous expression of Pf-PLB-P under variouscultivation conditions, including different pH values, temperatures,inoculation volumes, and methanol concentrations. The highestenzyme activity was observed at pH 6.5, as shown in Fig. 4a, whichincreased 29% and 31% compared with pH 5.5 and pH 7.5, respec-tively. Therefore, controlling the medium pH during the fermenta-tion process is necessary, the optimum pH may depends onindividual properties of the protein [22]. The highest enzyme activ-ity of 40 U/ml was detected at 26 �C, as shown in Fig. 4b. The tem-peratures for enzyme induction with methanol in the P. pastorisexpression system were different while most other proteinexpressed at about 30 �C [23–28]. A benefit of lowering the temper-ature is to reduce the proteolytic degradation of the recombinantprotein in the culture medium [22]. This phenomenon may resultfrom the poor stability of the recombinant protein and foldingproblems at high temperatures [25]. The optimum methanol con-centration of 1% (v/v) (Fig. 4c) and optimum inoculum volume of3% (v/v) (Fig. 4d) were determined by varying the methanol con-centration from 0.5% to 2% and inoculum volume from 1% to 5%.

Methanol (1%) was added to the culture for inducing enzymeexpression when P. pastoris had OD600 �4 under the optimum con-ditions (pH 6.5, 26 �C, 3% inoculation volume). Pf-PLB-P activitywas detected 12 h after adding the inducer. Results showed thatenzyme activity sharply increased in the logarithmic phase of cellgrowth with the total enzyme activity reaching the maximumvalue of 65 U/ml at 36 h, which was twice the value expressedby P. fluorescens BIT-18. In addition, the time that the PLB activity

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 ammoniumsulfate for Pf-PLB-P purification by anion-exchange chromatogra-phy and gel filtration. After the component with PLB activity wasapplied to a DEAE FF column, the targeted enzyme (Fig. 6c, lane2) was obtained after elution with 200 mM NaCl (Fig. 6a). The con-centrated enzyme solution was then fractionated by gel filtration

Table 1Purification of recombinant phospholipase B from Pichia pastoris.

Purification steps Total protein (mg) Total activity (U) Specific activity (U tmg�1) Recovery (%) Purification (fold)

The fermentation 52.4 3461 66 100.00 1.0(NH4)2SO4 precipitation 15.8 2815 178 81.35 2.7Anion-exchange chromatography 9.1 2448 268 70.74 4.1Gel filtration 6.3 1855 294 53.60 4.5

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.

Table 2Kinetic parameters of recombinant phospholipase B from Pseudomonas fluorescens BIT-18

Substrate Km (mM) Vmax (mmolSoybean phospholipids 4.75 98.67

Each value represents the mean of triplicate experiments.

Table 3Application of recombinant phospholipase B in both soybean and peanut oil degumming.

Enzyme Phosphorus content

Pf-PLB Soybean oil 121.23 ± 11.47Peanut oil 96.86 ± 10.5

Pf-PLB-P Soybean oil 121.23 ± 11.47Peanut oil 96.86 ± 10.5

S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28 27

chromatography using a Superdex 75 column (Fig. 6b). Highlypurified Pf-PLB-P was obtained after these steps as proved by thesingle band corresponding to a molecular mass around 46 kDa inthe SDS–PAGE gel (Fig. 6c, lane 1). The recovery and purificationfactors of Pf-PLB-P at different purification steps were summarizedin Table 1.

3.4. Enzymatic properties

The enzymatic properties of Pf-PLB-P are shown in Fig. 7. Theoptimum reaction temperature was determined to be 30 �C(Fig. 7a), which was similar to that of PLB from native P. fluorescensBIT-18 [9] and the one expressed by E. coli [12]. The optimum reac-tion pH was 6.5, similar to that of native PLB (pH 6.5) [9,10] but 0.5higher than that of PLB expressed by E. coli (pH 6.0) [12]. Addition-ally, >70% of enzyme activity remained with increased pH from 5.0to 7.5, which was almost the same as that of PLB expressed by E. coli.In conclusion, Pf-PLB-P displayed a slightly broader temperaturerange for soybean phospholipids hydrolysis than the wild type.

Table 2 shows that Km and Vmax of Pf-PLB-P for soybean phos-pholipids hydrolysis were 4.75 mM and 98.67 mmol/(L min),respectively. kcat was 36.54 s�1 and kcat/Km was 7.69 s�1/mM.Understanding the enzyme kinetic properties may help understandthe catalytic mechanism.

3.5. Application of Pf-PLB-P in degumming vegetable oil

The phosphorus contents of soybean and peanut oils decreasedfrom 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 inTable 3. This phosphorous level after degumming was acceptablefor industrial applications. These results exhibited that Pf-PLB-Phad similar degumming characteristics of soybean oil but showeda much higher enzyme productivity of 65 U/ml, which was twicehigher than native Pf-PLB [9].

In traditional chemical vegetable oil degumming process; dur-ing the non-hydratable phosphatides degumming acid is addedat temperatures between 85 and 90 �C and neutralized with base[29,30]. While enzymatic degumming requires enzymes (PLA1,

expressed in P. pastoris reacting with soybean phospholipids.

L�1 min�1) kcat (s�1) kcat/Km (s�1 mM�1)36.54 7.69

before degumming Phosphorus content after degumming

4.86 ± 0.623.73 ± 0.42

4.96 ± 0.593.54 ± 0.41

28 S. Huang et al. / Chemical Engineering Journal 237 (2014) 23–28

PLA2, PLB, etc.) at 40–50 �C to degum the non-hydratable phos-phatides of the vegetable oil [31,32]. If we draw a comparison be-tween these two degumming methods the cost of the enzymaticdegumming is the cost of the enzyme. Therefore, the cost of theenzymatic degumming could be significantly lowered by con-structing a strong PLB expression system and it will provide acheaper enzyme for industrial applications. The requirement ofthe small quantity of water, acid and base for enzymatic degum-ming also make it cost effective and industrially applicable.

4. Conclusions

PLB expressed by P. pastoris was successfully produced. Afteroptimization of the fermentation conditions, the enzyme activitywas twice that of native PLB from P. fluorescens BIT-18. High-purityPLB was obtained by ammonium sulfate fractionation, anion-exchange chromatography and gel filtration successively. Km ofthe enzyme for soybean phospholipid was 4.75 mM, and Vmax

was 98.67 mmol/(L min). The purified enzyme was used to degumsoybean 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 produc-tion of Pf-PLB for industrial application.

Acknowledgements

This work was financially supported by National Science Foun-dation of China (Nos. 21176028, 21276025, 21376028), DoctoralFund of Ministry of Education of China (Nos. 20091101110036,20121101110050), the Major State Basic Research DevelopmentProgram of China (973 Program) (No. 2013CB733900), and theNational High Technology Research and Development Program ofChina (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 etBiophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1761 (2006) 1391–1399.

[2] M.A. Ghannoum, Potential role of phospholipases in virulence and fungalpathogenesis, Clinical Microbiology Reviews 13 (2000) 122–143.

[3] J.L. Farn, R.A. Strugnell, P.A. Hoyne, W.P. Michalski, J.M. Tennent, Molecularcharacterization of a secreted enzyme with phospholipase B activity fromMoraxella bovis, Journal of Bacteriology 183 (2001) 6717–6720.

[4] S.C. Chen, L.C. Wright, J.C. Golding, T.C. Sorrell, Purification andcharacterization of secretory phospholipase B, lysophospholipase andlysophospholipase/transacylase from a virulent strain of the pathogenicfungus Cryptococcus neoformans, Biochemical Journal 347 (2000) 431.

[5] D.K. Kim, H.J. Lee, Y. Lee, Detection of two phospholipase A2 (PLA2) activities inleaves of higher plant Vicia faba and comparison with mammalian PLA2’s, FEBSLetters 343 (1994) 213.

[6] T. Lu, M. Ito, U. Tchoua, H. Takemori, M. Okamoto, H. Tojo, Identification ofessential 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 thecatalytic serine and the proregion involved in its processing and enzymaticactivity, Journal of Biological Chemistry 277 (2002) 44093–44099.

[8] S. Xu, L. Zhao, A. Larsson, P. Venge, The identification of a phospholipase Bprecursor in human neutrophils, FEBS Journal 276 (2009) 175–186.

[9] F. Jiang, J. Wang, I. Kaleem, D. Dai, X. Zhou, C. Li, Degumming of vegetable oilsby a novel phospholipase B from Pseudomonas fluorescens BIT-18, BioresourceTechnology 102 (2011) 8052–8056.

[10] F.y. Jiang, J.m. Wang, L.c. Ju, I. Kaleem, D.z. Dai, C. Li, Optimization ofdegumming process for soybean oil by phospholipase B, Journal of ChemicalTechnology and Biotechnology 86 (2011) 1081–1087.

[11] G.C. Gervajio, Bailey’s Industrial Oil and Fat Products, John Wiley & Sons, NewYork, 2005.

[12] F. Jiang, S. Huang, K. Imadad, C. Li, Cloning and expression of a gene withphospholipase B activity from Pseudomonas fluorescens in Escherichia coli,Bioresource Technology 104 (2012) 518–522.

[13] J.L. Cereghino, J.M. Cregg, Heterologous protein expression in themethylotrophic yeast Pichia pastoris, FEMS Microbiology Reviews 24 (2000)45–66.

[14] E. Paifer, E. Margolles, J. Cremata, R. Montesino, L. Herrera, J.M. Delgado,Efficient expression and secretion of recombinant alpha amylase in Pichiapastoris using two different signal sequences, Yeast 10 (1994) 1415–1419.

[15] C.H. Yang, Y.C. Huang, C.Y. Chen, C.Y. Wen, Expression of Thermobifida fuscathermostable raw starch digesting alpha-amylase in Pichia pastoris and itsapplication in raw sago starch hydrolysis, Journal of Industrial Microbiologyand Biotechnology 37 (2010) 401–406.

[16] T. Kim, X.G. Lei, Expression and characterization of a thermostable serineprotease (TfpA) from Thermomonospora fusca YX in Pichia pastoris, AppliedMicrobiology and Biotechnology 68 (2005) 355–359.

[17] J.A. Mertens, J.D. Braker, D.B. Jordan, Catalytic properties of two Rhizopusoryzae 99–880 glucoamylase enzymes without starch binding domainsexpressed in Pichia pastoris, Applied Biochemistry and Biotechnology 162(2010) 2197–2213.

[18] J.A. Vallejo, J.M. Ageitos, M. Poza, T.G. Villa, Cloning and expression of buffaloactive chymosin in Pichia pastoris, Journal of Agricultural and Food Chemistry56 (2008) 10606–10610.

[19] Y. Guo, F. Lu, H. Zhao, Y. Tang, Z. Lu, Cloning and heterologous expression ofglucose oxidase gene from Aspergillus niger Z-25 in Pichia pastoris, AppliedBiochemistry and Biotechnology 162 (2010) 498–509.

[20] U.K. Laemmli, Cleavage of structural proteins during the assembly of the headof bacteriophage T4, Nature 227 (1970) 680–685.

[21] M.M. Bradford, A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding, Analytical Biochemistry 72 (1976) 248–254.

[22] P. Li, A. Anumanthan, X.-G. Gao, K. Ilangovan, V.V. Suzara, N. Düzgünes�, V.Renugopalakrishnan, Expression of recombinant proteins in Pichia pastoris,Applied Biochemistry and Biotechnology 142 (2007) 105–124.

[23] K. Kobayashi, S. Kuwae, T. Ohya, T. Ohda, M. Ohyama, K. Tomomitsu, Additionof oleic acid increases expression of recombinant human serum albumin bythe AOX2 promoter in Pichia pastoris, Journal of Bioscience and Bioengineering89 (2000) 479–484.

[24] N. Koganesawa, T. Aizawa, H. Shimojo, K. Miura, A. Ohnishi, M. Demura, Y.Hayakawa, K. Nitta, K. Kawano, Expression and purification of a small cytokinegrowth-blocking peptide from armyworm Pseudaletia separata by anoptimized fermentation method using the methylotrophic yeast Pichiapastoris, Protein Expression and Purification 25 (2002) 416–425.

[25] H. Feng, N.Q. Meinander, L.J. Jönsson, Fermentation strategies for improvedheterologous expression of laccase in Pichia pastoris, Biotechnology andBioengineering 79 (2002) 438–449.

[26] Z. Li, F. Xiong, Q. Lin, M. d’Anjou, A.J. Daugulis, D.S. Yang, C.L. Hew, Low-temperature increases the yield of biologically active herring antifreezeprotein in Pichia pastoris, Protein Expression and Purification 21 (2001) 438–445.

[27] M.P. Sue, M.L. Fazenda, B. McNeil, L.M. Harvey, Heterologous proteinproduction using the Pichia pastoris expression system, Yeast 22 (2005) 249–270.

[28] D. Mattanovich, P. Branduardi, L. Dato, B. Gasser, M. Sauer, D. Porro,Recombinant Protein Production in Yeasts, in: Recombinant GeneExpression, Springer, 2012, pp. 329–358.

[29] A.J. Dijkstra, M. Van Opstal, The total degumming process, Journal of theAmerican Oil Chemists’ Society 66 (1989) 1002–1009.

[30] M.M.R. Khan, M. Tsukada, Y. Gotoh, H. Morikawa, G. Freddi, H. Shiozaki,Physical properties and dyeability of silk fibers degummed with citric acid,Bioresource Technology 101 (2010) 8439–8445.

[31] A.J. Dijkstra, Enzymatic degumming, European Journal of Lipid Science andTechnology 112 (2010) 1178–1189.

[32] L. De Maria, J. Vind, K. Oxenbøll, A. Svendsen, S. Patkar, Phospholipases andtheir industrial applications, Applied Microbiology and Biotechnology 74(2007) 290–300.