Abstract
A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 104 molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Recently, the development of yeast surface display system heralds a new era of combinatorial bioengineering by virtue of characteristics of well developed cultural technique, post-translational modification as well as the industrial application in the field of biotechnology (Kondo and Ueda 2004). In yeast surface display technology, proteins or enzymes are immobilized on the cell surface by genetic operation and could be used for a wide variety of biotechnological applications (Tanaka et al. 2010), such as the delivery of live vaccines (Georgiou et al. 1997; Liljeqvist et al. 1997), immobilized biocatalysts used bioconversions (Li et al. 2008; Lee et al. 2004; Tanino et al. 2009), bioremediation of heavy metals (Samuelson et al. 2000; Kuroda and Ueda 2006), and as biosensors(Li et al. 2008). As a common host, Pichia pastoris proved its competitive ability for appropriate N-glycosylation for heterologous protein, inducible expression regulated tightly by alcohol oxidase 1 promoter (pAOX1) as well as little own protein secretion in the supernatant when compared to other common yeasts such as Saccharomyces cerevisiae (Wang et al. 2010; Tanino et al. 2006).
To some extent, the efficiency of protein display or enzyme anchoring on the yeast surface which has not been accurately evaluated hinders the practical application of cell surface display systems. Shibasaki et al. (2001) calculated the number of enhanced green fluorescent protein (EGFP) displayed as α-agglutinin fused protein on the S. cerevisiae cell surface by measuring the fluorescence intensity of the cell pellet suspended in PBS buffer (pH 7.4). But few reports have described the quantification of foreign proteins/enzyme displayed on the P. pastoris cell surface for any anchor. In addition, due to differences between the two hosts, the method suggested by Shibasaki et al. (2001) was insufficiently accurate for quantifying surface displaying proteins.
FLO1p from S. cerevisiae is a lectin-like anchor protein that plays an important role in flocculation. FLO1p contains 1,200 amino acids and is composed of the following four domains: (1) secretion signal, (2) flocculation functional domain, (3) glycosylphosphatidylinositol (GPI)-anchor attachment signal, and (4) membrane-anchoring domain (FS anchor) (Kondo and Ueda 2004).The FS anchor of approx. 874 amino acids here contains the N-terminal flocculation functional domain (FFD) which can recognize and adhere noncovalently to cell-wall components such as mannan carbohydrates (Han et al. 2009; Kondo and Ueda 2004). Moreover, the FS anchor is especially suitable for displaying the enzymes which has their active sites near the C terminus such as Rhizopus oryzae lipase (ROL), Rhizomucor miehei lipase, RML (Han et al. 2010), so that it is becoming a popular anchor for yeast surface display (Matsumoto et al. 2002; Han et al. 2010). Tanino et al. (2006) displayed the Rhizopus oryzae lipase including a pro sequence (ProROL) on P. pastoris with FS as anchor, and demonstrated that the FS anchor system in P. pastoris is effective in the same manner as in S. cerevisiae or ever more competitive when taking account of the factor that P. pastoris can be cultivated at a higher cell density.
In this study, we used the FS anchor to display a fusion protein of EGFP and Candida antarctica lipase B (CALB) on the surface of P. pastoris. CALB catalyzes many esterification and trans esterification reactions (Jaeger and Eggert 2002). Here we examined the relationship between the fluorescence intensity of leaked fusion protein and its protein concentration in the culture medium of recombinant EGFP-CALB/CALB-EGFP displaying P. pastoris. Using the relationship between fluorescence intensity and protein concentration, we quantified the number of fusion displayed on the P. pastoris cell surface. This method can be extend to other CALB yeast surface displaying systems involving other anchor proteins, thus facilitating more efficient CALB displaying systems and enabling accurate evaluation of displaying efficiency of single host cell.
Materials and methods
Strains and media
Escherichia coli strain Top 10 was used as a host for recombinant DNA manipulation. P. pastoris strain GS115 was used to display the fuse protein of EGFP and CALB. E. coli cells were grown in LB medium (1 % tryptone, 0.5 % yeast extract, 0.5 % NaCl and 0.1 % glucose). P. pastoris was grown in BMGY medium(1 % yeast extract, 2 % peptone, 100 mM KH2PO4/K2HPO4 buffer (pH 6.0), 1.34 % yeast nitrogen base without amino acids (YNB), 4 × 10−5 % biotin, and 1 % v/v glycerol), and BMMY medium (1 % yeast extract, 2 % peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34 % yeast nitrogen base, 4 × 10−5 % biotin, and 0.5–2 % v/v methanol).
Construction of expression plasmids
The fluorescence positive control plasmid pKFS-EGFP was constructed as follows: a MluI–EcoRI fragment of the EGFP gene was prepared using PCR (primers 5′-ATGAACGCGTATGGTGAGCAAGGGCGAGG-3′ and 5′-CTGAGAATTCCTTGTACAGCTCGTCCATGC-3′) using pEGFP-N1 (GenBank Accession No.U55762) as a template, and inserted into the MluI–EcoRI site of the integration-type yeast cell-surface-displaying cassette vector pKFSR (stored in our laboratory; Han et al. 2009). The resulting plasmid was referred to as pKFS-EGFP. The pKFS-EGFP-CALB plasmid was constructed as follows: a MluI-ApaI fragment of the EGFP gene was prepared using PCR (primers 5′-ATGAACGCGTATGGTGAGCAAGGGCGAGG-3′ and 5′- TAGTGGGCCCCTTGTACAGCTCGTCCATGC-3′) using pEGFP-N1 (GenBank Accession No. U55762) as a template, and an ApaI–EcoRI fragment of the CALB gene was prepared using PCR (primers,5′-ATGTGGGCCCCTACCTTCCGGTTCG-3′and 5′- GTATGAATTCGGGGGTGACGATGC-3′) using pKNS-CALB (stored in our laboratory; Su et al. 2010) as a template. The two genes were linked through ApaI and inserted into the MluI–EcoRI site of the integration-type yeast cell-surface-displaying cassette vector pKFSR. The resulting plasmid was referred to as pKFS-EGFP-CALB. A pKFS-CALB-EGFP plasmid was constructed as follows: an ApaI–EcoRI fragment of the EGFP gene was prepared using PCR (primers, 5′-ACTTGGGCCCATGGTGAGCAAGGGCGAG-3′ and 5′-CTGAGAATTCCTTGTACAGCTCGTCCATGC-3′) using pEGFP-N1 (GenBank Accession No.U55762) as a template, and an MluI-ApaI fragment of the CALB gene was prepared using PCR (primers 5′-ATTGACGCGTCTACCTTCCGGTTCG-3′ and 5′-ATTAGGGCCCGGGGGTGACGATGC-3′) using pKNS-CALB as a template. The two genes were linked through ApaI and inserted into the MluI–EcoRI site of the integration-type yeast cell-surface-displaying cassette vector pKFSR. The resulting plasmid was referred to as pKFS-CALB-EGFP. The plasmids pKFS-EGFP, pKFS-EGFP-CALB, and pKFS-CALB-EGFP were transformed into P. pastoris using the lithium chloride method.
Fluorometric, microscopy and flow cytometry (FLC) assays
A fluorescence spectrophotometer (Tecan Infinite M200, San Jose, CA, USA) was used to measure the fluorescence of samples in Tris–HCl buffer (50 mM, pH8.0) on a 96-well plate. A filter with excitation at 480 nm and a filter for emission at 520 nm were used to detect fluorescence intensity. Fluorescence microscopy was carried out using an L710 confocal laser scanning microscope (CLSM) (Shibasaki et al. 2001).The 488 nm line of an argon/krypton ion laser was used for excitation. To measure the fluorescent signal of EGFP, the beam splitter and filter were set to 500–530 nm. The flow cytometer used for the analysis was a Flow Cytometry Quanta SC equipped with an argon ion laser (488 nm wavelength) capable of GFP excitation (Bahl et al. 2004).
Analysis of lipase activity
Lipase activity was measured using 4-nitrophenyl caprylate (pNPC; Sigma) and following the release of p-nitrophenol, at 405 nm. Free lipases and cell surface-displayed lipases were dissolved in Tris–HCl buffer (50 mM, pH 8.0), mixed with substrate solution (0.0625 mM pNPC, 0.1 % Triton X-100 in lipase assay buffer), and reacted for 5 min before measuring the A405 using a kinetic microplate reader. After centrifugation at 10,000×g for 1 min, 200 μl of the supernatant was added to a 96-well plate and absorbance was measured. Product yields were determined based on a standard curve prepared using p-nitrophenol as a standard. One unit of activity was defined as the amount of enzyme that released l μmol pNP per min from pNPC under the assay conditions. Each sample was assayed in triplicate and the average value was determined (Han et al. 2009).
Western blot analysis and protein analysis
SDS-PAGE using an 8 % (v/v) gel was carried out. The protein extracts separated on the gel were electroblotted on a nitrocellulose (NC) membrane (PALL Corporation, Port Washington, NY, USA) and allowed to react with the anti-GFP HRP-DirecT (MBL International, Woburn, MA, USA).A Bradford protein assay kit was used to determine protein concentration (Tanino et al. 2006; MBL International; Afanassiev et al. 2000).
Results
Detection the EGFP fluorescence intensity on cell surface
Using CLSM and FLC, the fluorescence emitted from the transformants GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP cultivated in BMGY and BMMY liquid medium were investigated. As a control, cells harboring the plasmid pKFS-EGFP (GS115/pKFS-EGFP) were used. After cultivation, all three strains showed obvious fluorescence when evaluate dusing CLSM and FLC. The light spots of GS115/pKFS-EGFP were much denser and brighter than the other two strains, GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP detected using CLSM (Fig. 1—2a, 2b, 2c). Additionally, the intensity of the light spots from GS115/pKFS-EGFP-CALB was similar to that of GS115/pKFS-CALB-EGFP (Fig. 1—2c), and the distribution of the light spots from the two strains were identical. Upon further analysis using FLC, the fluorescence histograms show the same trend as observed using CLSM; the signal of GS115/pKFS-EGFP was much stronger that for the other two plasmids (approx. 10 times), and the signal of the other two strains were similar to each other (Fig. 1—3a, 3b, 3c).
Detection of CALB activity
The CALB displayed on recombinant yeasts GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP were analyzed. As a negative control, GS115/PKFS-EGFP showed no CALB activity (date not shown) and the CALB activity displayed on the cell surface of GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP were similar (Table 1). The highest activity of the two strains obtained at 120 h induced by methanol was consistent (approx. 500 U/g). These results agree with those of Tanino et al. (2006) who showed that some protein leaked into the culture medium. Notably, leakage into the medium for both the GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP appeared to approach the same extent since the CALB activities in the supernatants were similar (Table 1, approx. 14 U/mL). Moreover, the OD600 of GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP revealed similar growth rates.
These results suggest that the position between EGFP and CALB in the fusion protein does not affect significantly the activity of CALB displayed on the cell surface or in the medium. A correlation between fluorescence intensity of the cells and lipase activity on the cell wall were expressed as Fig. 2. The fluorescence intensity and lipase activity went up at the same slope with the culture time increased. Besides, when the fluorescence intensity of the displaying yeast increased within the range of 4 × 107 to 108 RFU/g, the corresponding lipase activity displayed on the cell surface showed a coincident rise from 150 to 500 U/g dry cell wt.
SDS-PAGE and Western blot analysis
Proteins of interest were extracted from the yeast cell wall and medium and analyzed using SDS-PAGE and Western blot (Fig. 3). The result in SDS-PAGE showed that only one band appeared in the medium and it is of the same molecular size as the main band of extracts from recombinant yeast cell wall. When performed with Western Blot, both these proteins of same molecular mass recognized anti-EGFP antibody as well. As mentioned in section of ‘Detection of CALB activity’, we also detected certain activity of CALB and certain fluorescence of EGFP in the medium and on the cell wall. Since no other protein existed in the medium, the protein in the medium must have the peptides or structures of both CALB and EGFP. Taking the other factors that FS has been fused with CALB and EGFP by genetic engineering and FS is non-covalently adherent to the cell wall as well as the leakage in the medium based on FS surface displaying, the protein appeared in medium could be referred as same as that immobilized on the cell wall. The molecular weights of all fusion proteins displayed on the cell surface or leaked into the medium were similar (approx. 220 kDa) regardless of what strains were used; GS115/pKFS-EGFP, GS115/pKFS-EGFP-CALB as well as GS115/pKFS-CALB-EGFP. This high molecular weight may have been due to glycosylation events in P. pastoris (Ardo et al. 2012). Proteins displayed by FS often have O-glycosylated or have high-mannose and/or hybrid structures linked to FS fused protein so the molecular mass of the fusion protein is larger than the calculated value (Goossens et al. 2011; Tanino et al. 2006).
Fluorescence and quantification of protein in the medium
Since no other protein detected, this protein concentration in the supernatant was attributed to the fusion protein. The protein concentration of GS115/pKFS-EGFP-CALB always kept accordant to that of GS115/pKFS-CALB-EGFP during the regular culture process (Table 2). Additionally, fluorescence spectrophotometry indicated that the increased fluorescence intensity of the two strains were similar as well. In addition, the fluorescence intensity of the two strains increased proportionately and respectively with increased protein concentration.
Quantification of molecular number displayed on the cell surface
From the result indicated by Fig. 2, the relationship between the fluorescence intensity and protein concentration for corresponding strain could be described as the following linear formula. The linear formula of GS115/pKFS-EGFP-CALB is y = 50.85 + 8.44 × 10−5x, the linear formula of GS115/pKFS-CALB-EGFP is y = 48.53 + 8.76 × 10−5x, where x is fluorescence intensity (RFU/mL) and y is protein concentration (μg/mL).
In view of the alignment between the fusion protein anchored on the cell wall and the leakage into the medium, we extrapolated the formula above to quantify the protein quantification and calculate the number of molecules on the cell surface.
Paus et al. (2002) indicated that 1OD600 = ∼5 × 107 P. pastoris cells. Therefore from Avogadro’s number, one dalton (Da) has a value of 1.660538921 × 10−27 kg; thus, the number of fused protein molecules displayed per cell is \( y \times 6.02 \times 10^{23} /\left( {M/N} \right) \), where M is molecule weight (kDa) of fusion protein which is deemed to be 220 kDa because of glycosylation, N is cell number and y is protein concentration (μg/mL).
The numbers and trend of the fusion protein displayed on the P. pastoris cell surface during the fermentation process are indicated in Fig. 4. Compared with the number of displayed EGFP molecules per cell calculated by Shibasaki et al. (2001), an approx. order of magnitude as much as 104 per cell for the fusion protein displayed on P. pastoris surface based on FS anchor system could be obtained.
Discussion
Currently, yeast cell surface display systems have become effective tools for protein evolution and enzyme catalyst development. Many yeast surface systems have been developed using P. pastoris as a host (Kondo and Ueda 2004). However, there is no suitable method for quantifying displayed protein; thus, the efficiency of display systems cannot be enhanced and better enzyme display systems using yeast whole cell catalysts cannot be explored. Shibasaki et al. (2001) used S. cerevisiae as a host and a GPI-anchor system such as α-agglutinin. However, a new method for quantifying other displayed proteins in other display systems, particularly for P. pastoris, is necessary. In this work, we proposed a method for quantifying molecular numbers of a model enzyme such as CALB displayed on the cell surface of P. pastoris based on the FS anchor system.
In this study, we constructed strains GS115/pKFS-EGFP, GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP which were then used to display proteins on the P. pastoris cell surface. EGFP was used as a monitoring protein. The results from CLSM and FCL showed that the fluorescence of GS115/pKFS-EGFP is much brighter than that of GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP, but the fluorescence of GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP were similar. CALB activity and protein concentration analysis showed a similar trend for both GS115/pKFS-EGFP-CALB and GS115/pKFS-CALB-EGFP, which reached the same maximum value. Thus, the location of EGFP and CALB in the fused gene does not result in a distinct difference in CALB display.
SDS-PAGE and western blot results demonstrated that the protein displayed on the cell surface and that leaked into the medium had the same molecular weight of approx. 220 kDa and could recognize the EGFP antibody. Thus, the protein displayed on the cell surface and that leaked into the medium were the same and should have the same properties. Additionally, the results of SDS-PAGE combined with those of western blot analysis showed that only the target protein existed in the culture medium, so the fluorescence and protein concentration detected in the medium were due to EGFP-CALB leaked into the medium. During extraction and purification, the EGFP-CALB protein anchored to the cell wall may degenerate and emit green fluorescence. Thus, quantification of CALB displayed on the P. pastoris directly is quite difficult.
The relationship between fluorescence and the protein concentration of the fusion protein leaked into culture medium was linear. Using our designs and results above, the correlation between fluorescence intensity and protein concentration in the medium was found to be also appropriate for fused EGFP-CALB displayed on the cell surface. Based on our linear formula, we developed a method for quantifying protein displayed on the P. pastoris cell surface.
Compared to the results of Shibasaki et al. (2001), the number of the fusion proteins displayed on the surface of P. pastoris is at least in the same order of magnitude as the number of EGFP displayed on the surface of S. cerevisiae). Shibasaki et al. (2001) only observed a single EGFP protein without regard to whether this molecule was part of a fusion protein. Additionally, compared with S. cerevisiae, P. pastoris has more advantages, such as ease of growth to very high cell density and limited presence of other endogenous proteins in the culture medium (Monika et al. 2009). P. pastoris surface display systems appear more promising for industrial application. Quantification of proteins displayed on the P. pastoris surface may provide insight into the development of more efficient yeast surface display systems.
References
Afanassiev V, Sefton M, Anantachaiyong T, Barker G, Walmsley R, Wolfl S (2000) Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances. Mutat Res-gen Tox En 464:297–308
Ardo ML, Sceno D, Huang C-J, Batt CA (2012) Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93:31–39
Bahl MI, Hansen LH, Licht TR, Sørensen SJ (2004) In vivo detection and quantification of tetracycline by use of a whole-cell biosensor in the rat intestine. Antimicrob Agents Chemother 48:1112–1117
Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtiss R III (1997) Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol 15:29–34
Goossens KVY, Stassen C, Stals I et al (2011) The N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae binds specifically to mannose carbohydrates. Eukaryot Cell 10:110–117
Han ZL, Han SY, Zheng SP, Lin Y (2009) Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface. Appl Microbiol Biotechnol 85:117–126
Han SY, Han ZL, Lin Y, Zheng SP (2010) Construction of high efficiency Pichia pastoris surface display system based on Flo1 protein. Prog Biochem Biophys 37(1):1–8
Jaeger K, Eggert T (2002) Lipase for biotechnology. Curr Opin Biotechnol 13:390–397
Jin Z, Ntwali J, Han SY, Zheng SP, Lin Y (2012) Production of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in a batch reactor. J Biotechnol 159:108–114
Kondo A, Ueda M (2004) Yeast cell-surface display - applications of molecular display. Appl Microbiol Biotechnol 64:28–40
Lee SH, Choi JI, Park SJ, Lee SY, Park BC (2004) Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis. Appl Environ Microb 70:5074–5080
Li XY, Zhen MC, Lin W, Liu J, Catherine M, Li J, Wang XH (2008) Construction of a new plasmid for surface display on cells of Yarrowia lipolytica. J Microbiol Methods 72:116–123
Liljeqvist S, Samuelson P, Hansson M, Nguyen TN, Binz H, Ståhl S (1997) Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus. Appl Environ Microb 63:2481–2488
Matsumoto T, Fukuda H, Ueda M et al (2002) Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flo1p flocculation functional domain. Appl Environ Microb 68:4517–4522
Monika B, David R, Francisco V, Pau F (2009) Recent Patents on the Pichia Pastoris expression system: expanding the toolbox for recombinant protein production. Recent Pat Biotechnol 3:192–201
Paus EJ, Willey J, Ridge RJ, Legg CR, Finkelman MA, Novitsky TJ, Ketchum PA (2002) Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification. Protein Expres Purif 26:202–210
Samuelson P, Wernerus H, Svedberg M, Ståhl S (2000) Staphylococcal surface display of metal-binding polyhistidyl peptides. Appl Environ Microb 66:1243–1248
Shibasaki S, Ueda M, Iizuka T, Hirayama M, Ikeda Y, Kamasawa N, Osumi M, Tanaka A (2001) Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses. Appl Microbiol Biotechnol 55:471–475
Su GD, Huang DF, Han S, Yan Zheng S P, Lin Y (2010) Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis. Appl Microbiol Biotechnol 86:1493–1501
Tanaka T, Masunari S, Jun I, Wakamura K, Segawa M, Fukuda H, Kondo A (2010) Displaying non-natural, functional molecules on yeast surfaces via biotin–streptavidin interaction. J Biotechnol 145:79–83
Tanino T, Fukuda H, Kondo A (2006) Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system. Biotechnol Progr 22:989–993
Tanino T, Ohno T, Aoki T et al (2007) Development of yeast cells displaying Candida antarctica lipaseB and their application to ester synthesis reaction. Appl Microbiol Biotechnol 75:1319–1325
Tanino T, Aoki T, Chung W, Watanabe Y, Ogino C, Fukuda H, Kondo A (2009) Improvement of a Candida antarctica lipase B-displaying yeast whole-cell biocatalyst and its application to the polyester synthesis reaction. Appl Microbiol Biotechnol 82:59–66
Wang KC, Patel Chirag A, Wang J, Wang JQ, Wang XM, Luo PP, Zhong PY (2010) Yeast surface display of antibodies via the heterodimeric interaction of two coiled-coil adapters. J Immunol Methods 354:11–19
Acknowledgments
This work was supported by Supported by the grants from National Natural Science Foundation of China (30900017).
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Liang, Xx., Wang, Bb., Sun, Yf. et al. Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system. Biotechnol Lett 35, 367–374 (2013). https://doi.org/10.1007/s10529-012-1085-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-012-1085-2