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Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris
Accepted Manuscript Title: Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris Author: Hailin Yang Yitian Wang Ling Zhang Wei ShenThese authors contributed equally to this work.
S1871-6784(15)00087-4 http://dx.doi.org/doi:10.1016/j.nbt.2015.04.005 NBT 787
To appear in: Received date: Revised date: Accepted date:
29-12-2014 12-3-2015 30-4-2015
Please cite this article as: Yang, H., Wang, Y., Zhang, L., Shen, W.,Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris, New Biotechnology (2015), http://dx.doi.org/10.1016/j.nbt.2015.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris
Key Laboratory of Industrial Biotechnology of Ministry of Education; School of
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Hailin Yang1,*,†, Yitian Wang1,†, Ling Zhang1 and Wei Shen1,*
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Biotechnology; Jiangnan University; Wuxi 214122, China
These authors contributed equally to this work.
*
Corresponding authors: Hailin Yang, Tel.: +86-510-85918119, Fax: +86-510-85918119, E-mail:
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†
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[email protected]
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[email protected]; Wei Shen, Tel.: +86-510-85918122, Fax: +86-510-85918122, E-mail:
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Highlights: (1) This is the first paper on the heterologous expression of fructosyltransferase (FTase) from A. niger in P. pastoris.
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(3) The FTase had a high specific activity, which was 6.8 × 104 U/mg.
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(2) The yield of recombinant FTase was high, and reached 1,020.0 U/mL in a 5-L fermentor.
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(4) The highest yield of FOS reached 343.3 g/L (w/v).
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Abstract
In this work, the cDNA encoding fructosyltransferase (FTase) from Aspergillus niger YZ59
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(CICIM F0901) was obtained and expressed in the methylotrophic yeast Pichia pastoris strain GS115. The yield of recombinant FTase in a 5-L fermentor reached 1,020.0 U/mL after 96 h
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of induction, which was 1,160.4 times higher that of native FTase from A. niger YZ59. The
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specific activity of recombinant FTase was 6.8 × 104 U/mg. The optimum temperature and
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pH of the recombinant FTase were 55°C and 5.5, respectively. The recombinant FTase was stable below 40°C and at pH from 3.0 to 10.0. Using sucrose as the substrate, the Km and Vmax values of recombinant FTase were 159.8 g/L and 0.66 g/(L·min), respectively. The turnover number (kcat) and catalytic efficiency (kcat/Km) of recombinant FTase was 1.1 × 104
min−1 and 68.8 L/(g·min), respectively. The recombinant FTase was slightly activated by 5 mM Ni2+, Mg2+, K+, Fe3+, or Mn2+, but inhibited by all other metal ions (Na+, Li+, Ba2+, Ca2+, Zn2+, and Cu2+). The highest yield of fructooligosaccharides for purified FTase reached
approximately 343.3 g/L (w/v). This is the first study reporting the heterologous expression of FTases from A. niger in P. pastoris. This study plays an important role in the
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fructooligosaccharide synthesis industry by recombinant FTases.
Keywords: Aspergillus niger; Fructosyltransferase; Overexpression; Pichia pastoris;
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Characterization
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Introduction
Fructooligosaccharides (FOS) are composed of linear chains of fructose units, linked by beta
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(2-1) bonds, often terminating in a glucose unit. The main types of FOS are 1-kestose (GF2), nystose (GF3), and fructosylnystose (GF4) [1], composed, respectively, by two, three, or four
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fructosyl units (F) bound to a sucrose molecule (GF) by a β-(1,2) bond [2]. Wheat, garlic,
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banana, honey, and onion are the most common sources of FOS [3]. FOS selectively support
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the growth of beneficial gut microflora (e.g., Bifidobacterium and Lactobacillus) [4], and are therefore considered as prebiotics [5]. The synthesis of FOS is mainly catalyzed by the fructosyltransferases (FTases), which belong to the glycoside hydrolase 32 (GH32) and 68 (GH68) families [6].
FTases are found in plants, fungi, and bacteria [6] such as Asparagus officinalis [7],
Aspergillus niger [8], and Penicillium frequentans [9]. FTases from different sources have different physical and chemical properties, and industrial enzymes with specific properties have been selected based on specific needs. Microbial FTases have unique properties for synthesizing FOS, and have been used for the last 20 years [10-12]. However, the low yields
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obtained with wild-type FTases make them unsuitable for large-scale industrial application. An FTase from Lactobacillus reuteri has been heterologously expressed in Escherichia coli; however, its sucrose-hydrolyzing activity was only 1.1 U/mL [13]. A fructan:fructan
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6G-fructosyltransferase (6G-FFT) from A. officinalis has also been cloned and expressed in
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Pichia pastoris, and its enzymatic activity was 0.1 U/mL [7].
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The methylotrophic yeast P. pastoris can express heterologous proteins from different sources, either intracellularly or extracellularly, with high expression level and secretion
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efficiency [14]. Commonly used P. pastoris expression strains include protease-deficient strains (e.g., P. pastoris SMD1165) and auxotrophic mutant P. pastoris GS115 [15]. As a
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eukaryotic expression system, P. pastoris presents many advantages for highly efficient expression of heterologous proteins, such as folding, post-translational modifications (e.g.,
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glycosylation), highly efficient signal peptide cleavage, and high secretion capacity [16,17].
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Several enzymes such as alkaline polygalacturonate lyase and α-amylase have been
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heterologously overexpressed in P. pastoris [14,18]. Nevertheless, the expression of FTases
from A. niger in P. pastoris has not yet been reported. In this work, cDNA encoding the FTase gene from A. niger was purified and the protein
was heterologously expressed in P. pastoris. The enzyme yield, specific activity, optimal
temperature and pH, thermal and pH stability, catalytic efficiency, effects of inhibitor (glucose), substrate specificity, and effects of metal ions were investigated.
Materials and methods Strains, Vectors and Materials
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A. niger YZ59 (CICIM F0901) was obtained from The Culture & Information Center of Industrial Microorganism of China Universities. P. pastoris GS115 and plasmid pPIC9K were purchased from Invitrogen (Carlsbad, CA, USA). The Agarose Gel DNA Purification Kit,
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Plasmid Miniprep Kit, T4 DNA ligase, and restriction enzymes were obtained from TakaRa
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(Dalian, China). GF2, GF3, and GF4 were obtained from Wako Pure Chemical Industries, Ltd.
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(Osako, Japan). Other chemicals (e.g. sucrose) were obtained from Shanghai Sangon
Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). DNA primers were
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synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd.
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(Shanghai, China).
Cloning of A. niger FTase
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The A. niger FTase cDNA, without its introns, was synthesized by SuperScript III First-Strand
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Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). The cDNA obtained by RT-PCR
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was used as a template for PCR. The sequences of the primers were as follows: Forward primer, 5′-CCGGAATTCATGAAGCTTCAAACGGCTTC-3′; Reverse primer, 5′-ATAAGAATGCGGCCGCTTAGTGATGATGATGATGAGACTGACGATCCGGCCA-3′. The primer sequences contained the EcoRI (5′) and NotI (3′) restriction sites (italic). The PCR conditions were as follows: initial denaturation step at 95°C for 5 min; 30 cycles of denaturation (94°C for 40 s), annealing (60°C for 2 min), and extension (72°C for 5 min); and a final extension step at 72°C for 12 min.
Construction and transformation of the recombinant plasmid pPIC9K-fwt
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The mature FTase gene was obtained by RT-PCR and PCR, as described above. The PCR product and vector pPIC9K were digested with EcoRI and NotI and gel-purified. The digested PCR product was then ligated into the digested pPIC9K. The identity of recombinant plasmid
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pPIC9K-fwt was confirmed by restriction analysis and sequencing. pPIC9K-fwt was linearized
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with SacI and then transformed into P. pastoris GS115 by electroporation. A Gene Pulser
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system (Bio-Rad, Hercules, California, USA) was used for electroporation, and the
electroporation conditions used were 1.5 kV, 200 Ω, and 25 μF. Transformants were grown
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on minimal dextrose medium (MD) agar plates at 30°C for 3 d [14]. The transformants
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growing on YPD agar plates with G418 (3.0 mg/mL) were selected.
Expression of FTase in P. pastoris
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Seed cultures of the selected colonies were incubated at 30°C for 24 h in 50 mL YPD medium
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in the flask (500 mL, 200 rmp). Approximately 180 mL seed broth was inoculated into 1.8 L
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basic salt medium (BSM) in a 5-L fermentor (BLBIO, China). The recombinant P. pastoris was cultured at batch grown at 30°C and pH 6.0. The pH was automatically controlled by addition of ammonium hydroxide (25%, w/v). When the dry cell weight (DCW) reached 25.0 g/L, the cultures were continuously fed with glycerol (50%, v/v). Feeding was controlled by dissolved oxygen measurements (DO-stat). Dissolved oxygen (DO) level was controlled below 30%. When the DCW reached 111.0 g/L, protein expression was induced by the addition of pure methanol for 120 h, also controlled by DO-stat and with a DO level below 30%. The protein production was sampled every 8 h during the induction, and the volume of each sampling was 5 mL.
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Purification of FTase The supernatant of recombinant P. pastoris was obtained by centrifuging the cultures at
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10,000 ×g for 20 min at 4°C. The supernatant (10 mL) was filtered and loaded onto a Ni2+
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column (GE Healthcare, Houston, TX, USA). The 6His-tagged FTase was purified using an
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AKTA purifier (GE Healthcare, Houston, TX, USA). Equilibration buffer (A) was phosphate buffer (50 mM, pH 7.4) with 20 mM imidazole, and elution buffer (B) was phosphate buffer
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(50 mM, pH 7.4) with 250 mM imidazole. The flow rate of purification was 1.0 mL/min. The sample fractions were collected based on a linear elution by ramping 0~100% buffer B. The
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pooled FTase fractions obtained was dialyzed at 4°C in 100 mM phosphate-citrate buffer (pH 5.5). The protein concentration was determined by SpectraMax Plus (Molecular Devices,
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Enzyme assays
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Sunnyvale, California, USA) based on the Bradford method.
FTase activity was determined by measuring the amount of GF2 product in the reaction mixture by HPLC [19]. The reaction mixture contained sucrose (25%, w/v) as a substrate, dissolved in 100 mM phosphate-citrate buffer (pH 5.5). The reaction was incubated at 40°C for 15 min and stopped by heating at 100°C for 10 min [20]. One unit of FTase activity was
defined as the amount of enzyme that produced 1 μmol GF2 per min at the assay conditions.
Effects of temperature on FTase activity The optimal temperature of FTase was analyzed at temperatures ranging from 30°C to 70°C
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in phosphate-citrate buffer (100 mM, pH 5.5), using sucrose as a substrate. The highest activity was taken as 100% at different temperatures. The thermostability of FTase was determined at different temperatures (40°C, 50°C, and 60°C) in phosphate-citrate buffer
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(100 mM, pH 5.5). The initial activity before incubation at various temperatures was taken as
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100%. Three independent experiments were performed.
Effects of pH on FTase activity
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To estimate the optimal pH for FTase activity, the purified protein was incubated at pH 3.0~8.0 (phosphate-citrate buffer, 100 mM). The activity determined at pH 6.0
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(phosphate-citrate buffer, 100 mM) was taken as 100%. In order to determine the pH stability, the enzyme was pre-incubated in the various buffers at 25°C for 24 h, and then
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assayed for residual activity at pH 5.5. The buffers used for determination of pH stability
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were as follows: phosphate-citrate buffer (100 mM, pH 3.0~8.0), phosphate buffer (100 mM,
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pH 8.0~9.0), and glycine-sodium hydroxide buffer (100 mM, pH 9.0~11.0). The highest residual activity was taken as 100% at different pH values. Three independent experiments were performed.
Determination of kinetic parameters Enzyme assays were performed in phosphate-citrate buffer (100 mM, pH 5.5) at 40°C, using sucrose as a substrate. Substrate concentrations were in the range 20~600 g/L. The kinetic parameters Km and Vmax were calculated by Eadie–Hofstee plots [21]. Three independent experiments were performed.
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Effect of metal ions on FTase activity To determine the effect of metal ions on FTase activity, the enzyme was pre-incubated with
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0.5 mM and 5 mM metal ions (K+, Li+, Na+, Ca2+, Mg2+, Mn2+, Zn2+, Fe3+, Ba2+, Ni2+, and Cu2+)
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and ethylene diamine tetraacetic acid (EDTA), respectively. The relative activity of FTase was
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determined, and compared to the activity obtained in phosphate-citrate buffer (100 mM, pH
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5.5) without the addition of any ions. Three independent experiments were performed.
Synthesis of fructooligosaccharides and HPLC analysis
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FOS synthesis was carried out for 12 h at 40°C and pH 5.5 (phosphate-citrate buffer, 100 mM). The high concentration of sucrose (600 g/L, w/v) as the initial substrate was used to
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analyze the FOS synthesis ability of this FTase during FOS industrial production [22,23]. The
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amount of FTase used was 20 U/g (sucrose) during the FOS synthesis reaction. The final
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reaction volume for the FOS synthesis was 50 mL, and the volume of each sampling for HPLC analysis was 1 mL. The samples withdrawn were heated to stop the reaction at 100°C for 10 min. After cooling, the samples were filtered by microporous membrane (0.2 μm). The concentrations of FOS synthesized by FTase at different phases were detected by HPLC. The conditions of the HPLC analysis were as followings: Refractive Index (RI) detector, Waters XbridgeTM Amide column (5 μm, 4.6 × 250 mm), acetonitrile-water (70:30, v/v), 30°C, and 1.5 mL/min. Three independent experiments were performed.
Results and discussion
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Cloning and expression of recombinant FTase in P. pastoris The cDNA of mature FTase without its introns and signal peptide was obtained by RT-PCR from the total RNA of A. niger YZ59 (CICIM F0901), and cloned into the P. pastoris vector
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pPIC9K (Fig. 1A). Nucleotide sequence analysis revealed that the gene was 1,884 bp long
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(Supplementary material) and that the protein encoded consisted of 628 amino acids. The
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cDNA sequence of FTase from A. niger YZ59 was compared with that of A. niger β-D-fructofuranosidase (suc1) [24], which shared 99.6% homology with that of
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β-D-fructofuranosidase (suc1) (GenBank: L06844.1). The amino acid sequence shared 98% homology with that of β-D-fructofuranosidase [24].
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The target fragment was integrated into the plasmid pPIC9K and transformed into P. pastoris GS115. The transformants were grown in MD agar plates and selected in YPD agar
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plates with G418 (3.0 mg/mL). The insertion of FTase gene in recombinant P. pastoris was
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confirmed by PCR. The control was the P. pastoris GS115 transformed with plasmid pPIC9K.
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The recombinant transformants were induced in BMMY medium (25 mL) at 30°C for 120 h in 250 mL shaker flasks. No FTase activity was detected in the supernatant of the control strain. The FTase activity in the supernatant was 117.3 U/mL under the same conditions, which was 133.4 times higher than that of native FTase from A. niger YZ59. The yield of FTase from wild-type A. niger was low in most reported cases. For example, Lateef et al. obtained the
maximum activity (24.5 U/mL) of FTase from wild-type A. niger in submerged fermentation after 48 h of fermentation [25]. The recombinant FTase from the supernatant was purified by His-tag affinity chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1B), and the specific activity of purified FTase was 6.8 ×
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104 U/mg. The general mechanism of glycoside hydrolase is a double-displacement mechanism, including glycosylation and deglycosylation [26]. According to the FTase in this work, there might be only one substrate-binding site supporting this double-displacement
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mechanism, which was consistent with the catalytic mechanism of fructosyltransferase from
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Aspergillus japonicus reported [26].
FTase production by recombinant P. pastoris in a 5-L fermentor
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The expression levels of FTase in recombinant P. pastoris GS115 were further explored in a 5-L fermentor (BLBIO, China). Fig. 2 shows the time profiles of cell growth of recombinant P.
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pastoris and its FTase production. The DCW of recombinant P. pastoris reached 111.1 g/L at the end of the glycerol fed-batch phase (64 h), and the highest specific growth rate was 1.7
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h−1. After induction for 96 h, the highest FTase activity in the supernatant reached 1,020.0
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U/mL, which was 8.7-fold higher than that obtained in shaker flasks. The highest specific
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production rate was 69.2 U/(g·h). The high yield of FTase indicated that recombinant P.
pastoris was a suitable host for the efficient expression and industrial production of FTase.
Effect of temperature on FTase activity and stability As shown in Fig. 3A, the activity of FTase increased at temperatures between 30°C and 55°C and decreased at temperatures between 55°C to 70°C. The optimum temperature was 55°C, and the activity at optimum temperature was 3.6-fold higher than that at 30°C. Fig. 3B shows that the FTase retained approximately 60% of its activity after incubation at 50°C for 60 min; however, its activity declined rapidly at temperatures above 60°C. The FTase was
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stable at 40°C for 60 min, and retained approximately 83.1% of its activity after incubation at 40°C for 6 d.
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Effect of pH on FTase activity and stability
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As shown in Fig. 4A, the optimum pH of FTase was 5.5. FTase activity rapidly increased with
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an increase in pH from 3.0 to 5.5, and decreased with an increase in pH from 5.5 to 8.0. It exhibited its highest value (> 90% of maximal activity) between pH 4.5 and 6.5. The stability
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curve of FTase at different pH values is shown in Fig. 4B. FTase displayed optimal stability at pH 6.0, and retained more than 90% of maximum activity between pH 4.0 and 9.0 after
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incubation at 25°C for 24 h. It also retained approximately 80% of maximal activity at pH values lower than 2.0 or higher than 11.0. The recombinant FTase expressed in P. pastoris
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was stable over a broad pH range.
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Kinetic parameters of FTase
The kinetics of FTase was determined using sucrose as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) values were analyzed by nonlinear curve analysis based on Eadie–Hofstee plots. The Km and Vmax values were 159.8 g/L and 0.66 g/(L·min),
respectively (Fig. 5). The turnover number (kcat) and catalytic efficiency (kcat/Km) values of
FTase were 1.1 × 104 min−1 and 68.8 L/(g·min), respectively. Jung et al. determined the properties of the fructosyltransferase from Aureobasidium pullulans, and found that the Km value and Vmax value for sucrose were 330 g/L and 130 g/(L·h) [27]. The kinetic constants of a fructosyltransferase from Rhodotorula sp. were determinated, and the Km value for sucrose
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was 197.9 g/L [22]. It was indicated that the substrate affinity of these fructosyltransferase from A. pullulans and Rhodotorula sp. was lower than that of FTase heterologously expressed in P. pastoris in this work.
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Fructose with high concentration (˃ 50%, w/v) did not have a significant inhibitory effect
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on the kinetic parameters of FTase, the inhibition by which was considered negligible, but
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glucose has a high competitive inhibition [22]. In order to evaluate the inhibitory effect of glucose on the kinetic parameters of FTase, 20 g/L glucose was added to the enzyme
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reaction medium. As shown in Fig. 5, glucose acts as a competitive inhibitor for the catalytic reaction of FTase on sucrose. The Ki value for glucose (20 g/L) as an inhibitor was calculated
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by Eadie–Hofstee plots, and was found to be 45.0 g/L. Alvarado-Huallanco and Maugeri-Filho calculated the Ki,app value of glucose on fructosyltransferase from Rhodotorula sp. by
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Lineweaver-Burk’s technique, which were 54.79 g/L and 55.20 g/L for purified enzyme and
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partially purified enzyme, respectively [28].
Effect of metal ions on FTase activity Metal ions play a key role in the folding and catalysis of proteins [29]. The effects of metal ions on FTase activity are shown in Table 1. FTase activity was enhanced by 5 mM Ni2+ or Mg2+, indicating that the activity might be Ni2+- or Mg2+-dependent, with a relative activity of
118.0% or 109.9%, respectively. No inhibition or activation of FTase was observed with 5 mM K+, Fe3+, or Mn2+, with relative activities of 105.3%, 101.0%, and 101.8%, respectively. Other metal ions had slight inhibitory effects on FTase activity, as follows: Na+ (0.5 and 5 mM), K+ (0.5 mM), Li+ (0.5 and 5 mM), Ba2+ (0.5 and 5 mM), Ca2+ (0.5 and 5 mM), Fe3+ (0.5
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mM), Zn2+ (0.5 and 5 mM), Mn2+ (0.5 mM), Cu2+ (0.5 and 5 mM), Mg2+ (0.5 mM), and Ni2+ (0.5 mM). Low concentration of EDTA (0.5 mM) had slight inhibitory effects on FTase activity. But the FTase was completely inactivated by 5 mM EDTA, indicating that the FTase activity might
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be divalent ions-dependent. FTase from Aspergillus oryzae was inhibited by Cu2+ and Zn2+ (5
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mM), with a relative activity of 11% and 17%, respectively [12]. The activation or inhibition
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of metal ions on FTase from other sources was also observed [30,31].
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Fructooligosaccharide synthesis
FOS synthesis was carried out for 12 h using sucrose as an initial substrate (600 g/L) at 40°C
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and pH 5.5. The highest yield of FOS for purified recombinant FTase reached approximately 343.3 g/L (w/v) at 2 h (Fig. 6, Supplementary material). During the entire process, the
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maximum yields of GF2, GF3, and GF4 reached 246.0 g/L (w/v) at 1.5 h, 186.0 g/L (w/v) at 10 h,
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and 88.2 g/L (w/v) at 12 h, respectively. Previous studies have reported longer durations
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required to achieve the maximum FOS yield [27, 32]. Alvarado-Huallanco and Maugeri-Filho, using purified fructosyltransferase from Rhodotorula sp., obtained a FOS production of 46% from sucrose concentration of 500 g/L (w/v) after 96 h of synthesis, the final composition of which were GF2 33.92% (w/w), GF3 9.01% (w/w), and GF4 3.40% (w/w), respectively [27].
Ghazi et al. achieved maximum FOS production from sucrose (650 g/L) by using immobilized Aspergillus aculeatus FTase after a 36-h reaction [32]. A. niger consumed sucrose for FOS formation after exit of lag phase, and the average yield of FOS by A. niger was 59.71 ± 2.32% (w/w) by recycling of cell culture [33].
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Conclusions cDNA of FTase from A. niger YZ59 was obtained by RT-PCR and expressed in P. pastoris GS115. The yield of recombinant FTase in a 5-L fermentor reached 1,020.0 U/mL, which was
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1,160.4-fold higher than that of native FTase from A. niger YZ59. The specific activity of
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purified FTase was 6.8 × 104 U/mg. The optimal temperature of the recombinant FTase was
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55°C, and it was stable at temperatures below 40°C. The optimum pH of FTase was 5.5, and it was stable at pH ranging from 3.0 to 10.0. The Km, Vmax, kcat, and kcat/Km values of
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recombinant FTase were 159.8 g/L, 0.66 g/(L·min), 1.1 × 104 min-1, and 68.8 L/(g·min), respectively. The Ki value of FTase for glucose (20 g/L) was 45.0 g/L. The recombinant FTase
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was slightly activated by 5 mM Ni2+, Mg2+, K+, Fe3+, or Mn2+, but inhibited by other metal ions. The highest yield of FOS for FTase reached approximately 343.3 g/L (w/v) after 2 h of
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Acknowledgements
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synthesis based on 600 g/L sucrose as an initial substrate.
This project was financially supported by the Fundamental Research Funds for the Central Universities (JUSRP51402A), the 111 Project (111-2-06), the Fundamental Research Funds for the Central Universities (JUSRP11429), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu province "Collaborative Innovation Center for Advanced Industrial Fermentation" industry development program.
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[15] Liu L, Yang HQ, Shin H-d, Chen RR, Li JH, Du GC, Chen J. How to achieve high-level expression of microbial enzymes: Strategies and perspectives. Bioeng 2013;4:212-23.
[16] Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 2014;98:5301-17. [17] Vogl T, Hartner FS, Glieder A. New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris. Curr Opin Biotechnol 2013;24:1094-101. [18] Wang HL, Li JH, Liu L, Li X, Jia DX, Du GC, Chen J, Song JN. Increased production of alkaline
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polygalacturonate lyase in the recombinant Pichia pastoris by controlling cell concentration during continuous culture. Bioresour Technol 2012;124:338-46. [19] Arrizon J, Morel S, Gschaedler A, Monsan P. Fructanase and fructosyltransferase activity of
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non-Saccharomyces yeasts isolated from fermenting musts of Mezcal. Bioresour Technol
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2012;110:560-5.
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[20] Salinas MA, Perotti NI. Production of fructosyltransferase by Aureobasidium sp. ATCC 20524 in batch and two-step batch cultures. J Ind Microbiol Biotechnol 2009;36:39-43.
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[21] Yang HQ, Liu L, Li JH, Du GC, Chen J. Heterologous expression, biochemical characterization, and overproduction of alkaline alpha-amylase from Bacillus alcalophilus in Bacillus subtilis. Microb
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Cell Fact 2011;10.
[22] Alvarado M, Maugeri F. Kinetic modelling of fructooligosaccharide synthesis using a Rhodotorula
te
d
sp fructosyltransferase. J Biotechnol 2007;131:S91-2. [23] Ghazi I, De Segura AG, Fernandez-Arrojo L, Alcalde M, Yates M, Rojas-Cervantes ML, Plou FJ,
Ac ce p
Ballesteros A. Immobilisation of fructosyltransferase from Aspergillus aculeatus on
epoxy-activated Sepabeads EC for the synthesis of fructo-oligosaccharides. J Mol Catal B-Enzym 2005;35:19-27.
[24] Boddy LM, Berges T, Barreau C, Vainstein MH, Dobson MJ, Ballance DJ, Peberdy JF. Purification and Characterization of an Aspergillus niger invertase and its DNA-sequence. Curr Genet
1993;24:60-6. [25] Lateef A, Oloke JK, Gueguim-Kana EB, Raimi OR. Production of fructosyltransferase by Aspergillus niger in both submerged and solid substrate media. Acta Alimentaria 2012;41:100-17. [26] Chuankhayan P, Hsieh CY, Huang YC, Hsieh YY, Guan HH, Hsieh YC, Tien YC, Chen CD, Chiang CM,
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Chen CC. Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis. J Biol Chem 2010;285:23249-62.
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fructo-oligosaccharides from sucrose. Enzyme Microb Technol 1989;11:491-4.
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[27] Jung KH, Yun JW, Kang KR, Lim JY, Lee JH. Mathematical model for enzymatic production of
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[28] Alvarado-Huallanco MB, Maugeri-Filho F. Kinetics and modeling of fructo-oligosaccharide synthesis by immobilized fructosyltransferase from Rhodotorula sp. J Chem Technol Biotechnol
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2010;85:1654-62.
[29] Yang HQ, Liu L, Li JH, Du GC, Chen J. Cloning, heterologous expression, and comparative
Ann Microbiol 2012;62:1219-26.
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characterization of a mesophilic alpha-amylase gene from Bacillus subtilis JN16 in Escherichia coli.
te
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[30] Fujishima M, Sakai H, Ueno K, Takahashi N, Onodera S, Benkeblia N, Shiomi N. Purification and characterization of a fructosyltransferase from onion bulbs and its key role in the synthesis of
Ac ce p
fructo-oligosaccharides in vivo. New Phytol 2005;165:513-24.
[31] Ghazi I, Fernandez-Arrojo L, Garcia-Arellano H, Ferrer M, Ballesteros A, Plou FJ. Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus. J Biotechnol 2007;128:204-11.
[32] Ghazi I, De Segura AG, Fernandez-Arrojo L, Alcalde M, Yates M, Rojas-Cervantes ML, Plou FJ, Ballesteros
A.
Immobilisation
of fructosyltransferase
from Aspergillus aculeatus
on
epoxy-activated Sepabeads EC for the synthesis of fructo-oligosaccharides. J Mol Catal B-Enzym 2005;35:19-27. [33] Ganaie MA, Gupta US. Recycling of cell culture and efficient release of intracellular
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fructosyltransferase by ultrasonication for the production of fructooligosaccharides. Carbohydr
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cr
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Polym 2014;110:253-8.
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Figure legends:
Fig. 1: Map of recombinant vector and SDS-PAGE analysis. A: Map of recombinant vector
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pPIC9K-fwt. B: SDS-PAGE gel of the FTase purification. M: Maker, fragment indicated by arrow: FTase.
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Fig. 2: Time profiles of FTase production by recombinant P. pastoris in 5-L fermentor.
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Fig. 3: Effect of temperature on the activity and stability of FTase. A: Effect of temperature on
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the activity of FTase. For determination of optimum temperature of FTase, the reaction was conducted from 30 to 70°C. B: Effect of temperature on the stability of FTase. The insert presents the thermal stability of FTase at 40°C for 6 d. The thermal stability of FTase was determined at 40°C for 6 d. The thermal stability of FTase was determined at 50 or 60°C for 60 min, respectively.
Fig. 4: Effect of pH on the activity and stability of FTase. A: Effect of pH on the activity of FTase. The FTase activity was determined at pH 3.0~8.0, respectively. B: Effect of pH on the stability of FTase. The pH stability was determined at pH 3.0~11.0. Fig. 5: Eadie-Hofstee plots for using sucrose as substrate by FTase. In order to evaluate the
Page 20 of 28
inhibitory effect of glucose on the kinetic parameters of FTase, glucose (20 g/L) was added into the enzyme reaction medium. GF: sucrose as substrate; GF+G (20 g/L): 20 g/L glucose was added.
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Fig. 6: FOS production by FTase. The synthesis of FOS was carried out for 12 h by using
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sucrose as initial substrate (600 g/L) at 40°C and pH 5.5. G: glucose, GF: sucrose, GF2:
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M
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1-kestose, GF3: nystose, GF4: fructosylnystose, FOS: Fructooligosaccharides.
Page 21 of 28
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Table:
Concentration (mM)
0.5
Metal ions
Relative activity (%) 90.8±1.5 99.4±1.2 93.9±0.9 105.3±1.4 97.8±1.6 99.3±1.1 95.9±2.2 86.6±1.3 89.9±2.0 99.5±1.3 88.6±1.7 101.0±0.9 89.9±1.1 92.4±1.5 83.9±1.4 101.8±1.6 87.6±2.0 95.1±2.2 84.9±1.8 109.9±2.0 94.8±2.5 118.0±1.7 95.9±2.3 0.2±0.0 100.0 100.0
M d te
Ac ce p
Na K+ Li+ Ba2+ Ca2+ Fe3+ Zn2+ Mn2+ Cu2+ Mg2+ Ni2+ EDTA CK
5
an
+
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Table 1 Effect of metal ions on the activity of FTase
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Figure-1
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Figure-2
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Figure-5
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S1871-6784(15)00087-4 http://dx.doi.org/doi:10.1016/j.nbt.2015.04.005 NBT 787
To appear in: Received date: Revised date: Accepted date:
29-12-2014 12-3-2015 30-4-2015
Please cite this article as: Yang, H., Wang, Y., Zhang, L., Shen, W.,Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris, New Biotechnology (2015), http://dx.doi.org/10.1016/j.nbt.2015.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris
Key Laboratory of Industrial Biotechnology of Ministry of Education; School of
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1
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Hailin Yang1,*,†, Yitian Wang1,†, Ling Zhang1 and Wei Shen1,*
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Biotechnology; Jiangnan University; Wuxi 214122, China
These authors contributed equally to this work.
*
Corresponding authors: Hailin Yang, Tel.: +86-510-85918119, Fax: +86-510-85918119, E-mail:
M
an
†
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[email protected]
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[email protected]; Wei Shen, Tel.: +86-510-85918122, Fax: +86-510-85918122, E-mail:
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Highlights: (1) This is the first paper on the heterologous expression of fructosyltransferase (FTase) from A. niger in P. pastoris.
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(3) The FTase had a high specific activity, which was 6.8 × 104 U/mg.
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(2) The yield of recombinant FTase was high, and reached 1,020.0 U/mL in a 5-L fermentor.
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(4) The highest yield of FOS reached 343.3 g/L (w/v).
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Abstract
In this work, the cDNA encoding fructosyltransferase (FTase) from Aspergillus niger YZ59
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(CICIM F0901) was obtained and expressed in the methylotrophic yeast Pichia pastoris strain GS115. The yield of recombinant FTase in a 5-L fermentor reached 1,020.0 U/mL after 96 h
d
of induction, which was 1,160.4 times higher that of native FTase from A. niger YZ59. The
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specific activity of recombinant FTase was 6.8 × 104 U/mg. The optimum temperature and
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pH of the recombinant FTase were 55°C and 5.5, respectively. The recombinant FTase was stable below 40°C and at pH from 3.0 to 10.0. Using sucrose as the substrate, the Km and Vmax values of recombinant FTase were 159.8 g/L and 0.66 g/(L·min), respectively. The turnover number (kcat) and catalytic efficiency (kcat/Km) of recombinant FTase was 1.1 × 104
min−1 and 68.8 L/(g·min), respectively. The recombinant FTase was slightly activated by 5 mM Ni2+, Mg2+, K+, Fe3+, or Mn2+, but inhibited by all other metal ions (Na+, Li+, Ba2+, Ca2+, Zn2+, and Cu2+). The highest yield of fructooligosaccharides for purified FTase reached
approximately 343.3 g/L (w/v). This is the first study reporting the heterologous expression of FTases from A. niger in P. pastoris. This study plays an important role in the
Page 2 of 28
fructooligosaccharide synthesis industry by recombinant FTases.
Keywords: Aspergillus niger; Fructosyltransferase; Overexpression; Pichia pastoris;
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Characterization
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Introduction
Fructooligosaccharides (FOS) are composed of linear chains of fructose units, linked by beta
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(2-1) bonds, often terminating in a glucose unit. The main types of FOS are 1-kestose (GF2), nystose (GF3), and fructosylnystose (GF4) [1], composed, respectively, by two, three, or four
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fructosyl units (F) bound to a sucrose molecule (GF) by a β-(1,2) bond [2]. Wheat, garlic,
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banana, honey, and onion are the most common sources of FOS [3]. FOS selectively support
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the growth of beneficial gut microflora (e.g., Bifidobacterium and Lactobacillus) [4], and are therefore considered as prebiotics [5]. The synthesis of FOS is mainly catalyzed by the fructosyltransferases (FTases), which belong to the glycoside hydrolase 32 (GH32) and 68 (GH68) families [6].
FTases are found in plants, fungi, and bacteria [6] such as Asparagus officinalis [7],
Aspergillus niger [8], and Penicillium frequentans [9]. FTases from different sources have different physical and chemical properties, and industrial enzymes with specific properties have been selected based on specific needs. Microbial FTases have unique properties for synthesizing FOS, and have been used for the last 20 years [10-12]. However, the low yields
Page 3 of 28
obtained with wild-type FTases make them unsuitable for large-scale industrial application. An FTase from Lactobacillus reuteri has been heterologously expressed in Escherichia coli; however, its sucrose-hydrolyzing activity was only 1.1 U/mL [13]. A fructan:fructan
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6G-fructosyltransferase (6G-FFT) from A. officinalis has also been cloned and expressed in
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Pichia pastoris, and its enzymatic activity was 0.1 U/mL [7].
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The methylotrophic yeast P. pastoris can express heterologous proteins from different sources, either intracellularly or extracellularly, with high expression level and secretion
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efficiency [14]. Commonly used P. pastoris expression strains include protease-deficient strains (e.g., P. pastoris SMD1165) and auxotrophic mutant P. pastoris GS115 [15]. As a
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eukaryotic expression system, P. pastoris presents many advantages for highly efficient expression of heterologous proteins, such as folding, post-translational modifications (e.g.,
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glycosylation), highly efficient signal peptide cleavage, and high secretion capacity [16,17].
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Several enzymes such as alkaline polygalacturonate lyase and α-amylase have been
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heterologously overexpressed in P. pastoris [14,18]. Nevertheless, the expression of FTases
from A. niger in P. pastoris has not yet been reported. In this work, cDNA encoding the FTase gene from A. niger was purified and the protein
was heterologously expressed in P. pastoris. The enzyme yield, specific activity, optimal
temperature and pH, thermal and pH stability, catalytic efficiency, effects of inhibitor (glucose), substrate specificity, and effects of metal ions were investigated.
Materials and methods Strains, Vectors and Materials
Page 4 of 28
A. niger YZ59 (CICIM F0901) was obtained from The Culture & Information Center of Industrial Microorganism of China Universities. P. pastoris GS115 and plasmid pPIC9K were purchased from Invitrogen (Carlsbad, CA, USA). The Agarose Gel DNA Purification Kit,
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Plasmid Miniprep Kit, T4 DNA ligase, and restriction enzymes were obtained from TakaRa
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(Dalian, China). GF2, GF3, and GF4 were obtained from Wako Pure Chemical Industries, Ltd.
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(Osako, Japan). Other chemicals (e.g. sucrose) were obtained from Shanghai Sangon
Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). DNA primers were
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synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd.
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(Shanghai, China).
Cloning of A. niger FTase
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The A. niger FTase cDNA, without its introns, was synthesized by SuperScript III First-Strand
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Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). The cDNA obtained by RT-PCR
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was used as a template for PCR. The sequences of the primers were as follows: Forward primer, 5′-CCGGAATTCATGAAGCTTCAAACGGCTTC-3′; Reverse primer, 5′-ATAAGAATGCGGCCGCTTAGTGATGATGATGATGAGACTGACGATCCGGCCA-3′. The primer sequences contained the EcoRI (5′) and NotI (3′) restriction sites (italic). The PCR conditions were as follows: initial denaturation step at 95°C for 5 min; 30 cycles of denaturation (94°C for 40 s), annealing (60°C for 2 min), and extension (72°C for 5 min); and a final extension step at 72°C for 12 min.
Construction and transformation of the recombinant plasmid pPIC9K-fwt
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The mature FTase gene was obtained by RT-PCR and PCR, as described above. The PCR product and vector pPIC9K were digested with EcoRI and NotI and gel-purified. The digested PCR product was then ligated into the digested pPIC9K. The identity of recombinant plasmid
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pPIC9K-fwt was confirmed by restriction analysis and sequencing. pPIC9K-fwt was linearized
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with SacI and then transformed into P. pastoris GS115 by electroporation. A Gene Pulser
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system (Bio-Rad, Hercules, California, USA) was used for electroporation, and the
electroporation conditions used were 1.5 kV, 200 Ω, and 25 μF. Transformants were grown
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on minimal dextrose medium (MD) agar plates at 30°C for 3 d [14]. The transformants
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growing on YPD agar plates with G418 (3.0 mg/mL) were selected.
Expression of FTase in P. pastoris
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Seed cultures of the selected colonies were incubated at 30°C for 24 h in 50 mL YPD medium
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in the flask (500 mL, 200 rmp). Approximately 180 mL seed broth was inoculated into 1.8 L
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basic salt medium (BSM) in a 5-L fermentor (BLBIO, China). The recombinant P. pastoris was cultured at batch grown at 30°C and pH 6.0. The pH was automatically controlled by addition of ammonium hydroxide (25%, w/v). When the dry cell weight (DCW) reached 25.0 g/L, the cultures were continuously fed with glycerol (50%, v/v). Feeding was controlled by dissolved oxygen measurements (DO-stat). Dissolved oxygen (DO) level was controlled below 30%. When the DCW reached 111.0 g/L, protein expression was induced by the addition of pure methanol for 120 h, also controlled by DO-stat and with a DO level below 30%. The protein production was sampled every 8 h during the induction, and the volume of each sampling was 5 mL.
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Purification of FTase The supernatant of recombinant P. pastoris was obtained by centrifuging the cultures at
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10,000 ×g for 20 min at 4°C. The supernatant (10 mL) was filtered and loaded onto a Ni2+
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column (GE Healthcare, Houston, TX, USA). The 6His-tagged FTase was purified using an
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AKTA purifier (GE Healthcare, Houston, TX, USA). Equilibration buffer (A) was phosphate buffer (50 mM, pH 7.4) with 20 mM imidazole, and elution buffer (B) was phosphate buffer
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(50 mM, pH 7.4) with 250 mM imidazole. The flow rate of purification was 1.0 mL/min. The sample fractions were collected based on a linear elution by ramping 0~100% buffer B. The
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pooled FTase fractions obtained was dialyzed at 4°C in 100 mM phosphate-citrate buffer (pH 5.5). The protein concentration was determined by SpectraMax Plus (Molecular Devices,
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Enzyme assays
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Sunnyvale, California, USA) based on the Bradford method.
FTase activity was determined by measuring the amount of GF2 product in the reaction mixture by HPLC [19]. The reaction mixture contained sucrose (25%, w/v) as a substrate, dissolved in 100 mM phosphate-citrate buffer (pH 5.5). The reaction was incubated at 40°C for 15 min and stopped by heating at 100°C for 10 min [20]. One unit of FTase activity was
defined as the amount of enzyme that produced 1 μmol GF2 per min at the assay conditions.
Effects of temperature on FTase activity The optimal temperature of FTase was analyzed at temperatures ranging from 30°C to 70°C
Page 7 of 28
in phosphate-citrate buffer (100 mM, pH 5.5), using sucrose as a substrate. The highest activity was taken as 100% at different temperatures. The thermostability of FTase was determined at different temperatures (40°C, 50°C, and 60°C) in phosphate-citrate buffer
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(100 mM, pH 5.5). The initial activity before incubation at various temperatures was taken as
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100%. Three independent experiments were performed.
Effects of pH on FTase activity
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To estimate the optimal pH for FTase activity, the purified protein was incubated at pH 3.0~8.0 (phosphate-citrate buffer, 100 mM). The activity determined at pH 6.0
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(phosphate-citrate buffer, 100 mM) was taken as 100%. In order to determine the pH stability, the enzyme was pre-incubated in the various buffers at 25°C for 24 h, and then
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assayed for residual activity at pH 5.5. The buffers used for determination of pH stability
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were as follows: phosphate-citrate buffer (100 mM, pH 3.0~8.0), phosphate buffer (100 mM,
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pH 8.0~9.0), and glycine-sodium hydroxide buffer (100 mM, pH 9.0~11.0). The highest residual activity was taken as 100% at different pH values. Three independent experiments were performed.
Determination of kinetic parameters Enzyme assays were performed in phosphate-citrate buffer (100 mM, pH 5.5) at 40°C, using sucrose as a substrate. Substrate concentrations were in the range 20~600 g/L. The kinetic parameters Km and Vmax were calculated by Eadie–Hofstee plots [21]. Three independent experiments were performed.
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Effect of metal ions on FTase activity To determine the effect of metal ions on FTase activity, the enzyme was pre-incubated with
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0.5 mM and 5 mM metal ions (K+, Li+, Na+, Ca2+, Mg2+, Mn2+, Zn2+, Fe3+, Ba2+, Ni2+, and Cu2+)
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and ethylene diamine tetraacetic acid (EDTA), respectively. The relative activity of FTase was
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determined, and compared to the activity obtained in phosphate-citrate buffer (100 mM, pH
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5.5) without the addition of any ions. Three independent experiments were performed.
Synthesis of fructooligosaccharides and HPLC analysis
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FOS synthesis was carried out for 12 h at 40°C and pH 5.5 (phosphate-citrate buffer, 100 mM). The high concentration of sucrose (600 g/L, w/v) as the initial substrate was used to
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analyze the FOS synthesis ability of this FTase during FOS industrial production [22,23]. The
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amount of FTase used was 20 U/g (sucrose) during the FOS synthesis reaction. The final
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reaction volume for the FOS synthesis was 50 mL, and the volume of each sampling for HPLC analysis was 1 mL. The samples withdrawn were heated to stop the reaction at 100°C for 10 min. After cooling, the samples were filtered by microporous membrane (0.2 μm). The concentrations of FOS synthesized by FTase at different phases were detected by HPLC. The conditions of the HPLC analysis were as followings: Refractive Index (RI) detector, Waters XbridgeTM Amide column (5 μm, 4.6 × 250 mm), acetonitrile-water (70:30, v/v), 30°C, and 1.5 mL/min. Three independent experiments were performed.
Results and discussion
Page 9 of 28
Cloning and expression of recombinant FTase in P. pastoris The cDNA of mature FTase without its introns and signal peptide was obtained by RT-PCR from the total RNA of A. niger YZ59 (CICIM F0901), and cloned into the P. pastoris vector
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pPIC9K (Fig. 1A). Nucleotide sequence analysis revealed that the gene was 1,884 bp long
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(Supplementary material) and that the protein encoded consisted of 628 amino acids. The
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cDNA sequence of FTase from A. niger YZ59 was compared with that of A. niger β-D-fructofuranosidase (suc1) [24], which shared 99.6% homology with that of
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β-D-fructofuranosidase (suc1) (GenBank: L06844.1). The amino acid sequence shared 98% homology with that of β-D-fructofuranosidase [24].
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The target fragment was integrated into the plasmid pPIC9K and transformed into P. pastoris GS115. The transformants were grown in MD agar plates and selected in YPD agar
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plates with G418 (3.0 mg/mL). The insertion of FTase gene in recombinant P. pastoris was
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confirmed by PCR. The control was the P. pastoris GS115 transformed with plasmid pPIC9K.
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The recombinant transformants were induced in BMMY medium (25 mL) at 30°C for 120 h in 250 mL shaker flasks. No FTase activity was detected in the supernatant of the control strain. The FTase activity in the supernatant was 117.3 U/mL under the same conditions, which was 133.4 times higher than that of native FTase from A. niger YZ59. The yield of FTase from wild-type A. niger was low in most reported cases. For example, Lateef et al. obtained the
maximum activity (24.5 U/mL) of FTase from wild-type A. niger in submerged fermentation after 48 h of fermentation [25]. The recombinant FTase from the supernatant was purified by His-tag affinity chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1B), and the specific activity of purified FTase was 6.8 ×
Page 10 of 28
104 U/mg. The general mechanism of glycoside hydrolase is a double-displacement mechanism, including glycosylation and deglycosylation [26]. According to the FTase in this work, there might be only one substrate-binding site supporting this double-displacement
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mechanism, which was consistent with the catalytic mechanism of fructosyltransferase from
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Aspergillus japonicus reported [26].
FTase production by recombinant P. pastoris in a 5-L fermentor
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The expression levels of FTase in recombinant P. pastoris GS115 were further explored in a 5-L fermentor (BLBIO, China). Fig. 2 shows the time profiles of cell growth of recombinant P.
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pastoris and its FTase production. The DCW of recombinant P. pastoris reached 111.1 g/L at the end of the glycerol fed-batch phase (64 h), and the highest specific growth rate was 1.7
d
h−1. After induction for 96 h, the highest FTase activity in the supernatant reached 1,020.0
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U/mL, which was 8.7-fold higher than that obtained in shaker flasks. The highest specific
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production rate was 69.2 U/(g·h). The high yield of FTase indicated that recombinant P.
pastoris was a suitable host for the efficient expression and industrial production of FTase.
Effect of temperature on FTase activity and stability As shown in Fig. 3A, the activity of FTase increased at temperatures between 30°C and 55°C and decreased at temperatures between 55°C to 70°C. The optimum temperature was 55°C, and the activity at optimum temperature was 3.6-fold higher than that at 30°C. Fig. 3B shows that the FTase retained approximately 60% of its activity after incubation at 50°C for 60 min; however, its activity declined rapidly at temperatures above 60°C. The FTase was
Page 11 of 28
stable at 40°C for 60 min, and retained approximately 83.1% of its activity after incubation at 40°C for 6 d.
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Effect of pH on FTase activity and stability
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As shown in Fig. 4A, the optimum pH of FTase was 5.5. FTase activity rapidly increased with
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an increase in pH from 3.0 to 5.5, and decreased with an increase in pH from 5.5 to 8.0. It exhibited its highest value (> 90% of maximal activity) between pH 4.5 and 6.5. The stability
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curve of FTase at different pH values is shown in Fig. 4B. FTase displayed optimal stability at pH 6.0, and retained more than 90% of maximum activity between pH 4.0 and 9.0 after
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incubation at 25°C for 24 h. It also retained approximately 80% of maximal activity at pH values lower than 2.0 or higher than 11.0. The recombinant FTase expressed in P. pastoris
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was stable over a broad pH range.
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Kinetic parameters of FTase
The kinetics of FTase was determined using sucrose as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) values were analyzed by nonlinear curve analysis based on Eadie–Hofstee plots. The Km and Vmax values were 159.8 g/L and 0.66 g/(L·min),
respectively (Fig. 5). The turnover number (kcat) and catalytic efficiency (kcat/Km) values of
FTase were 1.1 × 104 min−1 and 68.8 L/(g·min), respectively. Jung et al. determined the properties of the fructosyltransferase from Aureobasidium pullulans, and found that the Km value and Vmax value for sucrose were 330 g/L and 130 g/(L·h) [27]. The kinetic constants of a fructosyltransferase from Rhodotorula sp. were determinated, and the Km value for sucrose
Page 12 of 28
was 197.9 g/L [22]. It was indicated that the substrate affinity of these fructosyltransferase from A. pullulans and Rhodotorula sp. was lower than that of FTase heterologously expressed in P. pastoris in this work.
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Fructose with high concentration (˃ 50%, w/v) did not have a significant inhibitory effect
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on the kinetic parameters of FTase, the inhibition by which was considered negligible, but
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glucose has a high competitive inhibition [22]. In order to evaluate the inhibitory effect of glucose on the kinetic parameters of FTase, 20 g/L glucose was added to the enzyme
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reaction medium. As shown in Fig. 5, glucose acts as a competitive inhibitor for the catalytic reaction of FTase on sucrose. The Ki value for glucose (20 g/L) as an inhibitor was calculated
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by Eadie–Hofstee plots, and was found to be 45.0 g/L. Alvarado-Huallanco and Maugeri-Filho calculated the Ki,app value of glucose on fructosyltransferase from Rhodotorula sp. by
d
Lineweaver-Burk’s technique, which were 54.79 g/L and 55.20 g/L for purified enzyme and
Ac ce p
te
partially purified enzyme, respectively [28].
Effect of metal ions on FTase activity Metal ions play a key role in the folding and catalysis of proteins [29]. The effects of metal ions on FTase activity are shown in Table 1. FTase activity was enhanced by 5 mM Ni2+ or Mg2+, indicating that the activity might be Ni2+- or Mg2+-dependent, with a relative activity of
118.0% or 109.9%, respectively. No inhibition or activation of FTase was observed with 5 mM K+, Fe3+, or Mn2+, with relative activities of 105.3%, 101.0%, and 101.8%, respectively. Other metal ions had slight inhibitory effects on FTase activity, as follows: Na+ (0.5 and 5 mM), K+ (0.5 mM), Li+ (0.5 and 5 mM), Ba2+ (0.5 and 5 mM), Ca2+ (0.5 and 5 mM), Fe3+ (0.5
Page 13 of 28
mM), Zn2+ (0.5 and 5 mM), Mn2+ (0.5 mM), Cu2+ (0.5 and 5 mM), Mg2+ (0.5 mM), and Ni2+ (0.5 mM). Low concentration of EDTA (0.5 mM) had slight inhibitory effects on FTase activity. But the FTase was completely inactivated by 5 mM EDTA, indicating that the FTase activity might
ip t
be divalent ions-dependent. FTase from Aspergillus oryzae was inhibited by Cu2+ and Zn2+ (5
cr
mM), with a relative activity of 11% and 17%, respectively [12]. The activation or inhibition
us
of metal ions on FTase from other sources was also observed [30,31].
an
Fructooligosaccharide synthesis
FOS synthesis was carried out for 12 h using sucrose as an initial substrate (600 g/L) at 40°C
M
and pH 5.5. The highest yield of FOS for purified recombinant FTase reached approximately 343.3 g/L (w/v) at 2 h (Fig. 6, Supplementary material). During the entire process, the
d
maximum yields of GF2, GF3, and GF4 reached 246.0 g/L (w/v) at 1.5 h, 186.0 g/L (w/v) at 10 h,
te
and 88.2 g/L (w/v) at 12 h, respectively. Previous studies have reported longer durations
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required to achieve the maximum FOS yield [27, 32]. Alvarado-Huallanco and Maugeri-Filho, using purified fructosyltransferase from Rhodotorula sp., obtained a FOS production of 46% from sucrose concentration of 500 g/L (w/v) after 96 h of synthesis, the final composition of which were GF2 33.92% (w/w), GF3 9.01% (w/w), and GF4 3.40% (w/w), respectively [27].
Ghazi et al. achieved maximum FOS production from sucrose (650 g/L) by using immobilized Aspergillus aculeatus FTase after a 36-h reaction [32]. A. niger consumed sucrose for FOS formation after exit of lag phase, and the average yield of FOS by A. niger was 59.71 ± 2.32% (w/w) by recycling of cell culture [33].
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Conclusions cDNA of FTase from A. niger YZ59 was obtained by RT-PCR and expressed in P. pastoris GS115. The yield of recombinant FTase in a 5-L fermentor reached 1,020.0 U/mL, which was
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1,160.4-fold higher than that of native FTase from A. niger YZ59. The specific activity of
cr
purified FTase was 6.8 × 104 U/mg. The optimal temperature of the recombinant FTase was
us
55°C, and it was stable at temperatures below 40°C. The optimum pH of FTase was 5.5, and it was stable at pH ranging from 3.0 to 10.0. The Km, Vmax, kcat, and kcat/Km values of
an
recombinant FTase were 159.8 g/L, 0.66 g/(L·min), 1.1 × 104 min-1, and 68.8 L/(g·min), respectively. The Ki value of FTase for glucose (20 g/L) was 45.0 g/L. The recombinant FTase
M
was slightly activated by 5 mM Ni2+, Mg2+, K+, Fe3+, or Mn2+, but inhibited by other metal ions. The highest yield of FOS for FTase reached approximately 343.3 g/L (w/v) after 2 h of
Ac ce p
Acknowledgements
te
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synthesis based on 600 g/L sucrose as an initial substrate.
This project was financially supported by the Fundamental Research Funds for the Central Universities (JUSRP51402A), the 111 Project (111-2-06), the Fundamental Research Funds for the Central Universities (JUSRP11429), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu province "Collaborative Innovation Center for Advanced Industrial Fermentation" industry development program.
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[13] van Hijum S, van Geel-Schutten GH, Rahaoui H, van der Maarel M, Dijkhuizen L. Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight
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[28] Alvarado-Huallanco MB, Maugeri-Filho F. Kinetics and modeling of fructo-oligosaccharide synthesis by immobilized fructosyltransferase from Rhodotorula sp. J Chem Technol Biotechnol
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[32] Ghazi I, De Segura AG, Fernandez-Arrojo L, Alcalde M, Yates M, Rojas-Cervantes ML, Plou FJ, Ballesteros
A.
Immobilisation
of fructosyltransferase
from Aspergillus aculeatus
on
epoxy-activated Sepabeads EC for the synthesis of fructo-oligosaccharides. J Mol Catal B-Enzym 2005;35:19-27. [33] Ganaie MA, Gupta US. Recycling of cell culture and efficient release of intracellular
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fructosyltransferase by ultrasonication for the production of fructooligosaccharides. Carbohydr
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cr
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Polym 2014;110:253-8.
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Figure legends:
Fig. 1: Map of recombinant vector and SDS-PAGE analysis. A: Map of recombinant vector
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pPIC9K-fwt. B: SDS-PAGE gel of the FTase purification. M: Maker, fragment indicated by arrow: FTase.
d
Fig. 2: Time profiles of FTase production by recombinant P. pastoris in 5-L fermentor.
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Fig. 3: Effect of temperature on the activity and stability of FTase. A: Effect of temperature on
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the activity of FTase. For determination of optimum temperature of FTase, the reaction was conducted from 30 to 70°C. B: Effect of temperature on the stability of FTase. The insert presents the thermal stability of FTase at 40°C for 6 d. The thermal stability of FTase was determined at 40°C for 6 d. The thermal stability of FTase was determined at 50 or 60°C for 60 min, respectively.
Fig. 4: Effect of pH on the activity and stability of FTase. A: Effect of pH on the activity of FTase. The FTase activity was determined at pH 3.0~8.0, respectively. B: Effect of pH on the stability of FTase. The pH stability was determined at pH 3.0~11.0. Fig. 5: Eadie-Hofstee plots for using sucrose as substrate by FTase. In order to evaluate the
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inhibitory effect of glucose on the kinetic parameters of FTase, glucose (20 g/L) was added into the enzyme reaction medium. GF: sucrose as substrate; GF+G (20 g/L): 20 g/L glucose was added.
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Fig. 6: FOS production by FTase. The synthesis of FOS was carried out for 12 h by using
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sucrose as initial substrate (600 g/L) at 40°C and pH 5.5. G: glucose, GF: sucrose, GF2:
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d
M
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1-kestose, GF3: nystose, GF4: fructosylnystose, FOS: Fructooligosaccharides.
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cr
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Table:
Concentration (mM)
0.5
Metal ions
Relative activity (%) 90.8±1.5 99.4±1.2 93.9±0.9 105.3±1.4 97.8±1.6 99.3±1.1 95.9±2.2 86.6±1.3 89.9±2.0 99.5±1.3 88.6±1.7 101.0±0.9 89.9±1.1 92.4±1.5 83.9±1.4 101.8±1.6 87.6±2.0 95.1±2.2 84.9±1.8 109.9±2.0 94.8±2.5 118.0±1.7 95.9±2.3 0.2±0.0 100.0 100.0
M d te
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Na K+ Li+ Ba2+ Ca2+ Fe3+ Zn2+ Mn2+ Cu2+ Mg2+ Ni2+ EDTA CK
5
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+
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Table 1 Effect of metal ions on the activity of FTase
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Figure-6
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