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Aqueous Two Phase (PEG4000/Na2SO4) Extraction and Characterization of an Acid Invertase from Potato Tuber (Solanum Tuberosum) a
Yonca Yuzugullu & Yonca Avcı Duman a
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Department of Biology, Kocaeli University, Kocaeli, Turkey
b
Department of Chemistry, Kocaeli University, Kocaeli, Turkey Accepted author version posted online: 15 Aug 2014.
To cite this article: Yonca Yuzugullu & Yonca Avcı Duman (2014): Aqueous Two Phase (PEG4000/Na2SO4) Extraction and Characterization of an Acid Invertase from Potato Tuber (Solanum Tuberosum), Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.943373 To link to this article: http://dx.doi.org/10.1080/10826068.2014.943373
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AQUEOUS TWO PHASE (PEG4000/Na2SO4) EXTRACTION AND CHARACTERIZATION OF AN ACID INVERTASE FROM POTATO TUBER (Solanum Tuberosum) Yonca Yuzugullu1, Yonca Avcı Duman2 Department of Biology, Kocaeli University, Kocaeli, Turkey, 2Department of Chemistry, Kocaeli University, Kocaeli, Turkey
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Correspondence: Email: [email protected]
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Abstract
Invertases are key metabolic enzymes that catalyze irreversible hydrolysis of sucrose into
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fructose and glucose. Plant invertases have essential roles in carbohydrate metabolism, plant development and stress responses. To study its isolation and purification from
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potato, an attractive system useful for the separation of biological molecules, aqueous two-phase system, was used. Influence of various system parameters such as type of phase forming salts, PEG molecular mass, salt and polymer concentration was investigated to obtain the highest recovery of enzyme. PEG4000 (12.5%, w/w) / Na2 SO4 (15%, w/w) system was found to be ideal for partitioning of invertase into the bottom salt
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We hereby declare that the work described has not been submitted for publication before or anywhere else, nor has it been published in whole or in part elsewhere.
rich phase. The addition of 3% MnSO4 (w/w) at pH 5.0 increased the purity by 5.11 fold with the recovered activity of 197%. The Km and Vmax on sucrose were 3.95 mM and 0.143 U mL-1 min-1 , respectively. Our data confirmed that the PEG4000/Na2SO4 aqueous two phase system combined with the presence of MnSO4 offers a low-cost purification of invertase from readily available potato tuber in a single step. The biochemical characteristics as temperature and pH stability of potato invertase prepared from an ATPS
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make the enzyme a good candidate for its potential use in many research and industrial applications.
invertase, purification, protein recovery.
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Invertase (β-D-fructofuronosidase; E.C. 3.2.1.26) is a glycoprotein that catalyzes the breakdown of terminal non-reducing β-fructofuronoside residues in β-D-
fructofuronosides, such as sucrose [1]. Invertase is mainly used in food industry for the production of fructose syrup from sucrose which has bitter taste and readily forms into
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crystals. It is also used in paper, pharmaceutical, and cosmetic industry and also for
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production of sucrose biosensors [2–3].
Invertases are widely produced by many plants including carrot, tomato, tobacco, sugar cane and bamboo [4–7]. Its presence has also been reported in different yeasts (Saccharomyces cerevisiae [8], Candida utilis [9], Pichia anomola [10]), and some fungi (mainly Neurospora sp. and Aspergillus niger) [11–12]. Plant invertases contain several
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INTRODUCTION
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KEYWORDS: Aqueous two phase system (ATPS), bottom phase, characterization,
invertase isoforms with different biochemical properties and subcellular localization [13]. In general, acid invertases have an optimum pH of 3.5-5.0, while optimal pH of alkaline invertases ranges from 7.0 to 8.0 [4–7].
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Invertase activity is present in fresh potato and shown to enhance during cold storage [14], which indicate potato tuber would be a good source to study purification and characterization of invertases. Although several methods available for purification of invertase from different plant sources, these methods are considered as time consuming
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and expensive [6, 15–17]. The conventional methods generally include at least four steps; each of which results in some degree of product and activity recovery loss. Therefore,
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techniques which are economic and give the highest level of purification in the fewest steps. Among them, aqueous two phase system (ATPS) is an attractive technology designed for this purpose. It was first established by Albertson in 1950’s to separate proteins, nucleic acids and cells [18]. The system has many advantages like simple and
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rapid separation with low denaturation, rapid mass transfer, selective partition and low
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cost. Therefore, it has been used in several fields of biotechnology [19–20].
An ATPS is formed either by mixing two incompatible polymers or one polymer and an inorganic salt. The basic ATPS separation strategy is based on partition of protein of interest predominantly in one phase while contaminants all in the other phase. In general, almost all smaller biomolecules tend to be present in more polar bottom phase (salt rich
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recent interest becoming widespread on protein purification is the application of
phase) whereas proteins remain in less polar top phase (PEG rich phase). However, this is not ideal since protein recovery from polymer phase requires additional steps like ultrafiltration and chromatography which would increase the cost of process. Various conditions can affect the tendency of partitioning of macromolecules such as size, charge, hydrophobicity, molecular mass, structure, viscosity and system pH
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[19–22]
.
Some successful applications of ATPS include amino acid isolation [23], protein extraction from corn extract [24], C-phycocyanin from spirulina [25], polyphenoloxidase from potato [26]
, phenylalanine ammonia-lyase from yeast Rhodotorula glutinis [27], bromelain from
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pineapple [28] and protein recovery from animal blood [29]. Purification of invertases from different sources has been extensively studied [30]. However, reports based on extraction
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aim of investigating the feasibility of invertase partitioned in an aqueous two phase
system, we have designed a system of PEG/Na 2SO 4 for isolation and purification and have determined the characteristic properties of the enzyme. Effect of phase composition, PEG molecular weight and concentration, inorganic salt structure and concentration,
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system pH and neutral salts on enzyme partition was studied. Beside this, pH and temperature effects on enzyme activity, stability tests and kinetic parameters, K m and
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Vmax, were also examined.
EXPERIMENTAL
Materials
Sucrose, Polyethylene glycol (PEG, mol. wt 1000, 2000, 3000, 4000, 6000, 8000), salts
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and purification invertase from Solanum tuberosum by ATPS is quite restricted. With the
and other chemicals were of analytical grade and purchased from Sigma Chem. Co. (St. Louis, MO, USA). Potato was obtained from local market in Turkey.
Methods Preparation Of Crude Extract
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Fresh potato roots were washed thoroughly with distilled water and then the peels were removed. 20 g roots were then cut in to small pieces and then blended in 50 mM pH 4.5 ice-cold citrate buffer at +4 ºC for a minute. The homogenate was filtered from five layers of cheese cloth, and then centrifuged at 10700 g for 20 min at +4 ºC. The clear
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supernatant was treated as the crude enzyme extract for the further study.
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ATPS was prepared in 12 mL centrifuge tubes by adding the appropriate quantities of stock solutions of PEG and salt and the total weight was made up to 5 g with crude extract, buffer and deionized water. To study the effect of different salts on invertase
partitioning, magnesium sulfate, ammonium sulfate, sodium sulfate, manganese sulfate,
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manganese chloride, and sodium citrate were evaluated by keeping the total level of PEG and salt in the system was set at 10%. The system parameters were selected based upon
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previous reports [24, 28, 31–33]. To avoid protein precipitation, PEG, salt, buffer and deionized water were first mixed before addition of 0.5 mL crude enzyme extract to the phase system (protein quantity and specific activity of crude extract 1.63 mg and 0.52 U/mg, respectively). The pH of the system was adjusted with concentrated NaOH or HCl to 5.0. The mixture was gently shaken for 30 min at room temperature and separated by
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Aqueous Two Phase Partitioning
centrifugation for 5 min at 1700 g. The upper phase was carefully separated from the lower phase by using a Pasteur pipette. The volumes of the separated phases were measured. Aliquots of the phases were analyzed for protein estimation and enzyme assay.
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The partition parameters including partition coefficient, specific activity, purification factor, and recovery were determined according to equations from previous reports [30–33]. The partition experiments were conducted in triplicate.
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The partition coefficient for invertase in the aqueous two phase systems is defined as the
CT CB
KE =
AT AB
(1)
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KP =
(2)
where CT and CB are the total protein concentrations in mg/mL of the top and bottom
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phases, AT and AB are the enzyme activities in U/mL of the top and bottom phases,
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respectively.
To evaluate the purification process, the enzyme-specific activity (SA, expressed in U/mg protein), the purification factor (PF) and the activity recovery (R) were also calculated according to the given equations [30]: SA =
AB CB
(3)
SA B SA i
(4)
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protein content (KP ) or enzyme activity (KE), as shown in Eqs. (1) or (2) [30]:
PF =
R % =
AB (5) Ai
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where AB and CB are the enzyme activity and protein concentration of the bottom phase, respectively. Ai is the activity and SAi is the specific activity of the crude extract. In Eq. (4), SAB represents the specific activity for the bottom phase.
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Enzyme Activity Assay
Plant-invertase activity was carried out according to the method of Miller [34]. The assay
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buffer) and 0.2 mL of suitably diluted enzyme was incubated at 37 ºC for 30 min. After
that, the reaction was stopped by adding 1 ml of DNS (3,5-Dinitrosalicylic acid) reagent and heated in a boiling water bath for 5 min. It was then cooled to room temperature in ice bath and the amount of reducing sugars was measured spectrophotometrically at 540
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nm [34]. One unit of invertase activity was defined as the amount of enzyme which released 1 µmole of glucose from sucrose per minute at pH 4.5 and 37 ºC. Specific
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activity is expressed as units per milligram of protein with sucrose as a substrate.
Protein Concentration Determination
Concentration of the protein was determined by Bradford method [35] using Coomassie Brilliant Blue G-250 dye as a reagent and bovine serum albumin (BSA) as standard, by
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mixture consisting of 0.8 mL of substrate (50 mM sucrose in 50 mM pH 4.5 citrate
measuring the absorbance at 595 nm at 25 ºC. Assays were performed in triplicate.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Molecular weight of purified invertase was determined by SDS-PAGE according to method of Laemmli [36] on a Mini Protean II gel electrophoresis unit (Bio-Rad
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Laboratories, Richmond, USA). Electrophoresis was carried out at a constant current of 100V, 400mA for about 2 h. The gel was stained with Coomassie Brilliant Blue R-250 for 1 hour for the detection of proteins and then destained using destaining solution
Kinetic Properties Of Potato Invertase Partitioned In ATPS
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Enzyme assays, described above, were conducted at temperatures from 25 to 65 ◦C to
find the optimum temperature for activity. Percent relative activities were calculated as the ratios of enzyme activities at different temperatures to the maximum enzyme activity, multiplied by 100. To determine thermostability, enzyme was incubated at 25–65 ◦C for
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up to 60 min. Percent residual enzyme activities were measured by the standard assay procedures and reported after dividing by the enzyme activity prior to heat treatment,
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multiplied by 100.
Effect Of Ph On Invertase Activity And Stability To investigate the effect of pH on invertase activity, assays were conducted at various pH values in a range of 4.0-8.0 using 25 mM of the following buffers: sodium acetate (pH
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Effect Of Temperature On Invertase Activity And Stability
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containing 40% (v/v) methanol and 10% (v/v) acetic acid for 3 h.
4.0-6.0), and sodium phosphate (pH 6.5-8.0). Enzyme concentration and temperature were kept constant as stated in standard assay conditions. Percent relative activity was calculated as described above. To determine pH stability of enzyme, enzyme samples were incubated for 60 min in a medium where pH ranged from 4.0 to 8.0. Residual activities were then determined under the standard assay conditions.
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Effect Of Substrate Concentration On Invertase Activity The influence of the substrate concentration on the invertase activity was carried out by determining the initial rates of the hydrolysis reaction with the initial concentration of
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sucrose ranging from 2 to 50 mM. The Michaelis-Menten constants (Km and Vmax) were
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RESULTS AND DISCUSSION
The remarkable demand for invertases in pure science and applied research has led scientist to explore efficient processes to purify the enzyme with high recovery. Several invertase isoforms have been isolated and purified from a number of plants by using
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ammonium sulfate precipitation and various chromatographic systems including ionexchange, gel filtration and affinity chromatography in a highly purified form [7, 15, 38–39].
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Despite the highest purity obtained at the end of the process, these conventional methods increases the overall cost. Considering this, it was intended to develop highly straightforward and cost-effective process for purification of plant invertase.
ATPS offers a very simple, benign and powerful method for the purification and recovery
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determined using Lineweaver-Burk plot [37].
of proteins [19]. ATPSs have many advantages in the downstream processing of biomolecules including characterization of the system with high-water content (70-80%) and subsequent low interfacial tension, low energy requirement, biocompatibility and minimized process cost. Moreover, the biphasic systems increase the enzyme recovery in the purified form [40]. For the enzymes used in industry-such as invertase, the biphasic
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systems therefore provide many benefits to both researchers and industrial companies. Among invertases from different sources, plant invertases possess great interest due to their significance for plant development, growth and carbon partitioning [41]. Despite various conventional methods available for purification of plant invertases, there are no
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reports presented in relation to isolation and extraction of acid invertase by ATPS and its
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ATPSs of PEG and salts were evaluated to find the best partitioning of invertase in one of two phases. PEG was chosen as phase forming polymer because of its low cost and ability to improve the refolding of proteins. The success of ATPS strongly depends on phase forming salt, molecular weight of PEG, relative proportion of each component and
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pH [42]. As there have multiple factors for the selection of the best phase system for the partitioning of invertase from the crude extract; optimization was performed step by step.
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The partition coefficients, recovery and purity of protein were determined to analyze the partitioning behavior of invertase in the two phases.
In almost all two phase systems, invertase from different sources exhibited greater tendency to participate in the salt-rich bottom phase, indicating the enzyme being highly
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biochemical characterization.
hydrophilic [30, 33, 43]. In fact, it is known that positively charged proteins tend to present in the bottom phase while negatively charged proteins would rather move into the top phase in ATPSs [18, 33]. It should be noted that invertase is known to be stable in the pH range of 3.5-5.5 [44]. Therefore, pH of the system was adjusted to 5.0. The plant invertase would be positively charged at pH 5.0 resulting in its separating to the salt-rich phase. Thus, the
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data for bottom phase are presented here to reveal invertase partitioning. Temperature used was kept constant in all systems.
Effect Of Salts On Phase Formation
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The partitioning of invertase from potato was studied in several biphasic systems of 10% PEG4000 with different salts, including magnesium sulfate, ammonium sulfate, sodium
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ones most commonly used in ATPSs. Sulfate salt was used due to its ability to promote hydrophobic interactions between proteins whereas citrate was chosen because of its
extraction capability from mother liquid and low environmental polluting properties [45].
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Among the phase systems used, no phase formation was observed in two systems of PEG4000/MgSO4 (10%, w/w) and PEG4000/sodium citrate (10%, w/w). In all systems,
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other than those reported above, phase formation was observed and the target protein appeared preferentially partitioned to the bottom, salt-rich phase (Table 1). The reason for extraction of the highest amount of potato invertase to the bottom phase can be explained by volume exclusion effect, namely an increase in polymer concentration or molecular weight reduces the space for biomolecules in the top phase and as a result, biomolecules
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sulfate and sodium citrate at various concentrations (Table 1). These four salts are the
tend to move to the bottom phase [46–48].
As shown in Table 1, specific enzyme activity, purification factor and recovery of invertase separated in PEG4000/salt systems were influenced by variety of salts tested. An increase in the concentration of ammonium sulfate resulted in a loss of enzyme
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recovery, and even at low concentrations the recovery obtained was very low. The reason for this result can be described by the denaturation of potato invertase caused by the salting-out effect [49]. On the other hand, sodium sulfate presented promising results with the highest specific activity (0.58 U/mg), purification fold (PF, 2.88) and activity
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recovery (R, 94%). Specific activity of potato invertase partitioned in the presence of Na2SO4 was lower than that of Baker’s yeast invertase in the same salt, while higher
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occurred in the bottom phase in the system composed of 10% PEG4000 and 15%
Na2SO4. Therefore, the PEG/Na2SO4 system was selected for further study. Reports on extraction and purification of various enzymes using PEG/Na 2 SO4 ATPS are available [27, .
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50–52]
Selection Of PEG Molecular Weight
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The partitioning of enzymes has been found to be strongly depended on polymer molecular mass [18, 41]. In general, an intermediate molecular weight of polymer is used to set up the biphase systems [26–27, 30].
To choose a suitable phase forming polymer molecular weight for the purification of
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purification fold was obtained in the presented study [4, 30]. The best enzyme partitioning
potato invertase, various biphase systems were produced with different molecular weights of PEG (MW 1000, 2000, 3000, 4000, 6000 and 8000). The potato invertase partitions in PEG/Na2SO4 systems with varying PEG molecular weights are shown in Fig. 1. Almost all invertases concentrated in the salt-rich phase while contaminants were transferred into the top phase with PEG4000. Above PEG4000, the partition coefficient
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of invertase was found to decrease from 1.44 to 1.03 with the increase in PEG molecular mass. The decrease of partition coefficient beyond PEG4000 is because of volume exclusion effect, where PEG chain progressively dominates more the top layer, leaving less space for contaminants [51]. The bottom phase therefore was achieving the solubility
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limit of the enzyme in the end and consequently the invertase activity was reducing above PEG 4000. As a synergistic effect, specific activity and enzyme recovery enhanced with
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in molecular mass. The maximum enzyme recovery (94%) and purification factor of 2.85 fold was detected at the PEG molecular mass of 4000. Thus, PEG4000/Na2SO4 system was selected for further studies.
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Effect Of PEG 4000 Concentration On Invertase Partitioning
The effect of PEG4000 concentration on phase formation was further investigated by
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using 10% (w/w) Na2SO 4 at pH 5.0. Results of activity recovery and purification fold versus different PEG concentrations are shown in Fig. 2. The results showed that increasing the concentration of PEG4000 decreased partitioning enzyme specific activity and recovery of invertase in the salt-rich phase. For 12.5% (w/w) concentration of PEG4000, the purification fold reached its highest value of 3.89-fold with the recovery of
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increasing polymer molecular mass till PEG4000 and then lessened with further increase
128%. Above this concentration, both purification fold and recovery were decreased to 2.81-fold and 90%, respectively at 20% (w/w) PEG4000. These findings suggested that increasing PEG concentration had a negative effect on distribution of invertase and other proteins in the crude extract to the bottom and top phases, respectively. An ATPS with
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the composition of 12.5% (w/w) PEG4000 and 15% (w/w) Na2 SO4 was thus selected for further studies.
Effect Of Salt Concentration
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A further key factor affecting the enzyme partition in biphase systems is the
concentration of salt used. In order to optimize the phase forming salt concentration, a
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employed. The effect of salt concentration on invertase separation is shown in Fig. 3. As is seen in the figure, sodium sulfate concentration did not significantly affect enzyme recovery while purification fold presented more sensitive behavior especially at salt concentrations between 14 and 16%. The maximum enzyme recovery (128%) with a
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purification factor of 3.89 fold was observed at Na 2SO4 concentration of 15%. Therefore,
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the PEG4000(12.5%)/Na2SO4(15%) system was chosen for further experiments.
Effect Of Neutral Salts
In general, neutral salts affect enzyme partitioning in aqueous two phase systems by altering the partition coefficient of proteins in relation to their charge. Proteins having predominantly hydrophobic anions or cations tend to be present in hydrophobic phase or
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series of solutions containing 12.5% PEG4000 and 13-20% (w/w) of Na2SO 4 were
vice versa [32]. Five different salts, KCl, NaCl, MgCl2, Na2CO3, MgSO 4, MnCl2 and MnSO4 were added into the 12.5%PEG4000/15%Na2SO4 system to probe their effects on invertase partition (Fig. 4A). Apart from MgSO4, MnCl2 and MnSO4, nearly all neutral salts had similar effects on phase separation (Fig. 4A). The recovery and purification fold of invertase indicate that partition of invertase into the top and bottom phase is not
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significantly changed with KCl, NaCl, MgCl2 or Na2CO3 addition to the PEG4000(12.5%)/Na2SO4(15%) system. The partition coefficient (KE) values of enzyme in the bottom phase were calculated as described in Experimental section and found to be 4.98, 4.52, 4.61 and 4.59 for KCl, NaCl, MgCl 2, Na2CO3, respectively. On the other
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hand, addition of MgSO4 into the same system increased the value of K E to 10.86.
However, the highest partition coefficient (KE) value was found as 11.57 while the lowest
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the recovery of invertase activity was previously reported in yeast cells [30]. When
compared with the partition coefficient values of five salts presented, it was noted that MnSO4 had the lowest KP indicating its promoting effect on transfer of contaminant proteins to top phase. Together with results of Karkaş et al. [30], it is suggested
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manganous salt is more effective than other neutral salts tested here for partitioning of
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potato invertase into the bottom and the other contaminants to the upper phase.
Once the best neutral salt determined for invertase purification, different concentrations of MnSO4 were further tested. Fig. 4B shows that the partitioning of enzyme obtains the highest purification fold (5.11) and recovery (197%) in the bottom phase at MnSO4 concentration of 3% (w/w). In consistent with our results, there are many reports also
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KP (protein content, 0.78) in the presence of MnSO4 . The stimulating effect of MnSO4 on
available in literature related to the use of neutral salts to improve the partitioning of different enzymes in different ATPSs [6, 25, 32, 52].
SDS-PAGE Analysis Of Plant Invertase
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The degree of purification of the resulting enzyme preparation after ATPS was analyzed by SDS-PAGE (Fig. 5). As shown in the figure, crude extract consisted of many proteins with various molecular weights (Fig. 5, lane 2). Subsequent extraction with aqueous two phase system resulted in the majority of the contaminant proteins partitioned to the
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polymer enriched phase (Fig. 5, lane 3). The molecular weight of plant invertase was estimated to be 60 kDa. The molecular weight of acidic plant invertases is mostly
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quite promising since it indicates an improvement in the purification of potato invertase (Fig. 5, lane 3).
Characterization Of Aqueous Two Phase Extracted Invertase
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Influence Of Temperature On The Activity And Stability Of Partitioned Invertase The temperature activity and stability profile of the enzyme is considered to be one
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important factor for industrial applications. Therefore, both invertase activity and stability over wide temperature range (25-65 ◦C) were investigated. The relative activity expressed in percentage of the maximum activity is presented in Fig. 6 as a function of temperature. As is also seen from the figure, relative activities were greater than 70% between 25 ◦C and 55 ◦C and maximal activity was at 37 ◦C for invertase activity (Fig. 6). The results
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differed between 55 and 70 kDa based on the subcellular localization [13, 44]. This result is
compare well with the previous reports. Generally, invertases from different plants show the highest activity at 37 ◦C [7, 38–39].
Thermal stability of potato invertase was analyzed over the temperature range of 25–65 ◦
C and presented in Fig. 6. After treatment of enzyme samples at various temperatures for
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1 hour, an increase in activity was detected at 37 ◦C. Invertase retained 65 % of its original activity at 65 ◦C indicating the thermal stability of invertase which would be a great advantage for its potential use in different fields of industry. Potato invertase appears to be more stable than Baker’s yeast invertase which retains almost 50% of its
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maximum activity at 60 °C [30]. The influence of temperature on invertase activity and stability has been studied and found to depend strongly on the enzyme localization,
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soluble invertase from wheat coleptiles showed maximum activity at 37 ◦C and the
enzyme retained 60% of its maximum activity after 4 min at 50 ◦C [38]. Another study of plant invertases reported by Singh et al. [39] explains how the thermal stability of different invertase forms in plants differs. The invertase 2 (pollen wall) has remained almost stable
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at temperatures above 40 ◦C while invertase 1 (cytoplasmic) showed rapid denaturation.
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Influence Of Ph On The Activity And Stability Of Partitioned Invertase The optimum pH on invertase activity was determined by changing the pH of the medium from 4.0 to 8.0 at room temperature. Over the pH range of 4.0–8.0, the highest activity was observed at pH 4.5 with a minor difference in activity within the wide range tested and more than 78% its initial activity was retained (Figure 7). The results agree with the
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incubation time, and temperature and tested medium as well. It’s been reported that the
similar previous works. The pH optimum of plant invertases changes in the range of pH 4.5–8 [7, 44]. The broad pH range offers an advantage in the application of the enzymes at lower pH, which can eliminate the possibility of microbial contamination during longterm operation [30]. Lee et al. [7] have reported that the activity of neutral invertase from carrot was optimum at pH 6.8 and that of alkaline invertase had a pH optimum at pH 8.0.
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The pH stability is one of the other key factors for selection of enzymes as biocatalysts in many industrial applications. The pH stability of the invertase was examined by preincubating the enzyme at various pH values from 4.0 to 8.0 at room temperature for 1 h
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and measuring the residual activity under the standard assay conditions at 50°C and pH
5.0. Accordingly, plant invertase was most stable at pH 4.5 (Fig. 7). More than 80% of its
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invertase was moderately stable in a pH range from 4.0 to 5.0 retaining about 50% of its initial activity. Above pH 5.0, enzyme activity showed gradual decrease and at pH 8
decreased to 35% initial activity. Similar results have been reported in different studies of [44]
.
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invertases. Generally, plant invertases are stable between pH 4.5 and 5.0
Kinetic Analysis Of Invertase Activity
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The effect of substrate concentration on invertase activity was examined with sucrose using the standard conditions of the assay described in the Materials and methods section (50 °C and pH 5.0). With the substrate sucrose the enzyme obeyed the Michaelis–Menten equation. The kinetic constants, Km and Vmax were calculated from Lineweaver–Burk graph as 3.95 mM sucrose and 0.143 U mL-1 min-1, respectively (Fig. 8). In general, acid
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initial activity remained after 60 min. Based on the results indicated in Fig. 7, the plant
invertases in plants have a Km for sucrose in the low mM range [44]. Matsushita et al. [15] have reported that, for sweet potato acid invertase the K m was determined as 4.5 mM for sucrose as substrate. The Km value observed was quite lower than that found for some other invertases produced from yeasts and plants [15, 30, 44]. Lower Km values reflect
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higher affinity between substrate and enzyme, indicating invertase from potato tubers having the highest affinity for sucrose among other invertases reported earlier.
CONCLUSION
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In this study, the purification and recovery of invertase from potato tuber by ATPS was introduced for the first time. The effects of some process parameters on invertase
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with 197% recovery and 5.11 purification fold from potato. The partition behavior of
invertase in PEG4000/Na2SO4 indicated that the plant enzyme can be extracted to the salt-rich bottom phase. PEG molecular weight, salt type and concentration were found to have noticeable effect on partitioning of invertase. The optimal system consisted of
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12.5% (w/w) PEG4000, 15% (w/w) Na 2SO4 and 3% (w/w) MnSO4 at system pH 5.0 and room temperature for invertase purification. When compared with other conventional
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purification methods, ATPS represents an inexpensive, straightforward, safe, and highly efficient way to purify proteins. Moreover, it was also intended to characterize the potato invertase partitioned by PEG/Na2 SO4 ATPS. The enzyme was highly active and had an excellent stability when purified by this aqueous two phase extraction system, which makes this potato invertase an attractive biocatalyst for its potential use in many
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extraction were explored. With an efficient and cheap technique, enzyme was purified
industrial applications. The optimized process is also expected to promote the applications of plant invertases in pure science and applied research for better understanding of fundamental processes in plants.
ACKNOWLEDGEMENTS
19
We would like to thank the Scientific Research Unit of Kocaeli University for funding (No. 2012/057).
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232.
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Table 1. Effect of different salt type and concentration on purification of invertase with
SA (U/mg)
PF (fold)
R (%)
10% PEG4000-10% Sodium sulfate
0.36
2
66
10% PEG4000-15% Sodium sulfate
0.58
2.88
94
10% PEG4000-10% Magnesium sulfate
-
-
-
10% PEG4000-15% Magnesium sulfate
0
0
0
10% PEG4000-10% Ammonium sulfate
0.042
0.23
8
10% PEG4000-10% Sodium citrate 10% PEG4000-15% Sodium citrate
0
0
0
-
-
-
0.47
2.61
69
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Result shows only bottom phase.
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10% PEG4000-15% Ammonium sulfate
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Phase composition (%, w/w)
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(-): Phase separation was not observed.
SA: specific activity, PF: purification fold, R: recovery. 0.5 mL crude enzyme of invertase with specific activity 0.52 U mg-1 and 1.63 mg protein.
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10% PEG4000 at pH 5.0
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Figure1. Effect of PEG molecular weight on purification of potato invertase in PEG/Na2 SO4 ATPS at pH 5.0 in the bottom phase. Phase compositions (w/w) (5 g) contain 10% PEG 1000-8000, 15% (w/w) Na2SO4 , and 0.5 ml of crude extract with a
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The partition experiments were performed in triplicate.
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plant invertase specific activity of 0.52 U/mg protein and a total protein mass of 1.63 mg.
28
Figure 2. Effect of PEG4000 concentration on purification of potato invertase in PEG/Na2 SO4 ATPS at pH 5.0 in the bottom phase. Phase compositions (w/w) (5 g) contain 10-20% PEG4000, 15% (w/w) Na 2SO 4, and 0.5 ml of crude extract with a plant
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partition experiments were performed in triplicate.
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invertase specific activity of 0.52 U/mg protein and a total protein mass of 1.63 mg. The
29
Figure 3. Effect of Na2SO4 concentration on purification of potato invertase in PEG/Na2 SO4 ATPS at pH 5.0 in the bottom phase. Phase compositions (w/w) (5 g) contain 12.5% PEG4000, 13-20% (w/w) Na2SO 4, and 0.5 ml of crude extract with a plant
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partition experiments were performed in triplicate.
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invertase specific activity of 0.52 U/mg protein and a total protein mass of 1.63 mg. The
30
Figure 4. (A) Influence of added neutral salts at 1% (w/w) and (B) Effect of MnSO4 concentration on purification of potato invertase (0.5 ml, specific activity of 0.52 U/mg, total protein mass of 1.63 mg) with 12.5% (w/w) PEG4000 and 15% (w/w) Na 2SO4 at pH
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5.0 in the bottom phase. The partition experiments were performed in triplicate.
31
Figure 5. Silver staining of SDS-PAGE gel showing the purity of invertase separated from potato extract by ATPS system. Electrophoresis was carried out at 100 mV for 120 min on 12% polyacrylamide gel system. For each lane, 20 µg protein with specific
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extract; lane 3: purified and recovered invertase.
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activity of 6.45 U/mg was applied. Lane 1: protein molecular mass marker; lane 2: crude
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Figure 6. The effect of temperature on activity and stability of potato tuber invertase (3.26 mg/ml) using 50 mM sucrose in 50 mM citrate buffer. Percent relative activity as a function of reaction temperature at pH 4.5. Percent residual activity at the end of 60 min
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All experiments were carried out in triplicate.
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pre-incubation at the stated temperature, followed by standard assay at 37°C and pH 4.5.
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Figure 7. The effect of pH on activity and stability of potato tuber invertase (3.26 mg/ml) using 50 mM sucrose in 50 mM citrate buffer. Percent relative activity as a function of reaction pH at 37°C in sodium acetate and sodium phosphate buffers. Percent residual activity after pre-incubation in 25 mM acetate buffer (pH 4.0-6.0) and phosphate buffer
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(pH 6.5–8.0) at 37°C for 60 min, followed by standard assay at 37°C and pH 4.5. All
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experiments were carried out in triplicate.
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Figure 8. Enzymatic reaction kinetics of potato invertase towards sucrose. LineweaverBurk double reciprocal plot showing 1/V versus 1/[S] (R2=0.948), V represents initial reaction rate, and [S] represents substrate concentration. All experiments were carried out
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in triplicate.
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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Aqueous Two Phase (PEG4000/Na2SO4) Extraction and Characterization of an Acid Invertase from Potato Tuber (Solanum Tuberosum) a
Yonca Yuzugullu & Yonca Avcı Duman a
b
Department of Biology, Kocaeli University, Kocaeli, Turkey
b
Department of Chemistry, Kocaeli University, Kocaeli, Turkey Accepted author version posted online: 15 Aug 2014.
To cite this article: Yonca Yuzugullu & Yonca Avcı Duman (2014): Aqueous Two Phase (PEG4000/Na2SO4) Extraction and Characterization of an Acid Invertase from Potato Tuber (Solanum Tuberosum), Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.943373 To link to this article: http://dx.doi.org/10.1080/10826068.2014.943373
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AQUEOUS TWO PHASE (PEG4000/Na2SO4) EXTRACTION AND CHARACTERIZATION OF AN ACID INVERTASE FROM POTATO TUBER (Solanum Tuberosum) Yonca Yuzugullu1, Yonca Avcı Duman2 Department of Biology, Kocaeli University, Kocaeli, Turkey, 2Department of Chemistry, Kocaeli University, Kocaeli, Turkey
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Correspondence: Email: [email protected]
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Abstract
Invertases are key metabolic enzymes that catalyze irreversible hydrolysis of sucrose into
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fructose and glucose. Plant invertases have essential roles in carbohydrate metabolism, plant development and stress responses. To study its isolation and purification from
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potato, an attractive system useful for the separation of biological molecules, aqueous two-phase system, was used. Influence of various system parameters such as type of phase forming salts, PEG molecular mass, salt and polymer concentration was investigated to obtain the highest recovery of enzyme. PEG4000 (12.5%, w/w) / Na2 SO4 (15%, w/w) system was found to be ideal for partitioning of invertase into the bottom salt
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We hereby declare that the work described has not been submitted for publication before or anywhere else, nor has it been published in whole or in part elsewhere.
rich phase. The addition of 3% MnSO4 (w/w) at pH 5.0 increased the purity by 5.11 fold with the recovered activity of 197%. The Km and Vmax on sucrose were 3.95 mM and 0.143 U mL-1 min-1 , respectively. Our data confirmed that the PEG4000/Na2SO4 aqueous two phase system combined with the presence of MnSO4 offers a low-cost purification of invertase from readily available potato tuber in a single step. The biochemical characteristics as temperature and pH stability of potato invertase prepared from an ATPS
1
make the enzyme a good candidate for its potential use in many research and industrial applications.
invertase, purification, protein recovery.
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Invertase (β-D-fructofuronosidase; E.C. 3.2.1.26) is a glycoprotein that catalyzes the breakdown of terminal non-reducing β-fructofuronoside residues in β-D-
fructofuronosides, such as sucrose [1]. Invertase is mainly used in food industry for the production of fructose syrup from sucrose which has bitter taste and readily forms into
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crystals. It is also used in paper, pharmaceutical, and cosmetic industry and also for
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production of sucrose biosensors [2–3].
Invertases are widely produced by many plants including carrot, tomato, tobacco, sugar cane and bamboo [4–7]. Its presence has also been reported in different yeasts (Saccharomyces cerevisiae [8], Candida utilis [9], Pichia anomola [10]), and some fungi (mainly Neurospora sp. and Aspergillus niger) [11–12]. Plant invertases contain several
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INTRODUCTION
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KEYWORDS: Aqueous two phase system (ATPS), bottom phase, characterization,
invertase isoforms with different biochemical properties and subcellular localization [13]. In general, acid invertases have an optimum pH of 3.5-5.0, while optimal pH of alkaline invertases ranges from 7.0 to 8.0 [4–7].
2
Invertase activity is present in fresh potato and shown to enhance during cold storage [14], which indicate potato tuber would be a good source to study purification and characterization of invertases. Although several methods available for purification of invertase from different plant sources, these methods are considered as time consuming
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and expensive [6, 15–17]. The conventional methods generally include at least four steps; each of which results in some degree of product and activity recovery loss. Therefore,
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techniques which are economic and give the highest level of purification in the fewest steps. Among them, aqueous two phase system (ATPS) is an attractive technology designed for this purpose. It was first established by Albertson in 1950’s to separate proteins, nucleic acids and cells [18]. The system has many advantages like simple and
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rapid separation with low denaturation, rapid mass transfer, selective partition and low
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cost. Therefore, it has been used in several fields of biotechnology [19–20].
An ATPS is formed either by mixing two incompatible polymers or one polymer and an inorganic salt. The basic ATPS separation strategy is based on partition of protein of interest predominantly in one phase while contaminants all in the other phase. In general, almost all smaller biomolecules tend to be present in more polar bottom phase (salt rich
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recent interest becoming widespread on protein purification is the application of
phase) whereas proteins remain in less polar top phase (PEG rich phase). However, this is not ideal since protein recovery from polymer phase requires additional steps like ultrafiltration and chromatography which would increase the cost of process. Various conditions can affect the tendency of partitioning of macromolecules such as size, charge, hydrophobicity, molecular mass, structure, viscosity and system pH
3
[19–22]
.
Some successful applications of ATPS include amino acid isolation [23], protein extraction from corn extract [24], C-phycocyanin from spirulina [25], polyphenoloxidase from potato [26]
, phenylalanine ammonia-lyase from yeast Rhodotorula glutinis [27], bromelain from
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pineapple [28] and protein recovery from animal blood [29]. Purification of invertases from different sources has been extensively studied [30]. However, reports based on extraction
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aim of investigating the feasibility of invertase partitioned in an aqueous two phase
system, we have designed a system of PEG/Na 2SO 4 for isolation and purification and have determined the characteristic properties of the enzyme. Effect of phase composition, PEG molecular weight and concentration, inorganic salt structure and concentration,
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system pH and neutral salts on enzyme partition was studied. Beside this, pH and temperature effects on enzyme activity, stability tests and kinetic parameters, K m and
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Vmax, were also examined.
EXPERIMENTAL
Materials
Sucrose, Polyethylene glycol (PEG, mol. wt 1000, 2000, 3000, 4000, 6000, 8000), salts
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and purification invertase from Solanum tuberosum by ATPS is quite restricted. With the
and other chemicals were of analytical grade and purchased from Sigma Chem. Co. (St. Louis, MO, USA). Potato was obtained from local market in Turkey.
Methods Preparation Of Crude Extract
4
Fresh potato roots were washed thoroughly with distilled water and then the peels were removed. 20 g roots were then cut in to small pieces and then blended in 50 mM pH 4.5 ice-cold citrate buffer at +4 ºC for a minute. The homogenate was filtered from five layers of cheese cloth, and then centrifuged at 10700 g for 20 min at +4 ºC. The clear
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supernatant was treated as the crude enzyme extract for the further study.
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ATPS was prepared in 12 mL centrifuge tubes by adding the appropriate quantities of stock solutions of PEG and salt and the total weight was made up to 5 g with crude extract, buffer and deionized water. To study the effect of different salts on invertase
partitioning, magnesium sulfate, ammonium sulfate, sodium sulfate, manganese sulfate,
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manganese chloride, and sodium citrate were evaluated by keeping the total level of PEG and salt in the system was set at 10%. The system parameters were selected based upon
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previous reports [24, 28, 31–33]. To avoid protein precipitation, PEG, salt, buffer and deionized water were first mixed before addition of 0.5 mL crude enzyme extract to the phase system (protein quantity and specific activity of crude extract 1.63 mg and 0.52 U/mg, respectively). The pH of the system was adjusted with concentrated NaOH or HCl to 5.0. The mixture was gently shaken for 30 min at room temperature and separated by
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Aqueous Two Phase Partitioning
centrifugation for 5 min at 1700 g. The upper phase was carefully separated from the lower phase by using a Pasteur pipette. The volumes of the separated phases were measured. Aliquots of the phases were analyzed for protein estimation and enzyme assay.
5
The partition parameters including partition coefficient, specific activity, purification factor, and recovery were determined according to equations from previous reports [30–33]. The partition experiments were conducted in triplicate.
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The partition coefficient for invertase in the aqueous two phase systems is defined as the
CT CB
KE =
AT AB
(1)
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KP =
(2)
where CT and CB are the total protein concentrations in mg/mL of the top and bottom
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phases, AT and AB are the enzyme activities in U/mL of the top and bottom phases,
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respectively.
To evaluate the purification process, the enzyme-specific activity (SA, expressed in U/mg protein), the purification factor (PF) and the activity recovery (R) were also calculated according to the given equations [30]: SA =
AB CB
(3)
SA B SA i
(4)
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protein content (KP ) or enzyme activity (KE), as shown in Eqs. (1) or (2) [30]:
PF =
R % =
AB (5) Ai
6
where AB and CB are the enzyme activity and protein concentration of the bottom phase, respectively. Ai is the activity and SAi is the specific activity of the crude extract. In Eq. (4), SAB represents the specific activity for the bottom phase.
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Enzyme Activity Assay
Plant-invertase activity was carried out according to the method of Miller [34]. The assay
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buffer) and 0.2 mL of suitably diluted enzyme was incubated at 37 ºC for 30 min. After
that, the reaction was stopped by adding 1 ml of DNS (3,5-Dinitrosalicylic acid) reagent and heated in a boiling water bath for 5 min. It was then cooled to room temperature in ice bath and the amount of reducing sugars was measured spectrophotometrically at 540
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nm [34]. One unit of invertase activity was defined as the amount of enzyme which released 1 µmole of glucose from sucrose per minute at pH 4.5 and 37 ºC. Specific
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activity is expressed as units per milligram of protein with sucrose as a substrate.
Protein Concentration Determination
Concentration of the protein was determined by Bradford method [35] using Coomassie Brilliant Blue G-250 dye as a reagent and bovine serum albumin (BSA) as standard, by
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mixture consisting of 0.8 mL of substrate (50 mM sucrose in 50 mM pH 4.5 citrate
measuring the absorbance at 595 nm at 25 ºC. Assays were performed in triplicate.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Molecular weight of purified invertase was determined by SDS-PAGE according to method of Laemmli [36] on a Mini Protean II gel electrophoresis unit (Bio-Rad
7
Laboratories, Richmond, USA). Electrophoresis was carried out at a constant current of 100V, 400mA for about 2 h. The gel was stained with Coomassie Brilliant Blue R-250 for 1 hour for the detection of proteins and then destained using destaining solution
Kinetic Properties Of Potato Invertase Partitioned In ATPS
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Enzyme assays, described above, were conducted at temperatures from 25 to 65 ◦C to
find the optimum temperature for activity. Percent relative activities were calculated as the ratios of enzyme activities at different temperatures to the maximum enzyme activity, multiplied by 100. To determine thermostability, enzyme was incubated at 25–65 ◦C for
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up to 60 min. Percent residual enzyme activities were measured by the standard assay procedures and reported after dividing by the enzyme activity prior to heat treatment,
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multiplied by 100.
Effect Of Ph On Invertase Activity And Stability To investigate the effect of pH on invertase activity, assays were conducted at various pH values in a range of 4.0-8.0 using 25 mM of the following buffers: sodium acetate (pH
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Effect Of Temperature On Invertase Activity And Stability
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containing 40% (v/v) methanol and 10% (v/v) acetic acid for 3 h.
4.0-6.0), and sodium phosphate (pH 6.5-8.0). Enzyme concentration and temperature were kept constant as stated in standard assay conditions. Percent relative activity was calculated as described above. To determine pH stability of enzyme, enzyme samples were incubated for 60 min in a medium where pH ranged from 4.0 to 8.0. Residual activities were then determined under the standard assay conditions.
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Effect Of Substrate Concentration On Invertase Activity The influence of the substrate concentration on the invertase activity was carried out by determining the initial rates of the hydrolysis reaction with the initial concentration of
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sucrose ranging from 2 to 50 mM. The Michaelis-Menten constants (Km and Vmax) were
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RESULTS AND DISCUSSION
The remarkable demand for invertases in pure science and applied research has led scientist to explore efficient processes to purify the enzyme with high recovery. Several invertase isoforms have been isolated and purified from a number of plants by using
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ammonium sulfate precipitation and various chromatographic systems including ionexchange, gel filtration and affinity chromatography in a highly purified form [7, 15, 38–39].
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Despite the highest purity obtained at the end of the process, these conventional methods increases the overall cost. Considering this, it was intended to develop highly straightforward and cost-effective process for purification of plant invertase.
ATPS offers a very simple, benign and powerful method for the purification and recovery
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determined using Lineweaver-Burk plot [37].
of proteins [19]. ATPSs have many advantages in the downstream processing of biomolecules including characterization of the system with high-water content (70-80%) and subsequent low interfacial tension, low energy requirement, biocompatibility and minimized process cost. Moreover, the biphasic systems increase the enzyme recovery in the purified form [40]. For the enzymes used in industry-such as invertase, the biphasic
9
systems therefore provide many benefits to both researchers and industrial companies. Among invertases from different sources, plant invertases possess great interest due to their significance for plant development, growth and carbon partitioning [41]. Despite various conventional methods available for purification of plant invertases, there are no
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reports presented in relation to isolation and extraction of acid invertase by ATPS and its
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ATPSs of PEG and salts were evaluated to find the best partitioning of invertase in one of two phases. PEG was chosen as phase forming polymer because of its low cost and ability to improve the refolding of proteins. The success of ATPS strongly depends on phase forming salt, molecular weight of PEG, relative proportion of each component and
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pH [42]. As there have multiple factors for the selection of the best phase system for the partitioning of invertase from the crude extract; optimization was performed step by step.
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The partition coefficients, recovery and purity of protein were determined to analyze the partitioning behavior of invertase in the two phases.
In almost all two phase systems, invertase from different sources exhibited greater tendency to participate in the salt-rich bottom phase, indicating the enzyme being highly
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biochemical characterization.
hydrophilic [30, 33, 43]. In fact, it is known that positively charged proteins tend to present in the bottom phase while negatively charged proteins would rather move into the top phase in ATPSs [18, 33]. It should be noted that invertase is known to be stable in the pH range of 3.5-5.5 [44]. Therefore, pH of the system was adjusted to 5.0. The plant invertase would be positively charged at pH 5.0 resulting in its separating to the salt-rich phase. Thus, the
10
data for bottom phase are presented here to reveal invertase partitioning. Temperature used was kept constant in all systems.
Effect Of Salts On Phase Formation
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The partitioning of invertase from potato was studied in several biphasic systems of 10% PEG4000 with different salts, including magnesium sulfate, ammonium sulfate, sodium
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ones most commonly used in ATPSs. Sulfate salt was used due to its ability to promote hydrophobic interactions between proteins whereas citrate was chosen because of its
extraction capability from mother liquid and low environmental polluting properties [45].
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Among the phase systems used, no phase formation was observed in two systems of PEG4000/MgSO4 (10%, w/w) and PEG4000/sodium citrate (10%, w/w). In all systems,
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other than those reported above, phase formation was observed and the target protein appeared preferentially partitioned to the bottom, salt-rich phase (Table 1). The reason for extraction of the highest amount of potato invertase to the bottom phase can be explained by volume exclusion effect, namely an increase in polymer concentration or molecular weight reduces the space for biomolecules in the top phase and as a result, biomolecules
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sulfate and sodium citrate at various concentrations (Table 1). These four salts are the
tend to move to the bottom phase [46–48].
As shown in Table 1, specific enzyme activity, purification factor and recovery of invertase separated in PEG4000/salt systems were influenced by variety of salts tested. An increase in the concentration of ammonium sulfate resulted in a loss of enzyme
11
recovery, and even at low concentrations the recovery obtained was very low. The reason for this result can be described by the denaturation of potato invertase caused by the salting-out effect [49]. On the other hand, sodium sulfate presented promising results with the highest specific activity (0.58 U/mg), purification fold (PF, 2.88) and activity
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recovery (R, 94%). Specific activity of potato invertase partitioned in the presence of Na2SO4 was lower than that of Baker’s yeast invertase in the same salt, while higher
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occurred in the bottom phase in the system composed of 10% PEG4000 and 15%
Na2SO4. Therefore, the PEG/Na2SO4 system was selected for further study. Reports on extraction and purification of various enzymes using PEG/Na 2 SO4 ATPS are available [27, .
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50–52]
Selection Of PEG Molecular Weight
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The partitioning of enzymes has been found to be strongly depended on polymer molecular mass [18, 41]. In general, an intermediate molecular weight of polymer is used to set up the biphase systems [26–27, 30].
To choose a suitable phase forming polymer molecular weight for the purification of
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purification fold was obtained in the presented study [4, 30]. The best enzyme partitioning
potato invertase, various biphase systems were produced with different molecular weights of PEG (MW 1000, 2000, 3000, 4000, 6000 and 8000). The potato invertase partitions in PEG/Na2SO4 systems with varying PEG molecular weights are shown in Fig. 1. Almost all invertases concentrated in the salt-rich phase while contaminants were transferred into the top phase with PEG4000. Above PEG4000, the partition coefficient
12
of invertase was found to decrease from 1.44 to 1.03 with the increase in PEG molecular mass. The decrease of partition coefficient beyond PEG4000 is because of volume exclusion effect, where PEG chain progressively dominates more the top layer, leaving less space for contaminants [51]. The bottom phase therefore was achieving the solubility
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limit of the enzyme in the end and consequently the invertase activity was reducing above PEG 4000. As a synergistic effect, specific activity and enzyme recovery enhanced with
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in molecular mass. The maximum enzyme recovery (94%) and purification factor of 2.85 fold was detected at the PEG molecular mass of 4000. Thus, PEG4000/Na2SO4 system was selected for further studies.
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Effect Of PEG 4000 Concentration On Invertase Partitioning
The effect of PEG4000 concentration on phase formation was further investigated by
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using 10% (w/w) Na2SO 4 at pH 5.0. Results of activity recovery and purification fold versus different PEG concentrations are shown in Fig. 2. The results showed that increasing the concentration of PEG4000 decreased partitioning enzyme specific activity and recovery of invertase in the salt-rich phase. For 12.5% (w/w) concentration of PEG4000, the purification fold reached its highest value of 3.89-fold with the recovery of
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increasing polymer molecular mass till PEG4000 and then lessened with further increase
128%. Above this concentration, both purification fold and recovery were decreased to 2.81-fold and 90%, respectively at 20% (w/w) PEG4000. These findings suggested that increasing PEG concentration had a negative effect on distribution of invertase and other proteins in the crude extract to the bottom and top phases, respectively. An ATPS with
13
the composition of 12.5% (w/w) PEG4000 and 15% (w/w) Na2 SO4 was thus selected for further studies.
Effect Of Salt Concentration
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A further key factor affecting the enzyme partition in biphase systems is the
concentration of salt used. In order to optimize the phase forming salt concentration, a
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employed. The effect of salt concentration on invertase separation is shown in Fig. 3. As is seen in the figure, sodium sulfate concentration did not significantly affect enzyme recovery while purification fold presented more sensitive behavior especially at salt concentrations between 14 and 16%. The maximum enzyme recovery (128%) with a
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purification factor of 3.89 fold was observed at Na 2SO4 concentration of 15%. Therefore,
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the PEG4000(12.5%)/Na2SO4(15%) system was chosen for further experiments.
Effect Of Neutral Salts
In general, neutral salts affect enzyme partitioning in aqueous two phase systems by altering the partition coefficient of proteins in relation to their charge. Proteins having predominantly hydrophobic anions or cations tend to be present in hydrophobic phase or
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series of solutions containing 12.5% PEG4000 and 13-20% (w/w) of Na2SO 4 were
vice versa [32]. Five different salts, KCl, NaCl, MgCl2, Na2CO3, MgSO 4, MnCl2 and MnSO4 were added into the 12.5%PEG4000/15%Na2SO4 system to probe their effects on invertase partition (Fig. 4A). Apart from MgSO4, MnCl2 and MnSO4, nearly all neutral salts had similar effects on phase separation (Fig. 4A). The recovery and purification fold of invertase indicate that partition of invertase into the top and bottom phase is not
14
significantly changed with KCl, NaCl, MgCl2 or Na2CO3 addition to the PEG4000(12.5%)/Na2SO4(15%) system. The partition coefficient (KE) values of enzyme in the bottom phase were calculated as described in Experimental section and found to be 4.98, 4.52, 4.61 and 4.59 for KCl, NaCl, MgCl 2, Na2CO3, respectively. On the other
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hand, addition of MgSO4 into the same system increased the value of K E to 10.86.
However, the highest partition coefficient (KE) value was found as 11.57 while the lowest
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the recovery of invertase activity was previously reported in yeast cells [30]. When
compared with the partition coefficient values of five salts presented, it was noted that MnSO4 had the lowest KP indicating its promoting effect on transfer of contaminant proteins to top phase. Together with results of Karkaş et al. [30], it is suggested
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manganous salt is more effective than other neutral salts tested here for partitioning of
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potato invertase into the bottom and the other contaminants to the upper phase.
Once the best neutral salt determined for invertase purification, different concentrations of MnSO4 were further tested. Fig. 4B shows that the partitioning of enzyme obtains the highest purification fold (5.11) and recovery (197%) in the bottom phase at MnSO4 concentration of 3% (w/w). In consistent with our results, there are many reports also
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KP (protein content, 0.78) in the presence of MnSO4 . The stimulating effect of MnSO4 on
available in literature related to the use of neutral salts to improve the partitioning of different enzymes in different ATPSs [6, 25, 32, 52].
SDS-PAGE Analysis Of Plant Invertase
15
The degree of purification of the resulting enzyme preparation after ATPS was analyzed by SDS-PAGE (Fig. 5). As shown in the figure, crude extract consisted of many proteins with various molecular weights (Fig. 5, lane 2). Subsequent extraction with aqueous two phase system resulted in the majority of the contaminant proteins partitioned to the
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polymer enriched phase (Fig. 5, lane 3). The molecular weight of plant invertase was estimated to be 60 kDa. The molecular weight of acidic plant invertases is mostly
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quite promising since it indicates an improvement in the purification of potato invertase (Fig. 5, lane 3).
Characterization Of Aqueous Two Phase Extracted Invertase
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Influence Of Temperature On The Activity And Stability Of Partitioned Invertase The temperature activity and stability profile of the enzyme is considered to be one
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important factor for industrial applications. Therefore, both invertase activity and stability over wide temperature range (25-65 ◦C) were investigated. The relative activity expressed in percentage of the maximum activity is presented in Fig. 6 as a function of temperature. As is also seen from the figure, relative activities were greater than 70% between 25 ◦C and 55 ◦C and maximal activity was at 37 ◦C for invertase activity (Fig. 6). The results
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differed between 55 and 70 kDa based on the subcellular localization [13, 44]. This result is
compare well with the previous reports. Generally, invertases from different plants show the highest activity at 37 ◦C [7, 38–39].
Thermal stability of potato invertase was analyzed over the temperature range of 25–65 ◦
C and presented in Fig. 6. After treatment of enzyme samples at various temperatures for
16
1 hour, an increase in activity was detected at 37 ◦C. Invertase retained 65 % of its original activity at 65 ◦C indicating the thermal stability of invertase which would be a great advantage for its potential use in different fields of industry. Potato invertase appears to be more stable than Baker’s yeast invertase which retains almost 50% of its
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maximum activity at 60 °C [30]. The influence of temperature on invertase activity and stability has been studied and found to depend strongly on the enzyme localization,
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soluble invertase from wheat coleptiles showed maximum activity at 37 ◦C and the
enzyme retained 60% of its maximum activity after 4 min at 50 ◦C [38]. Another study of plant invertases reported by Singh et al. [39] explains how the thermal stability of different invertase forms in plants differs. The invertase 2 (pollen wall) has remained almost stable
M
at temperatures above 40 ◦C while invertase 1 (cytoplasmic) showed rapid denaturation.
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Influence Of Ph On The Activity And Stability Of Partitioned Invertase The optimum pH on invertase activity was determined by changing the pH of the medium from 4.0 to 8.0 at room temperature. Over the pH range of 4.0–8.0, the highest activity was observed at pH 4.5 with a minor difference in activity within the wide range tested and more than 78% its initial activity was retained (Figure 7). The results agree with the
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incubation time, and temperature and tested medium as well. It’s been reported that the
similar previous works. The pH optimum of plant invertases changes in the range of pH 4.5–8 [7, 44]. The broad pH range offers an advantage in the application of the enzymes at lower pH, which can eliminate the possibility of microbial contamination during longterm operation [30]. Lee et al. [7] have reported that the activity of neutral invertase from carrot was optimum at pH 6.8 and that of alkaline invertase had a pH optimum at pH 8.0.
17
The pH stability is one of the other key factors for selection of enzymes as biocatalysts in many industrial applications. The pH stability of the invertase was examined by preincubating the enzyme at various pH values from 4.0 to 8.0 at room temperature for 1 h
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and measuring the residual activity under the standard assay conditions at 50°C and pH
5.0. Accordingly, plant invertase was most stable at pH 4.5 (Fig. 7). More than 80% of its
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invertase was moderately stable in a pH range from 4.0 to 5.0 retaining about 50% of its initial activity. Above pH 5.0, enzyme activity showed gradual decrease and at pH 8
decreased to 35% initial activity. Similar results have been reported in different studies of [44]
.
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invertases. Generally, plant invertases are stable between pH 4.5 and 5.0
Kinetic Analysis Of Invertase Activity
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The effect of substrate concentration on invertase activity was examined with sucrose using the standard conditions of the assay described in the Materials and methods section (50 °C and pH 5.0). With the substrate sucrose the enzyme obeyed the Michaelis–Menten equation. The kinetic constants, Km and Vmax were calculated from Lineweaver–Burk graph as 3.95 mM sucrose and 0.143 U mL-1 min-1, respectively (Fig. 8). In general, acid
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initial activity remained after 60 min. Based on the results indicated in Fig. 7, the plant
invertases in plants have a Km for sucrose in the low mM range [44]. Matsushita et al. [15] have reported that, for sweet potato acid invertase the K m was determined as 4.5 mM for sucrose as substrate. The Km value observed was quite lower than that found for some other invertases produced from yeasts and plants [15, 30, 44]. Lower Km values reflect
18
higher affinity between substrate and enzyme, indicating invertase from potato tubers having the highest affinity for sucrose among other invertases reported earlier.
CONCLUSION
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In this study, the purification and recovery of invertase from potato tuber by ATPS was introduced for the first time. The effects of some process parameters on invertase
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with 197% recovery and 5.11 purification fold from potato. The partition behavior of
invertase in PEG4000/Na2SO4 indicated that the plant enzyme can be extracted to the salt-rich bottom phase. PEG molecular weight, salt type and concentration were found to have noticeable effect on partitioning of invertase. The optimal system consisted of
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12.5% (w/w) PEG4000, 15% (w/w) Na 2SO4 and 3% (w/w) MnSO4 at system pH 5.0 and room temperature for invertase purification. When compared with other conventional
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purification methods, ATPS represents an inexpensive, straightforward, safe, and highly efficient way to purify proteins. Moreover, it was also intended to characterize the potato invertase partitioned by PEG/Na2 SO4 ATPS. The enzyme was highly active and had an excellent stability when purified by this aqueous two phase extraction system, which makes this potato invertase an attractive biocatalyst for its potential use in many
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extraction were explored. With an efficient and cheap technique, enzyme was purified
industrial applications. The optimized process is also expected to promote the applications of plant invertases in pure science and applied research for better understanding of fundamental processes in plants.
ACKNOWLEDGEMENTS
19
We would like to thank the Scientific Research Unit of Kocaeli University for funding (No. 2012/057).
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Yücekan, İ.; Önal, S. Partitioning of invertase from tomato in poly(ethylene
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232.
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Table 1. Effect of different salt type and concentration on purification of invertase with
SA (U/mg)
PF (fold)
R (%)
10% PEG4000-10% Sodium sulfate
0.36
2
66
10% PEG4000-15% Sodium sulfate
0.58
2.88
94
10% PEG4000-10% Magnesium sulfate
-
-
-
10% PEG4000-15% Magnesium sulfate
0
0
0
10% PEG4000-10% Ammonium sulfate
0.042
0.23
8
10% PEG4000-10% Sodium citrate 10% PEG4000-15% Sodium citrate
0
0
0
-
-
-
0.47
2.61
69
M
Result shows only bottom phase.
an us
10% PEG4000-15% Ammonium sulfate
cr ip t
Phase composition (%, w/w)
ep te d
(-): Phase separation was not observed.
SA: specific activity, PF: purification fold, R: recovery. 0.5 mL crude enzyme of invertase with specific activity 0.52 U mg-1 and 1.63 mg protein.
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10% PEG4000 at pH 5.0
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Figure1. Effect of PEG molecular weight on purification of potato invertase in PEG/Na2 SO4 ATPS at pH 5.0 in the bottom phase. Phase compositions (w/w) (5 g) contain 10% PEG 1000-8000, 15% (w/w) Na2SO4 , and 0.5 ml of crude extract with a
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The partition experiments were performed in triplicate.
cr ip t
plant invertase specific activity of 0.52 U/mg protein and a total protein mass of 1.63 mg.
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Figure 2. Effect of PEG4000 concentration on purification of potato invertase in PEG/Na2 SO4 ATPS at pH 5.0 in the bottom phase. Phase compositions (w/w) (5 g) contain 10-20% PEG4000, 15% (w/w) Na 2SO 4, and 0.5 ml of crude extract with a plant
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partition experiments were performed in triplicate.
cr ip t
invertase specific activity of 0.52 U/mg protein and a total protein mass of 1.63 mg. The
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Figure 3. Effect of Na2SO4 concentration on purification of potato invertase in PEG/Na2 SO4 ATPS at pH 5.0 in the bottom phase. Phase compositions (w/w) (5 g) contain 12.5% PEG4000, 13-20% (w/w) Na2SO 4, and 0.5 ml of crude extract with a plant
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partition experiments were performed in triplicate.
cr ip t
invertase specific activity of 0.52 U/mg protein and a total protein mass of 1.63 mg. The
30
Figure 4. (A) Influence of added neutral salts at 1% (w/w) and (B) Effect of MnSO4 concentration on purification of potato invertase (0.5 ml, specific activity of 0.52 U/mg, total protein mass of 1.63 mg) with 12.5% (w/w) PEG4000 and 15% (w/w) Na 2SO4 at pH
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5.0 in the bottom phase. The partition experiments were performed in triplicate.
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Figure 5. Silver staining of SDS-PAGE gel showing the purity of invertase separated from potato extract by ATPS system. Electrophoresis was carried out at 100 mV for 120 min on 12% polyacrylamide gel system. For each lane, 20 µg protein with specific
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extract; lane 3: purified and recovered invertase.
cr ip t
activity of 6.45 U/mg was applied. Lane 1: protein molecular mass marker; lane 2: crude
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Figure 6. The effect of temperature on activity and stability of potato tuber invertase (3.26 mg/ml) using 50 mM sucrose in 50 mM citrate buffer. Percent relative activity as a function of reaction temperature at pH 4.5. Percent residual activity at the end of 60 min
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All experiments were carried out in triplicate.
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pre-incubation at the stated temperature, followed by standard assay at 37°C and pH 4.5.
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Figure 7. The effect of pH on activity and stability of potato tuber invertase (3.26 mg/ml) using 50 mM sucrose in 50 mM citrate buffer. Percent relative activity as a function of reaction pH at 37°C in sodium acetate and sodium phosphate buffers. Percent residual activity after pre-incubation in 25 mM acetate buffer (pH 4.0-6.0) and phosphate buffer
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(pH 6.5–8.0) at 37°C for 60 min, followed by standard assay at 37°C and pH 4.5. All
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experiments were carried out in triplicate.
34
Figure 8. Enzymatic reaction kinetics of potato invertase towards sucrose. LineweaverBurk double reciprocal plot showing 1/V versus 1/[S] (R2=0.948), V represents initial reaction rate, and [S] represents substrate concentration. All experiments were carried out
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in triplicate.
35
