Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=lsst20 Download by: [37.140.189.245] Date: 14 February 2017, At: 00:45 Separation Science and Technology ISSN: 0149-6395 (Print) 1520-5754 (Online) Journal homepage: http://www.tandfonline.com/loi/lsst20 Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS) Yonca Avcı Duman, Arda Acemi, Yonca Yuzugullu & Fazıl Özen To cite this article: Yonca Avcı Duman, Arda Acemi, Yonca Yuzugullu & Fazıl Özen (2017) Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS), Separation Science and Technology, 52:4, 691-699, DOI: 10.1080/01496395.2016.1253588 To link to this article: http://dx.doi.org/10.1080/01496395.2016.1253588 Accepted author version posted online: 04 Nov 2016. Published online: 04 Nov 2016. Submit your article to this journal Article views: 19 View related articles View Crossmark data
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Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=lsst20
Download by: [37.140.189.245] Date: 14 February 2017, At: 00:45
To cite this article: Yonca Avcı Duman, Arda Acemi, Yonca Yuzugullu & Fazıl Özen (2017)Separation of catalase from Amsonia orientalis with single step by aqueous two-phasepartitioning system (ATPS), Separation Science and Technology, 52:4, 691-699, DOI:10.1080/01496395.2016.1253588
To link to this article: http://dx.doi.org/10.1080/01496395.2016.1253588
Accepted author version posted online: 04Nov 2016.Published online: 04 Nov 2016.
Separation of catalase from Amsonia orientalis with single step by aqueoustwo-phase partitioning system (ATPS)Yonca Avcı Dumana, Arda Acemib, Yonca Yuzugullub, and Fazıl Özenb
aDepartment of Chemistry, Faculty of Arts and Sciences, Kocaeli University, Kocaeli, Turkey; bDepartment of Biology, Faculty of Arts andSciences, Kocaeli University, Kocaeli, Turkey
ABSTRACTCatalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared againsthydrophobic interaction chromatography. Activity recovery and purification fold of purifiedcatalase by ATPS were examined under varying experimental conditions. The effects of variousfactors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH andtemperature effects on partitioning were investigated. The highest activity recovery (156%) andpurification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g)PEG4000, 15% (g/g) Na2SO4 at pH 6.0 and room temperature. In hydrophobic interaction chro-matography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weightof catalase was determined as 75 kDa by SDS-PAGE.
ARTICLE HISTORYReceived 15 March 2016Accepted 24 October 2016
Catalase (hydrogen peroxide oxidoreductase;EC.1.11.1.6), as an antioxidant enzyme, is widely distrib-uted in nature, especially in animals, plants and almost allaerobic organisms. Catalases are capable of protectingthe cells from the toxic effect of hydrogen peroxide bycatalyzing H2O2 into molecular oxygen and water with-out production of free radicals.[1] They are mainly usedin many applications including food, textile and phar-maceutical industries, biosensor systems as well as cancertreatment during anti-metastatic therapy.[2–5]
Purification of catalase by conventional methods suchas salting out and chromatographic techniques is time-consuming and difficult to scale up, requires someexpensive reagents and devices, thus contributing toraise the cost of downstream processing, and increasesmacromolecular unfolding, leading to biological activityloss.[6–10] Aqueous two-phase system (ATPS) is an alter-native method for separation and recovery of biomole-cules with minimum number of steps and thus reducesthe overall cost. This extraction method consists of twowater-soluble polymers (e.g., polyethylene glycol/dex-tran) or a polymer and a salt (e.g., polyethylene glycol/phosphate, sulfate, citrate, etc.) and useful for biotechno-logical application such as separation and purification ofproteins, enzymes, nucleic acids, virus, antibodies andcell organelles.[11,12] It has many advantages for
industrial applications like simple and benign technique(presence of more than 80% water in both phases), rapidseparation with little denaturation (volatile organic com-ponents are not used), rapid mass transfer (low interfa-cial tension), selective separation (affinity partition) andeasy scale-up.[13] The amount of partitioned of a biomo-lecule between two phases can bemodulated by changingthe system conditions such as the type and concentrationof salt, environmental conditions (pH and temperature),characteristic features (molar mass, shape, charge andspecific binding sites) and surface properties of thebiomolecules.[10] ATPS has been successfully used forpartitioning and recovering various enzymes such asD-galactose dehydrogenase,[14] lipase,[15] urease[16] andpapain.[17] To the best of our knowledge, there is onlyone report about partitioning of catalase by ATPS fromPhanerochaete chrysosporium[18] but none from plants.Amsonia orientalis belonging to Apocynaceae family isan endangeredmedicinal and ornamental plant and con-sidered as medicinally important plant due to its alkaloidcontent and antimicrobial activity.[19] The main aim ofthis work was to purify and recover catalase enzymedirectly from the crude extract of callus cultures of A.orientalis by PEG/salt ATPS. For this purpose, we haveidentified the optimal levels of pH, temperature, PEGmolar mass and concentration, salt type andconcentration.
CONTACT Yonca Avcı Duman [email protected] Department of Chemistry, Faculty of Arts and Sciences, Kocaeli University, Kocaeli41380, Turkey.Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsst.
SEPARATION SCIENCE AND TECHNOLOGY2017, VOL. 52, NO. 4, 691–699http://dx.doi.org/10.1080/01496395.2016.1253588
PEG of average molecular weight (PEG 600, 1000, 2000,3000, 4000, 6000 and 8000) was purchased from SigmaChemical Co. (St. Louis, MO, USA) and used withoutfurther purification. All the other reagents were ofanalytical quality and thus were used without furtherpurification. Distilled and deionized water was used inthe experiments.
Healthy shoots, 10 cm in length, were collected from8-year-old field-grown individuals of A. orientalisbefore flowering in May 2013, and all leaves werecut off. These shoots were washed under tap waterfor 15 minutes, and then for easier manageability,they were cut into 1- to 2-cm-long segments thathad at least one node (single node explants).Explants were disinfected by dipping in 70% (v/v)ethyl alcohol (EtOH) for 2 min and 1% (v/v) sodiumhypochlorite (NaOCl) for 12 min, respectively.Disinfected explants were subsequently washed withsterile water three times and then inoculated verti-cally in culture vessels containing 40 ml of Murashigeand Skoog’s[20] medium (MS) supplemented with 6-benzylaminopurine (BAP; 1.0 mg l−1) as performedpreviously.[21] Single node explants from in vitroraised shoots were cultured in the same medium toobtain desired amount of plant material for callusinduction. The leaves of these shoots were collectedand inoculated onto a new callus induction mediumsupplemented with the auxin 2,4-dichlorophenoxya-cetic acid (2,4-D; 0.5 mg l−1) and BAP (0.5 mg l−1) asdescribed previously.[22] At the end of the incubationperiod, developed callus was excised from the leafexplants and cultured further on the same fresh cal-lus induction medium.
Preparation of crude catalase extract from callus ofA. orientalis
Ten gram of callus was homogenized with 50 mMphosphate buffer (pH 7.0). The homogenate wasfiltered from rough filter paper and then centrifugedat 10000 rpm for 15 min at 4 °C (Sigma 4–16 K).The clear supernatant was represented as ‘‘crudecatalase extract’’ and used for further purificationsteps.
Partitioning of catalase
ATPS was performed according to the report byAlbertsson.[23] Stock solutions of 50% (w/w) PEG600, 1000, 2000, 3000, 4000, 6000, 8000 and 40%(w/w) manganese sulfate, sodium citrate, ammoniumsulfate and 30% (w/w) sodium sulfate were prepared.Partitioning experiments were prepared by mixingthe appropriate stock solutions, 50 mM pH 7.0 phos-phate buffer, NaCl and crude catalase extract sample(protein quantity and specific activity of crude extractwere 0.612 mg and 0.15 U/mg, respectively), in atotal weight of 5 g. To avoid protein precipitation,PEG, salt and buffer were mixed before adding of 0.5mL crude enzyme extract. The pH of the system wasadjusted with 0.5M HCl to 6.0. The contents weremixed for 30 min with magnetic stirrer and allowedto phase separate for 30 min at room temperature.Complete separation was done by low-speed centri-fugation for 5 min at 4000 rpm. Top phase andbottom phase were separated by using glass Pasteurpipette, dialyzed overnight against to 50 mM pH 7.0phosphate buffer. Volumes, activity and proteinquantity of each phase were measured carefully. Allexperiments were performed in duplicate, and theresults represented the mean values of two indepen-dent experiments
Calculations of the partitioning parameters weredone according to the below:
Ke ¼ At=Ab (1)
Kp ¼ Ct=Cb (2)
where At and Ab are the total enzyme activities, and Ct
and Cb are the total protein concentrations of thebottom and top phases, respectively.
SAb¼Ab=Cb (3)
PFb¼ SAð Þb= SAð Þi (4)
Yb ¼ 100� Vt � Keð Þ½ �= Vt � Keð Þ þ Vb½ �: (5)
Rb¼Ab=Ai (6)
SA: Specific activity of the enzyme (U/mg protein), thepurification fold (PF), the yield (Yb) and the recovery(Rb) in the bottom phase was also calculated accordingto the given equations above. Ai and SAi are theactivity and the specific activity of initial crudeenzyme, respectively.
692 Y. A. DUMAN ET AL.
Purification of catalase by HIC columnchromatography
Catalase purification was performed with a Bio-Radfraction collector 2110 and Econopump system. Allthe steps of purification were performed at 4°C.Crude enzyme solution as mentioned in Section 2.3was fractionated by HIC on Phenyl Sepharose 6 fastflow high sub. (GE Healthcare, Sweden) The gel waspacked in a column (2.5 x 10 cm) and equilibrated withthe mobile phase (50 mM, pH 7.0 phosphate bufferwith 3 M NaCl) at a flow rate of 2 mL/min. The enzymesolution (15 mL) was applied and the stepwise elutionprofile obtained by continuous measurement of theabsorbance at 280 nm. Fractions of 1 mL were col-lected. The protein concentration and the activitytoward H2O2 were determined.
Catalase activity and total protein assay
Catalase activity was performed by measuring thedecrease in absorbance at 240 nm. The reaction mixturecontained suitably diluted 100 µL enzyme and 10 mM30% (v/v) H2O2 in 50 mM pH 7.0 phosphate buffer in atotal volume of 3 mL. The data presented for all catalaseactivity determinations are mean values of duplicateassay. One unit of activity was defined as the amountof enzyme catalyzing the decomposition of 1µmol H2O2
per min and calculated from the extinction coefficientfor H2O2 at 240 nm of 0.039 cm2µmol−1.[24,25]
Protein contents were measured with Bradford[26]
protein assay with the appropriate dilution of samples.
Electrophoretic analysis
Electrophoretic analysis of catalase was determined bySDS-PAGE according to the method of Laemmli[27] ona Mini Protean II gel electrophoresis unit (Bio-RadLaboratories, Richmond, CA). Protein solutions weremixed at 1:5 (v/v) ratio with the 0.125 mM Tris–basecontaining Coomassie Brilliant Blue R-250, 10% (v/v)2-mercaptoethanol, 40% (v/v) glycerol and 10% (w/v)SDS and boiled for 5 min. The samples were thenloaded onto the gel made of 4% stacking and 12%separating gels and subjected to electrophoresis at cur-rent of 100 V for first 10 min then 120 V for 2 h. Bio-Rad Precision Plus Protein™ Unstained Standard wasused as molecular weight marker. Gels were stainedusing silver stain[28] and photographed. Activity stain-ing of catalase was carried out by the method describedby Manchenko.[29] Accordingly, the gel was soaked in50 mM pH 7.0 phosphate buffer containing 10 mMH2O2 for 30 min then washed with phosphate buffer
(pH 7.0) for 1 min and immerged in the solutioncontaining 30 mM TEMED, 2.5 mM NBT and 5 mMriboflavin with gentle agitation. Achromatic bands wereappeared on blue-dark blue gel.
Results and discussion
Aqueous two-phase systems can be classified into the fourfollowing categories: polymer-based ATPS, ionic liquid(IL)-based ATPS, surfactant-based ATPS and hydrophilicalcohols-based ATPS.[30] The success of ATPS depends onthe choice of the phase system used. The most significantand difficult step in ATPS is to define the appropriate typeand the composition of the system in terms of achievingefficient extraction of the biomolecule.[31] Polymer/salt sys-tems have superiority to polymer/polymer systems due tolarger differences in density, greater selectivity, lower visc-osity, lower cost and the larger relative size of the drops.[13]
In general, polyethylene glycol (PEG) is used as one ofthe phase-forming polymers in ATPS, because it provideslow cost and forms a two-phase system with other neutralpolymers as well as salts. Besides, PEG has a positive effecton the refolding of proteins to recover the activity.[32]
Using of polypropylene glycol (PPG) instead of PEGmay lead to enzyme activity lost.[18] Sulfates are the com-monly used as salts in polymer/salt systems. These ionshave positive effect on hydrophobic interactions betweenthe proteins, which determine the success of ATPS systemand affected by charge, molecular weight, number ofhydrogen bonds and steric effects of biomolecule.[33,34]
But citrate salts are also used as a phase-forming compo-nent with PEG due to being environmental friendly andbiodegradable.[13,35,36]
As there are multiple factors for the selection of thebest phase system for the partitioning of catalase fromthe crude extract, we have studied all parameters one byone and determined the partition coefficients, activityrecovery value and purification factor to analyze thepartitioning behavior of catalase in the top and bottomphases obtained from ATPS system.
Determination of molecular weight concentrationof PEG and type concentration of salt
PEG molecular weight is known to have a great effecton the partitioning behavior of biomolecules. The lowermolecular weight PEG has a hydrophilic end groupwith shorter polymer chains that reduces the hydro-phobicity, but higher molecular weights of PEG haveless coefficient factor; so it appears that lower polymerconcentration is needed for high separation. This maybe due to the low interfacial tension that lower mole-cular weights of PEG have.[36,37]
SEPARATION SCIENCE AND TECHNOLOGY 693
The A. orientalis catalase partition profile in PEG/sodium citrate systems with varying PEG molecularweights is shown in Table 1. Accordingly, the lowestmolecular weights of PEG (600 and 1000) were unableto create two phases in the presence of salt. It has beenreported that the low molecular weights of PEG maypush all proteins to the top phase, so this may cause theweak separation and purification of target molecule.[38]
In our study, although PFt value was higher than PFbvalue (4.43 vs. 2.70) in PEG4000 system, Ab was 0.1 Uwhile At 0.04 U. This result indicates the most ofcatalase partitioned to the bottom phase but still someexisted in top phase at 10% PEG4000/sodium citrate15% (w/w) system. When we compared the activityrecovery of catalase for both phases, the highest activityrecovery of catalase was observed at PEG4000 with theYt of 89% in the bottom phase. As shown in Table 1, Yt
increased with the increase of PEG MW until PEG4000but decreased at the PEG6000 and PEG8000. The low-est Yt was found as 23% in the system consisting of 10%PEG8000 (w/w) and sodium citrate 15% (w/w).
It is noted that the free volume in the top phasesignificantly decreases with an increase in molecularweight of PEG by increasing the chain length of thePEG polymer, resulting in selective partitioning of the
biomolecules to the bottom phase.[39] Moreover, thesolubility is governed by salting out effect in salt-richbottom phase.[40] This result overlaps with the generalrule that the partitioning of the target protein in the topphase decreases with higher molecular weight of PEGresulting from prevailing volume exclusion effect oversalting out.[18]
Table 1 shows that all the Ke and Kp in the systemare bigger than 1 (except Ke of PEG2000). The high Kp
value indicates the most proteins partitioning to the topphase.[41] For successful purification of catalase fromthe contaminant proteins, Kp value should be reduced,on the other hand purification fold and activity recov-ery of catalase be enhanced by the system. Type of salthas a direct effect on the separation and purification ofa protein in ATPS. To investigate the influence of salton the extraction and the partitioning of catalase fromA. orientalis, system was carried out using variousphase-forming salts at a constant concentration, 15%including sodium sulfate, manganese sulfate, sodiumcitrate and ammonium sulfate with 10% (w/w)PEG4000. The effect of salt type on purification andpartitioning of catalase is shown in Fig. 1. It has beenfound that sodium sulfate was the favorable salt for thepartitioning catalase in this system. This may be due toits ability to promote hydrophobic interactions betweenproteins.[33] Although the highest activity recovery ofenzyme was observed in the top phase of 15% manga-nese sulfate and 10% PEG4000 system, Kp value wascalculated as 1.62. On the other hand, a phase systemcontaining 15% sodium sulfate and 10% PEG4000 gavethe highest purification fold (2.64), specific activity(1.40 U/mg), activity recovery, (112%) and the lowestKp (0.82). This system was selected for further studies.
Another factor, besides PEG molecular weight andsalt type, the concentrations of those should have a greateffect on partitioning of catalase. Therefore, to deter-mine the effect of phase-forming salt concentration on
Table 1. The effect of PEG molecular weight in ATPS on thepartitioning of catalase at 25°C, each tube containing crudeextract (protein quantity and specific activity of 0.612 mg and0.15 U/mg, respectively), PEG10% (w/w) and sodium citrate15% (w/w) in a total weight 5 g.Type of PEG, 10(%)
Activity recovery, top phase Activity recovery, bottom phase
Purification factor, top phase Purification factor, bottom phase
Figure 1. The effect of salt type for partitioning of A. orientalis catalase in ATPS at 25 ◦C, each tube containing crude extract (proteinquantity and specific activity of 0.612 mg and 0.15 U/mg, respectively), 10% PEG4000 (w/w) and magnesium sulfate, sodium citrate,ammonium sulfate, sodium sulfate 15% (w/w) in a total weight 5 g.
694 Y. A. DUMAN ET AL.
partitioning of catalase, assays were performed in differ-ent phase systems at the different salt concentrationsvarying from 7 to 20% (w/w) with the constantPEG4000 concentration (10% (w/w)). The results areshown in Fig. 2 A&B. From the figure, it can be seenthat the catalase activity recovery and purification foldwere the highest at 15% (w/w) sodium sulfate concen-tration in the bottom phase (Fig. 2A). The Ke was foundincreased (especially 15% (w/w) sodium sulfate concen-tration) with increasing salt concentration. These resultspointed relative decrease in catalase activity in the topphase compared to that of the bottom phase, at increas-ing salt concentrations. In the case of total proteins, theKp was observed decreased with increasing salt concen-tration at 15% (w/w) sodium sulfate concentration asseen from Fig. 2B. This can be due to the presence ofhigher amounts of protein in the top phase then thebottom phase. It has been previously reported thatincreasing salt concentrations cause decrease in the Kp
value but increase in the Ke value at the bottom phasemeaning that the contaminant protein load in top phaseresults in improved purification factor.[39,42] Accordingto Babu et al.,[42] in PEG/salt systems, the partitioning of
the biomolecules depends on the volume exclusion effectof the polymer in the polymer-rich (top) phase andsalting out effect in the salt-rich (bottom) phase. Theauthors stated that with an increased salt concentrationin the salt-rich (bottom) phase, the solubility of biomo-lecules decreases, which results in increased partitioningof biomolecules to the top phase and is inferred as “salt-ing out effect.” In this study, the solubility limit wasdetected at 15% sodium sulfate; thus, maximum enzymerecovery (113%) with a purification factor of 2.66-foldwas observed at 15% (w%w) sodium sulfateconcentration.
The effect of PEG4000 concentrations on partition-ing of catalase was carried out in different phase sys-tems of PEG4000 (7.5–20% w/w) while maintainingsodium sulfate constant at 15% (w/w). Figure 3A&Bshows the results indicating activity recovery and pur-ification fold of catalase in the bottom phase decreasedwith an increase in PEG4000 till 10% (w/w)concentration.
As can be seen from Fig. 3A, the highest activityrecovery and purification fold were observed at 10%PEG4000 concentration as 119% and 2.70, respectively.From Fig. 3B, Ke and Kp values showed positive and
A
0.0000.0200.0400.0600.0800.1000.1200.1400.160
0.60
0.70
0.80
0.90
1.00
1.10
1.20
7.5 10 12.5 15 17.5 20
Kp
Ke
Sodium Sulfate Concentration, (%)
Ke KpB
Figure 2. (A) The effect of sodium sulfate concentration onpurification fold and activity recovery of A. orientalis catalasein ATPS at 25◦C, each tube containing crude extract (proteinquantity and specific activity of 0.612 mg and 0.15 U/mg,respectively), 10% PEG4000 (w/w) and increasing concentra-tions of sodium sulfate (7–20% (w/w)) in a total weight 5 g.(B) Protein content and activity of catalase at the conditions asdesribed in Fig.2.A.
B
A
Figure 3. (A), The effect of PEG4000 concentration on purifica-tion fold and activity recovery of A. orientalis catalase in ATPS at25◦C, each tube containing crude extract (protein quantity andspecific activity of 0.612 mg and 0.15 U/mg, respectively), 15%sodium sulfate (w/w) and increasing concentrations of PEG4000(7–20% (w/w)) in a total weight 5 g.(B), Protein content andactivity of catalase at the conditions as desribed in Fig.3A.
SEPARATION SCIENCE AND TECHNOLOGY 695
negative effects, with increase in PEG4000 concentra-tion. Hence, it can be said that the highest enzymeactivity and the lowest protein account were detectedat 10% PEG4000. For this reason, PEG4000 10% andNa2SO4 15% system was selected further more.
pH and temperature effect on partitioning ofcatalase
As protein molecules are ionic molecules due to aminoacid residues, partitioning of enzymes is dependent onpH (Table 2). As a general rule, negatively chargedproteins prefer the top phase in PEG/salt systems,while positively charged ones normally partition inthe bottom phase due to the electrostatic attractionresulting from charge distribution.[18] The isoelectricpoint is the pH at which protein has a net charge ofzero. At higher pH, the protein is more negativelycharged; at low pH, the proteins have a net positivecharge. In this study, increased purification fold andactivity recovery of catalase at pH 6.0 might be relatedto pI value of enzyme. So, it would become inevitablethat negatively charged catalase at lower pH valuespartitioned to the bottom phase.
The partition of catalase by aqueous two-phasesystem was assayed at two different temperatures: 25(room temperature) and 37°C (catalase activity assaytemperature). Temperature effect on the phase com-position is complicated due to the fact that theelectrostatic interactions and the hydrophobic inter-actions are all interacted to the temperature. Somereports have described increasing or decreasingeffects of temperature on partition coefficient,while others have found that the partition coeffi-cient showed no temperature dependence.[43] Inour study, we have also observed no market effectof temperature on catalase partitioning. This may bebecause there was no change to phase compositionin the PEG4000/Na2SO4 (10–15% (w/w)) at pH 6system when temperature was changed. Therefore,room temperature was selected for further studies(Table 3).
Catalase purification
To compare the purification efficiency of ATPS withchromatography technique, catalase was purifiedfrom the same crude A. orientalis extract via sin-gle-step hydrophobic interaction chromatographyand eluted with a stepwise NaCl gradient (Fig. 4).The chromatographic profile is presented in Fig. 4where three major protein peaks were observed, butonly one peak contained the active protein. HIC
Table 2. The effect of pH on partitioning of A. orientalis catalasein ATPS at 25°C, each tube containing crude extract (proteinquantity and specific activity of 0.612 mg and 0.15 U/mg,respectively), 10% PEG4000 (w/w), 15% sodium sulfate (w/w)in a total weight 5 g.
Table 3. The effect of temperature on partitioning of A. orientaliscatalase in ATPS at 25°C, pH 6.0, each tube containing crude extract(protein quantity and specific activity of 0.612 mg and 0.15 U/mg,respectively), 10% PEG4000 (w/w) 15% sodium sulfate (w/w) in atotal weight 5 g.
Figure 4. Amsonia orientalis catalase purification by using Phenyl Sepharose high-performance column at pH 7, 3 M NaCl. Elutionwas carried out by stepwise in 3.0 M to 0 M NaCl.
696 Y. A. DUMAN ET AL.
gave 12.54-fold purified protein with a final yield of57.5% and an observed specific activity of 1.76 U/mg. Partitioning and purification profiles of A.orientalis catalase by ATPS and HIC column chro-matography are summarized in Table 4. Comparedto HIC, the ATPS proves to be a useful system forthe recovery, purification and concentration ofcatalase.
SDS-PAGE analysis of catalase
The purity of partitioned catalase was analyzed by SDS-PAGE. Figure 4 shows the corresponding pattern. Asshown in the figure, crude extract consisted of manyproteins with various molecular weights. Extractionwith the aqueous two-phase system resulted in themajority of the contaminant proteins being partitionedto the polymer-enriched phase (Figure 4, lane 3).According to activity staining analysis, molecularweight of catalase was estimated as 75 kDa whichagreed well with the literature.[44–46]
Conclusions
In this study, the partitioning of catalase fromAmsonia orientalis by ATPS was introduced for thefirst time. The effects of extraction parameters onthe partitioning of catalase were determined. Withan efficient and cheap technique, the enzyme waspurified with 156% recovery and 8.67 purificationfold plant. The partition behavior of catalase inPEG4000/Na2SO4 (10–15% (w/w)) at pH 6.0 androom temperature indicated that the plant enzymecan be extracted to the salt-rich bottom phase. PEGmolecular weight, salt type and concentration werefound to have a noticeable effect on the partitioningof catalase. When compared with other conventionalpurification methods, ATPS represents an inexpen-sive, straightforward, safe and highly efficient way topurify proteins. Moreover, it was also determinedthe molecular weight of catalase as 75 kDa withsingle subunit. Purification of the enzyme with thismethod might be useful in many industrialapplications.
Funding
This work was supported by The Scientific and TechnologicalResearch Council of Turkey (TÜBİTAK) under Grant(Number 113Z609).
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