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Three-phase partitioning of protease from Calotropis procera latex
Biochemical Engineering Journal 50 (2010) 145–149
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Three-phase partitioning of protease from Calotropis procera latex Saroat Rawdkuen a,∗ , Phanuphong Chaiwut b , Punyawatt Pintathong b , Soottawat Benjakul c a
Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand School of Cosmetic Science, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand c Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand b
a r t i c l e
i n f o
Article history: Received 12 March 2010 Received in revised form 7 April 2010 Accepted 17 April 2010
Keywords: Calotropis procera Latex Protease Three-phase partitioning t-Butanol
a b s t r a c t Three-phase partitioning (TPP) was used to partially purify protease from the latex of Calotropis procera (C. procera). To optimize the TPP for protease isolation a ratio of crude extract to t-butanol, percent saturation of (NH4 )2 SO4 , and the cycle of TPP was required. The highest proteolytic recovery (first cycle) of 182% with a purification of 0.95 folds was obtained at the interphase of the system comprising the ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 50% (NH4 )2 SO4 . The second cycle of TPP was prepared by adding of (NH4 )2 SO4 up to 65% (w/v) to the bottom phase obtained from 30% (NH4 )2 SO4 –1.0:0.5 system of the first TPP. A purification of 6.92-fold was achieved with about 132% activity recovery. SDS-PAGE and zymography profiles revealed the substantial isolation of protease from C. procera latex by the TPP. The molecular weight of major protease was found to be around 28 kDa. The present study shows high interesting outcomes and could be used as a primary purification process in comparison with existing literature’s values. © 2010 Published by Elsevier B.V.
1. Introduction Three-phase partitioning (TPP) is a relatively recent bioseparation technique, which employs collective operation of principles involved in numerous techniques for protein precipitation. The TPP has widely been used for the extraction and purification of various proteins [1–5]. It uses (NH4 )2 SO4 with certain saturation to precipitate the protein, and t-butanol was added to make three-phase layers and to remove some small molecular weight compounds such as lipids, phenolics and some detergents [6]. In general, biomolecules are recovered in a purified form at the interphase, while the contaminants mostly partition in t-butanol (top phase) and aqueous phase (bottom phase) [3]. This method was scalable and could be used directly with the crude suspensions [4]. Proteases from plant sources have received special attention from the pharmaceutical industry and by food biotechnology because their properties of activity over wide range of temperature and pHs. They have been exploited commercially in the food industry such as papain for meat tenderizing and ficin and bromelain for brewing [7]. Although recently enzymes from microorganism have been widely commercially produced, consumer still aware for used it as an edible ingredients into their meals. Therefore, natural sources from both plant and animal tissues still desired.
∗ Corresponding author. Tel.: +66 5391 6752; fax: +66 5391 6739. E-mail address: [email protected] (S. Rawdkuen). 1369-703X/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.bej.2010.04.007
Calotropis procera is a plant found in tropical and sub-tropical regions. It is well-known for its great capacity of producing latex which exudates from the green damaged parts. Scientific reports have mentioned various medicinal activities of C. procera latex, such as insecticidal [8], anti-fungal [9] and wound healing [10]. Some biochemical properties of the enzyme containing in the latex of C. procera have been documented and named as procerain [11]. It has been reported that C. procera latex is a potential material for enzyme purification [12,13]. Consequently, scientific researches of protease extraction from C. procera have recently been reported. Protease activity recovery of 74.6% with 4.08-fold of purification was obtained from C. procera latex by using aqueous two-phase system [13]. However, there is no report of using TPP as the single method to separate the protease from this potential source. Therefore, the aim of this study was to optimize the separation process of protease from the latex of C. procera by using TPP technique. 2. Materials and methods 2.1. Chemicals and raw materials Polyethylene glycol (PEG), sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), casein and l-cysteine were obtained from Fluka (Buchs, Switzerland). Beta-mercaptoethanol (-ME) and Coomassie Brilliant Blue G-250 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ammonium sulfate ((NH4 )2 SO4 ),
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magnesium sulfate (MgSO4 ), trichloroacetic acid (TCA), tertbutanol (t-butanol) and other chemicals with analytical grade were obtained from Merck (Darmstadt, Germany). Latex of C. procera was collected from Nayong, Trang Province, Thailand.
2.5. Protein determination
2.2. Latex preparation
2.6. Electrophoresis
Latex was collected in a clean tube by breaking the C. procera stems and then diluted with distilled water (1:1, v/v). After mixing, it was centrifuged at 8000 × g at 4 ◦ C for 10 min. The obtained supernatant was referred to as the “crude extract” [protein content: 3.49 mg/ml, activity: 1138 unit/ml, specific activity: 326 unit/mg protein] and used for further study.
2.6.1. SDS-PAGE SDS-PAGE of the samples was performed according to the method of Laemmli [15] with slight modification. Protein solutions were mixed at a 1:1 (v/v) ratio with the sample buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 20% glycerol). The samples (10 and 2 g protein for protein and activity staining, respectively) were loaded onto the gel made of 4% stacking and 15% separating gels. They were subjected to an electrophoresis set at a constant current of 20 mA/gel. For protein staining, the gel obtained after electrophoresis was stained overnight with a solution of 0.02% (w/v) Coomassie Brilliant Blue R-250 in 50% (v/v) methanol, and 7.5% (v/v) acetic acid. Gels were destained with 50% (v/v) methanol and 7.5% (v/v) acetic acid for 40 min, followed by 5% (v/v) methanol and 7.5% (v/v) acetic acid for 20 min. Pierce blue prestained protein molecular weight marker mix consists of (kDa) myosin (215), phosphorylase B (120), BSA (84), ovalbumin (60), carbonic anhydrase (39.2), trypsin inhibitor (28) and lysozyme (18.3) was used.
2.3. Three-phase partitioning 2.3.1. Effect of crude extract to t-butanol ratio on protease partitioning The TPP was carried out as described by Roy and Gupta [3]. The effect of the ratio of crude extract to t-butanol was studied. Firstly, t-butanol was added to the crude extract at the ratios of crude extract to t-butanol of 1.0:0.5, 1.0:1.0, 1.0:1.5, and 1.0:2.0 (v/v) with a constant (NH4 )2 SO4 saturation of 30%. The mixture was mixed thoroughly and then allowed to stand for 60 min before subjecting to centrifuge at 5000 × g for 10 min to facilitate the separation of phases. The lower aqueous layer and the interfacial precipitate were collected and dialyzed against water overnight at 4 ◦ C. After dialysis, the samples were analyzed for protease activity and total protein content. The ratio employing the highest enzyme recovery was chosen for further study. 2.3.2. Effect of ammonium sulfate saturation on protease partitioning The (NH4 )2 SO4 saturations at 20, 30, 40, and 50% affecting the partitioning of protease was also investigated by using the ratio of crude extract to t-butanol (with the highest enzyme recovery) obtained from 2.3.1. The lower aqueous layer and the interfacial precipitate were collected and dialyzed against water overnight at 4 ◦ C. After dialysis, the samples were analyzed for protease activity and total protein content. The system providing the highest protease recovery was chosen for further study. 2.3.3. Optimization of TPP for proteases recovery The phase obtained from the first TPP that gave the highest activity recovery was chosen as the starting material for optimization in the second TPP. The selected phase (without dialysis) was mixed with the t-butanol in ratio of 1.0:0.5 and (NH4 )2 SO4 was added to the mixture to obtain the final saturations of 50, 55, 60, and 65%. After the complete phase separation, the phases were collected as previously mentioned. The dialyzed phases from the second TPP were subjected to protease activity and total protein content analysis. 2.4. Caseinolytic activity assay An enzyme sample of 0.10 ml was mixed with 1.10 ml of 1% (w/v) casein in 0.10 M Tris–HCl (pH 8.0) containing 12 mM cysteine. The reaction was started by incubation the mixture at 37 ◦ C for 20 min. The reaction was stopped by adding 1.8 ml of 5% TCA. After centrifugation at 3000 × g for 15 min, the absorption of the soluble peptides in supernatant was measured at 280 nm. One of caseinolytic activity units is defined as the amount of enzymes needed to produce an increment of 0.01 absorbance unit per minute at the assayed condition [13].
Protein concentrations were measured by Bradford method using BSA as a protein standard [14].
2.6.2. Protease activity staining The protease separated on the gel was verified by using activity staining as done in Garcia-Carreno et al. [16]. The gel was immersed in 50 ml of 2% (w/v) casein in 50 mM Tris–HCl buffer, pH 8.0 containing 12 mM cysteine for 30 min with constant agitation at 4 ◦ C. The reaction was generated by incubation the gel at 37 ◦ C for 15 min. The treated gel was then stained and destained as described above. The development of a clear band on the dark background indicated the caseinolytic activity of protease from C. procera latex. 3. Results and discussion 3.1. Effect of the ratio of crude extract to t-butanol on protease partitioning The effect of crude extract to t-butanol ratio for protease partitioning in the first TPP was firstly investigated. Different TPP experiments with various extract to t-butanol ratios (1.0:0.5, 1.0:1.0, 1.0:1.5 and 1.0:2.0) at 30% (w/v) ammonium sulfate saturation were performed. The highest protease recovery (99.3%) and purification fold (1.13-fold) were obtained from the bottom phase of a TPP system with the ratio of 1.0:0.5. An increase in t-butanol volume resulted in a decrease in activity recovery and purification fold values of the bottom phase. This may be attributed to the synergistic effects of the increase in concentration of t-butanol and decrease in saturation of ammonium sulfate [5]. Dennison and Lovrien [6] reported that only 0.2–0.5 ml of t-butanol usually is required per milliliter of beginning aqueous sample to precipitate out protein. The best results were obtained when the ratio was 1.0:1.0 used for partitioning of ␣-galactosidase from fermented media of Aspergillus oryzae with 60% (NH4 )2 SO4 saturation [4]. The ratio of sample to t-butanol content is higher than one, the denaturation of the protein is more likely [4]. The partitioning of protease in the interphase was contrary with the bottom phase. However, the yield obtained was still lower than 30% in all ratios tested. As the results, most protease was still remained in the aqueous phase rather than in the interphase. Therefore, the ratio of crude extract to t-butanol of 1.0:0.5 which provided the highest recovery
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was selected for investigating the effect of (NH4 )2 SO4 saturation on partitioning of C. procera protease. 3.2. Effect of ammonium sulfate saturation on protease partitioning The study was performed by maintaining the ratio of crude extract to t-butanol at 1.0:0.5 and varying the (NH4 )2 SO4 saturation in the ranges of 20, 30, 40, and 50%. Generally, one starts with a minimum salt saturation of 20% and optimizes this so as to obtain the maximum amount of the desired protein in the interfacial precipitate [1,2]. It was found that the protease recovery of 182% yield with 0.95 folds of purification was obtained in the interphase of the system contained 50% (NH4 )2 SO4 . Increase of (NH4 )2 SO4 saturation resulted in increase of protease recovery in the interphase. However, the purity of enzyme seemed to be reduced when (NH4 )2 SO4 saturation was raised. Narayan et al. [5] reported that the increasing in concentration of (NH4 )2 SO4 , the degree of purification decreased significantly. As in conventional salting out with (NH4 )2 SO4 , the extent of protein precipitation in TPP is a function of the (NH4 )2 SO4 concentration [17]. The“salting out effect” plays a major role in the system contained high salt concentration. The principle of sulfate ion for salting out protein has been viewed in five different ways namely, ionic strength effects, kosmotropy, cavity surface tension and enhancement osmotic stressor (dehydration), exclusion crowding agent, and binding of sulfate ion to cationic sites of protein [5]. As the result obtained, the system consisted of 50% (NH4 )2 SO4 was seemed to be the optimum condition for protease partitioning due to its highest protease yield at the interphase. However, this fraction gave low enzyme purity. While the highest purity was found in the bottom phase of the system contained 30% (NH4 )2 SO4 . This system was also belonged to the highest yield among all of the bottom phase. Pike and Dennison [18] also reported that 30% (w/v) (NH4 )2 SO4 is the best salt concentration for carrying out TPP efficient. 3.3. Optimization of TPP for recovery of protease from C. procera latex The system condition from the first TPP (1.0:0.5 extract: tbutanol (v/v), 30% (NH4 )2 SO4 ) saturation) providing the highest protease recovery in the aqueous phase was used as the starting
Fig. 1. Optimization of (NH4 )2 SO4 saturation for protease partitioning by the second step three-phase partitioning. The bottom phase of the system of 1.0:0.5 extract: t-butanol in the presence 30% (NH4 )2 SO4 was used as the starting material for the second TPP. Bar [] and line [–] represent the protease recovery and purification fold, respectively. I-phase: interphase, B-phase: bottom phase and P-phase: precipitate.
material for optimization of enzyme partitioning in the second TPP. The reason for carrying out the second cycle of TPP was that sometimes the first TPP cycle could not remove the contaminant proteins efficiently. It was expected that the target protease will be more partitioned to the interphase with the highest yield and purity. Saxena et al. [19] reported that the aqueous phase containing most of the desired protein when subjected to a second cycle of TPP results in considerable purification of the target protein. Therefore, in this study, (NH4 )2 SO4 was added to the aqueous phase of the first TPP fraction to obtain the final saturation of 50, 55, 60, and 65%. Interestingly, it was found that the precipitated phase was also observed as the fourth phase. This can be explained by increase of salt addition provided over salting out effect and protein subsequently precipitated as indicated by the fourth phase occurred. As shown in Fig. 1, increasing of purification fold in the precipitate as well as in the bottom phase was found when the salt content increased. At the salt content of 65%, most protease was recovered in the bottom phase (202.7%) with a little remaining in the interphase (1.1%). However, residue protease was precipitated (35.2%) with the highest purification of 12.79 folds. Kiss et al. [20] concluded that the amount of protein precipitated in the interphase was delineated as a function of the composition of the partitioning system as well as the initial protein concentration.
Fig. 2. Protein pattern (A, 10 g protein) and activity staining (B, 2 g protein) of Calotropis procera latex and their fractions from three-phase partitioning. M: molecular weight marker, 1: crude latex, 2: interphase of 1.0:0.5 t-butanol–50% (NH4 )2 SO4 , 3: interphase of 60% (NH4 )2 SO4 , 4: precipitate of 60% (NH4 )2 SO4 , 5: bottom phase of 1.0:0.5 t-butanol–50% (NH4 )2 SO4 , and 6: bottom phase of 60% (NH4 )2 SO4 .
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Table 1 Recovery of protease from Calotropis procera latex in the interphase (I) of TPP. Sample
Total activity (U)
Total protein (mg)
Crude latex I-1st TPP-50% ASa I-2nd TPP-60% ASb
5,633 ± 107 10,252 ± 244 7,445 ± 505
18.48 ± 1.34 35.40 ± 0.32 3.53 ± 0.05
Specific activity (U/mg) 304.82 ± 20.54 289.58 ± 4.33 2,110.00 ± 110.00
Purification (fold)
Recovery (%)
1.00 ± 0.00 0.95 ± 0.03 6.92 ± 0.34
100 ± 0.0 182 ± 2.1 132 ± 8.9
a
The system consists of the ratio of crude extract to t-butanol of 1.0:0.5–50% (NH4 )2 SO4 . The system consists of the ratio of bottom phase of the first TPP (1.0:0.5 t-butanol, 30%, saturation of (NH4 )2 SO4 ) with the addition of (NH4 )2 SO4 to 60%. I: interphase, AS: (NH4 )2 SO4 . b
Increasing activity recovery was found in the interphase of the system containing (NH4 )2 SO4 less than 60% (Fig. 1). As expected, the highest yield (132%) and high enzyme purity (6.92 folds) was found in the interphase of the system added with 60%(NH4 )2 SO4 . In view of interest, the second TPP system containing the crude extract to t-butanol ratio of 1.0:0.5 with the addition of (NH4 )2 SO4 up to 60% was efficient to partial purified the protease from C. procera. Narayan et al. [5] reported that the optimum condition for extraction peroxidase from the leave of Ipomoea palmate consisted of the crude extract to t-butanol of 1.0:1.0 and 30% (NH4 )2 SO4 . By using this condition resulted in about 160% activity recover and 2-fold of purification in the aqueous phase of the first TPP. Protein patterns of the crude extract and their fractions from the first TPP and second TPP are shown in Fig. 2A. The major protein bands with the molecular weight (MW) of around 39–50 and less than 18.3 kDa were found as the major component in the crude extract (lane 1A). It was observed that the protein pattern of the precipitate fraction (lane 4A) was present as higher band intensity than those of the crude extract and other fractions. For the first TPP containing the crude extract to t-butanol at the ratio of 1.0:0.5 with 50% (NH4 )2 SO4 , a numbers of protein band mostly presented in the bottom phase (lane 5A) than in the interphase (lane 2A). There are previous reports that the maximum proteolytic activity of C. procera protein found a protein band possessing around 30 kDa [20,21]. It was reported that the purified protease from C. procera appeared as a single band at around 29 kDa in a reducing condition of SDS-PAGE [11]. Fig. 2B shows the activity staining (zymography) of the crude extract and the fractions from TPP optimization. Clear zone on the dark background was remarked as the activity of protease against casein substrate on the gel. A protein band with the molecular weight of ∼30 kDa was clearly observed in all fractions. Depending on the intensity of the clear zone, the fraction of the interphase provided interesting protease activity (lanes 2B and 3B) as expected. The results revealed that TPP was able to partition the proteases into the interphase of system in which the salt and t-butanol content was optimized. 3.4. Overall partial purification of protease from C. procera latex by TPP The isolation of protease from C. procera latex (by the first TPP with 50% (w/v) (NH4 )2 SO4 and 1.0:0.5 ratio of the crude extract to t-butanol) gave the highest protease recovery (182%) in the interphase (Table 1). However, for further optimization of enzyme partitioning in the second TPP, the bottom phase of the system containing 30% saturated (NH4 )2 SO4 was used as it provided the highest yield among the bottom phases of all conditions tested. Performing the second TPP with 60% saturated (NH4 )2 SO4 resulted in 132% recovery in the interphase with higher purity (6.92 folds). High protease recovery obtained may probably due to the TPP led to simultaneous activation of the enzyme which results of such an apparently observed value more than 100%. Singh et al. [22] reported that the tertiary structure of serin protease (proteinase K) was altered by TPP, reflecting remarkable enhancement of the enzyme activity. Dennison and Lovrein [6] reported that Bacillus
subtilis protease, Saccharomyces cerevisiae invertase, Candida cylindracea lipase gave a yield of 300, 100 and 900%, respectively, after TPP. These unusual percentage yields were believed to be due to adopting a number of amino acid residues on the enzyme molecule. Sharma and Gupta [1,2] also reported that enzyme activation frequently observed during TPP may be a result of increased flexibility in the enzyme molecule. From the second TPP, the overall result of protease activity recovery, as well as of final purity, is considerably better than the first TPP. Roy and Gupta [3] reported that using TPP for biological activity recovery provided the best results with high recovery activity (90–98%) and high purification fold (65–100-fold). Increasing of purification fold of 18 times with 81% activity recovery was achieved with the second TPP. Saxena et al. [19] also found that by using the second TPP can increase the purification fold of amylase inhibitor and trypsin inhibitor from 8.9 and 8.65-fold to 20.1 and 16-fold purification, respectively. To compare between the first and second TPP in terms of recovery and purity of enzyme from C. procera, the first TPP possessed higher protease yield of 182% than that of the second TPP (132%). In addition, purity of enzyme was increased from 0.95 folds in the first step to 6.92 folds in the second step. This indicates that purification of enzyme was greatly obtained during operation of the second TPP. It can be pointed that only first step was sufficient when consideration of enzyme recovery. However, when regarding to the purity of the enzyme, the second step should be needed. Thus procedures utilizing the two cycles of TPP constitute a fairly simple and efficient isolation process for protease and they also yield a partial purified protein. The availability of this simple partial purification strategy should be employed in production of protease with a more efficient and economic way. 4. Conclusion The TPP was a useful technique for partial purification of protease from C. procera. It is a simple, quick and economical technique and scaling up is convenient. The TPP system consists of the ratio of crude extract to t-butanol of 1.0:0.5 and 50% (w/v) (NH4 )2 SO4 gave the highest yield but rather low purity in the interphase. Increasing the (NH4 )2 SO4 saturation of the bottom phase to the first TPP up to 60% could improve the purity of the enzyme up to 6.92 folds. Acknowledgements The authors would like to express their sincere thanks to Mae Fah Luang University, Thailand and The Thailand Research Funds under The Senior Research Scholar program for the financial support. References [1] A. Sharma, M.N. Gupta, Purification of pectinases by three-phase partitioning, Biotechnol. Lett. 23 (2001) 1625–1627. [2] A. Sharma, M.N. Gupta, Three phase partitioning as a large-scale separation method for purification of a wheat germ bifunctional protease/amylase inhibitor, Process Biochem. 37 (2001) 193–196.
S. Rawdkuen et al. / Biochemical Engineering Journal 50 (2010) 145–149 [3] I. Roy, M.N. Gupta, Three-phase affinity partitioning of proteins, Anal. Biochem. 300 (2002) 11–14. [4] S.K. Dhananjay, V.H. Mulimani, Three-phase partitioning of ␣-galactosidase from fermented media of Aspergillus oryzae and comparison with conventional purification techniques, J. Ind. Microbiol. Biotechnol. 36 (2009) 123–128. [5] A.V. Narayan, M.C. Madhusudhan, K.S.M.S. Raghavarao, Extraction and purification of Ipomoea peroxidase employing three-phase partitioning, Appl. Biochem. Biotechnol. 151 (2008) 263–272. [6] C. Dennison, R. Lovrein, Three phase partitioning: concentration and purification of proteins, Prot. Exp. Pur. 11 (1997) 149–161. [7] G. Walsh, Proteins: Biochemistry and Biotechnology, John Wiley & Sons, Ltd., New York, 2002. [8] M.V. Ramos, C.D.T. Freitas, F. Staniscuaski, L.L.P. Macedo, M.P.S. Sales, D.P. Sousa, C.R. Carlini, Performance of distinct crop pests reared on diets enriched with latex proteins from Calotropis procera: role of laticifer proteins in plant defense, Plant Sci. 173 (2007) 349–357. [9] R. Sehgal, S. Arya, V.L. Kumar, Inhibitory effect of extracts of latex of Calotropis procera against Candida albicans: a preliminary study, Ind. J. Pharmacol. 37 (2005) 334–335. [10] A.M. Rasik, R. Raghubir, A. Gupta, A. Shukla, M.P. Dubey, S. Srivastava, H.K. Jain, D.K. Kulshrestha, Healing potential of Calotropis procera on dermal wounds in guinea pigs, J. Ethnopharmacol. 68 (1999) 261–266. [11] V.K. Dubey, M.V. Jagannadham, Procerain, a stable cysteine protease from the latex of Calotropis procera, Phytochemistry 62 (2003) 1057–1071. [12] S.O. Kareem, I. Akpan, M.B. Osho, Calotropis procera (Sodom apple)—a potential material for enzyme purification, Bioresour. Technol. 87 (2003) 133–135. [13] S. Rawdkuen, P. Pintathong, P. Chaiwut, S. Benjakul, The partitioning of protease from Calotropis procera latex by aqueous two phase system and its
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Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Three-phase partitioning of protease from Calotropis procera latex Saroat Rawdkuen a,∗ , Phanuphong Chaiwut b , Punyawatt Pintathong b , Soottawat Benjakul c a
Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand School of Cosmetic Science, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand c Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand b
a r t i c l e
i n f o
Article history: Received 12 March 2010 Received in revised form 7 April 2010 Accepted 17 April 2010
Keywords: Calotropis procera Latex Protease Three-phase partitioning t-Butanol
a b s t r a c t Three-phase partitioning (TPP) was used to partially purify protease from the latex of Calotropis procera (C. procera). To optimize the TPP for protease isolation a ratio of crude extract to t-butanol, percent saturation of (NH4 )2 SO4 , and the cycle of TPP was required. The highest proteolytic recovery (first cycle) of 182% with a purification of 0.95 folds was obtained at the interphase of the system comprising the ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 50% (NH4 )2 SO4 . The second cycle of TPP was prepared by adding of (NH4 )2 SO4 up to 65% (w/v) to the bottom phase obtained from 30% (NH4 )2 SO4 –1.0:0.5 system of the first TPP. A purification of 6.92-fold was achieved with about 132% activity recovery. SDS-PAGE and zymography profiles revealed the substantial isolation of protease from C. procera latex by the TPP. The molecular weight of major protease was found to be around 28 kDa. The present study shows high interesting outcomes and could be used as a primary purification process in comparison with existing literature’s values. © 2010 Published by Elsevier B.V.
1. Introduction Three-phase partitioning (TPP) is a relatively recent bioseparation technique, which employs collective operation of principles involved in numerous techniques for protein precipitation. The TPP has widely been used for the extraction and purification of various proteins [1–5]. It uses (NH4 )2 SO4 with certain saturation to precipitate the protein, and t-butanol was added to make three-phase layers and to remove some small molecular weight compounds such as lipids, phenolics and some detergents [6]. In general, biomolecules are recovered in a purified form at the interphase, while the contaminants mostly partition in t-butanol (top phase) and aqueous phase (bottom phase) [3]. This method was scalable and could be used directly with the crude suspensions [4]. Proteases from plant sources have received special attention from the pharmaceutical industry and by food biotechnology because their properties of activity over wide range of temperature and pHs. They have been exploited commercially in the food industry such as papain for meat tenderizing and ficin and bromelain for brewing [7]. Although recently enzymes from microorganism have been widely commercially produced, consumer still aware for used it as an edible ingredients into their meals. Therefore, natural sources from both plant and animal tissues still desired.
∗ Corresponding author. Tel.: +66 5391 6752; fax: +66 5391 6739. E-mail address: [email protected] (S. Rawdkuen). 1369-703X/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.bej.2010.04.007
Calotropis procera is a plant found in tropical and sub-tropical regions. It is well-known for its great capacity of producing latex which exudates from the green damaged parts. Scientific reports have mentioned various medicinal activities of C. procera latex, such as insecticidal [8], anti-fungal [9] and wound healing [10]. Some biochemical properties of the enzyme containing in the latex of C. procera have been documented and named as procerain [11]. It has been reported that C. procera latex is a potential material for enzyme purification [12,13]. Consequently, scientific researches of protease extraction from C. procera have recently been reported. Protease activity recovery of 74.6% with 4.08-fold of purification was obtained from C. procera latex by using aqueous two-phase system [13]. However, there is no report of using TPP as the single method to separate the protease from this potential source. Therefore, the aim of this study was to optimize the separation process of protease from the latex of C. procera by using TPP technique. 2. Materials and methods 2.1. Chemicals and raw materials Polyethylene glycol (PEG), sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), casein and l-cysteine were obtained from Fluka (Buchs, Switzerland). Beta-mercaptoethanol (-ME) and Coomassie Brilliant Blue G-250 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ammonium sulfate ((NH4 )2 SO4 ),
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magnesium sulfate (MgSO4 ), trichloroacetic acid (TCA), tertbutanol (t-butanol) and other chemicals with analytical grade were obtained from Merck (Darmstadt, Germany). Latex of C. procera was collected from Nayong, Trang Province, Thailand.
2.5. Protein determination
2.2. Latex preparation
2.6. Electrophoresis
Latex was collected in a clean tube by breaking the C. procera stems and then diluted with distilled water (1:1, v/v). After mixing, it was centrifuged at 8000 × g at 4 ◦ C for 10 min. The obtained supernatant was referred to as the “crude extract” [protein content: 3.49 mg/ml, activity: 1138 unit/ml, specific activity: 326 unit/mg protein] and used for further study.
2.6.1. SDS-PAGE SDS-PAGE of the samples was performed according to the method of Laemmli [15] with slight modification. Protein solutions were mixed at a 1:1 (v/v) ratio with the sample buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 20% glycerol). The samples (10 and 2 g protein for protein and activity staining, respectively) were loaded onto the gel made of 4% stacking and 15% separating gels. They were subjected to an electrophoresis set at a constant current of 20 mA/gel. For protein staining, the gel obtained after electrophoresis was stained overnight with a solution of 0.02% (w/v) Coomassie Brilliant Blue R-250 in 50% (v/v) methanol, and 7.5% (v/v) acetic acid. Gels were destained with 50% (v/v) methanol and 7.5% (v/v) acetic acid for 40 min, followed by 5% (v/v) methanol and 7.5% (v/v) acetic acid for 20 min. Pierce blue prestained protein molecular weight marker mix consists of (kDa) myosin (215), phosphorylase B (120), BSA (84), ovalbumin (60), carbonic anhydrase (39.2), trypsin inhibitor (28) and lysozyme (18.3) was used.
2.3. Three-phase partitioning 2.3.1. Effect of crude extract to t-butanol ratio on protease partitioning The TPP was carried out as described by Roy and Gupta [3]. The effect of the ratio of crude extract to t-butanol was studied. Firstly, t-butanol was added to the crude extract at the ratios of crude extract to t-butanol of 1.0:0.5, 1.0:1.0, 1.0:1.5, and 1.0:2.0 (v/v) with a constant (NH4 )2 SO4 saturation of 30%. The mixture was mixed thoroughly and then allowed to stand for 60 min before subjecting to centrifuge at 5000 × g for 10 min to facilitate the separation of phases. The lower aqueous layer and the interfacial precipitate were collected and dialyzed against water overnight at 4 ◦ C. After dialysis, the samples were analyzed for protease activity and total protein content. The ratio employing the highest enzyme recovery was chosen for further study. 2.3.2. Effect of ammonium sulfate saturation on protease partitioning The (NH4 )2 SO4 saturations at 20, 30, 40, and 50% affecting the partitioning of protease was also investigated by using the ratio of crude extract to t-butanol (with the highest enzyme recovery) obtained from 2.3.1. The lower aqueous layer and the interfacial precipitate were collected and dialyzed against water overnight at 4 ◦ C. After dialysis, the samples were analyzed for protease activity and total protein content. The system providing the highest protease recovery was chosen for further study. 2.3.3. Optimization of TPP for proteases recovery The phase obtained from the first TPP that gave the highest activity recovery was chosen as the starting material for optimization in the second TPP. The selected phase (without dialysis) was mixed with the t-butanol in ratio of 1.0:0.5 and (NH4 )2 SO4 was added to the mixture to obtain the final saturations of 50, 55, 60, and 65%. After the complete phase separation, the phases were collected as previously mentioned. The dialyzed phases from the second TPP were subjected to protease activity and total protein content analysis. 2.4. Caseinolytic activity assay An enzyme sample of 0.10 ml was mixed with 1.10 ml of 1% (w/v) casein in 0.10 M Tris–HCl (pH 8.0) containing 12 mM cysteine. The reaction was started by incubation the mixture at 37 ◦ C for 20 min. The reaction was stopped by adding 1.8 ml of 5% TCA. After centrifugation at 3000 × g for 15 min, the absorption of the soluble peptides in supernatant was measured at 280 nm. One of caseinolytic activity units is defined as the amount of enzymes needed to produce an increment of 0.01 absorbance unit per minute at the assayed condition [13].
Protein concentrations were measured by Bradford method using BSA as a protein standard [14].
2.6.2. Protease activity staining The protease separated on the gel was verified by using activity staining as done in Garcia-Carreno et al. [16]. The gel was immersed in 50 ml of 2% (w/v) casein in 50 mM Tris–HCl buffer, pH 8.0 containing 12 mM cysteine for 30 min with constant agitation at 4 ◦ C. The reaction was generated by incubation the gel at 37 ◦ C for 15 min. The treated gel was then stained and destained as described above. The development of a clear band on the dark background indicated the caseinolytic activity of protease from C. procera latex. 3. Results and discussion 3.1. Effect of the ratio of crude extract to t-butanol on protease partitioning The effect of crude extract to t-butanol ratio for protease partitioning in the first TPP was firstly investigated. Different TPP experiments with various extract to t-butanol ratios (1.0:0.5, 1.0:1.0, 1.0:1.5 and 1.0:2.0) at 30% (w/v) ammonium sulfate saturation were performed. The highest protease recovery (99.3%) and purification fold (1.13-fold) were obtained from the bottom phase of a TPP system with the ratio of 1.0:0.5. An increase in t-butanol volume resulted in a decrease in activity recovery and purification fold values of the bottom phase. This may be attributed to the synergistic effects of the increase in concentration of t-butanol and decrease in saturation of ammonium sulfate [5]. Dennison and Lovrien [6] reported that only 0.2–0.5 ml of t-butanol usually is required per milliliter of beginning aqueous sample to precipitate out protein. The best results were obtained when the ratio was 1.0:1.0 used for partitioning of ␣-galactosidase from fermented media of Aspergillus oryzae with 60% (NH4 )2 SO4 saturation [4]. The ratio of sample to t-butanol content is higher than one, the denaturation of the protein is more likely [4]. The partitioning of protease in the interphase was contrary with the bottom phase. However, the yield obtained was still lower than 30% in all ratios tested. As the results, most protease was still remained in the aqueous phase rather than in the interphase. Therefore, the ratio of crude extract to t-butanol of 1.0:0.5 which provided the highest recovery
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was selected for investigating the effect of (NH4 )2 SO4 saturation on partitioning of C. procera protease. 3.2. Effect of ammonium sulfate saturation on protease partitioning The study was performed by maintaining the ratio of crude extract to t-butanol at 1.0:0.5 and varying the (NH4 )2 SO4 saturation in the ranges of 20, 30, 40, and 50%. Generally, one starts with a minimum salt saturation of 20% and optimizes this so as to obtain the maximum amount of the desired protein in the interfacial precipitate [1,2]. It was found that the protease recovery of 182% yield with 0.95 folds of purification was obtained in the interphase of the system contained 50% (NH4 )2 SO4 . Increase of (NH4 )2 SO4 saturation resulted in increase of protease recovery in the interphase. However, the purity of enzyme seemed to be reduced when (NH4 )2 SO4 saturation was raised. Narayan et al. [5] reported that the increasing in concentration of (NH4 )2 SO4 , the degree of purification decreased significantly. As in conventional salting out with (NH4 )2 SO4 , the extent of protein precipitation in TPP is a function of the (NH4 )2 SO4 concentration [17]. The“salting out effect” plays a major role in the system contained high salt concentration. The principle of sulfate ion for salting out protein has been viewed in five different ways namely, ionic strength effects, kosmotropy, cavity surface tension and enhancement osmotic stressor (dehydration), exclusion crowding agent, and binding of sulfate ion to cationic sites of protein [5]. As the result obtained, the system consisted of 50% (NH4 )2 SO4 was seemed to be the optimum condition for protease partitioning due to its highest protease yield at the interphase. However, this fraction gave low enzyme purity. While the highest purity was found in the bottom phase of the system contained 30% (NH4 )2 SO4 . This system was also belonged to the highest yield among all of the bottom phase. Pike and Dennison [18] also reported that 30% (w/v) (NH4 )2 SO4 is the best salt concentration for carrying out TPP efficient. 3.3. Optimization of TPP for recovery of protease from C. procera latex The system condition from the first TPP (1.0:0.5 extract: tbutanol (v/v), 30% (NH4 )2 SO4 ) saturation) providing the highest protease recovery in the aqueous phase was used as the starting
Fig. 1. Optimization of (NH4 )2 SO4 saturation for protease partitioning by the second step three-phase partitioning. The bottom phase of the system of 1.0:0.5 extract: t-butanol in the presence 30% (NH4 )2 SO4 was used as the starting material for the second TPP. Bar [] and line [–] represent the protease recovery and purification fold, respectively. I-phase: interphase, B-phase: bottom phase and P-phase: precipitate.
material for optimization of enzyme partitioning in the second TPP. The reason for carrying out the second cycle of TPP was that sometimes the first TPP cycle could not remove the contaminant proteins efficiently. It was expected that the target protease will be more partitioned to the interphase with the highest yield and purity. Saxena et al. [19] reported that the aqueous phase containing most of the desired protein when subjected to a second cycle of TPP results in considerable purification of the target protein. Therefore, in this study, (NH4 )2 SO4 was added to the aqueous phase of the first TPP fraction to obtain the final saturation of 50, 55, 60, and 65%. Interestingly, it was found that the precipitated phase was also observed as the fourth phase. This can be explained by increase of salt addition provided over salting out effect and protein subsequently precipitated as indicated by the fourth phase occurred. As shown in Fig. 1, increasing of purification fold in the precipitate as well as in the bottom phase was found when the salt content increased. At the salt content of 65%, most protease was recovered in the bottom phase (202.7%) with a little remaining in the interphase (1.1%). However, residue protease was precipitated (35.2%) with the highest purification of 12.79 folds. Kiss et al. [20] concluded that the amount of protein precipitated in the interphase was delineated as a function of the composition of the partitioning system as well as the initial protein concentration.
Fig. 2. Protein pattern (A, 10 g protein) and activity staining (B, 2 g protein) of Calotropis procera latex and their fractions from three-phase partitioning. M: molecular weight marker, 1: crude latex, 2: interphase of 1.0:0.5 t-butanol–50% (NH4 )2 SO4 , 3: interphase of 60% (NH4 )2 SO4 , 4: precipitate of 60% (NH4 )2 SO4 , 5: bottom phase of 1.0:0.5 t-butanol–50% (NH4 )2 SO4 , and 6: bottom phase of 60% (NH4 )2 SO4 .
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Table 1 Recovery of protease from Calotropis procera latex in the interphase (I) of TPP. Sample
Total activity (U)
Total protein (mg)
Crude latex I-1st TPP-50% ASa I-2nd TPP-60% ASb
5,633 ± 107 10,252 ± 244 7,445 ± 505
18.48 ± 1.34 35.40 ± 0.32 3.53 ± 0.05
Specific activity (U/mg) 304.82 ± 20.54 289.58 ± 4.33 2,110.00 ± 110.00
Purification (fold)
Recovery (%)
1.00 ± 0.00 0.95 ± 0.03 6.92 ± 0.34
100 ± 0.0 182 ± 2.1 132 ± 8.9
a
The system consists of the ratio of crude extract to t-butanol of 1.0:0.5–50% (NH4 )2 SO4 . The system consists of the ratio of bottom phase of the first TPP (1.0:0.5 t-butanol, 30%, saturation of (NH4 )2 SO4 ) with the addition of (NH4 )2 SO4 to 60%. I: interphase, AS: (NH4 )2 SO4 . b
Increasing activity recovery was found in the interphase of the system containing (NH4 )2 SO4 less than 60% (Fig. 1). As expected, the highest yield (132%) and high enzyme purity (6.92 folds) was found in the interphase of the system added with 60%(NH4 )2 SO4 . In view of interest, the second TPP system containing the crude extract to t-butanol ratio of 1.0:0.5 with the addition of (NH4 )2 SO4 up to 60% was efficient to partial purified the protease from C. procera. Narayan et al. [5] reported that the optimum condition for extraction peroxidase from the leave of Ipomoea palmate consisted of the crude extract to t-butanol of 1.0:1.0 and 30% (NH4 )2 SO4 . By using this condition resulted in about 160% activity recover and 2-fold of purification in the aqueous phase of the first TPP. Protein patterns of the crude extract and their fractions from the first TPP and second TPP are shown in Fig. 2A. The major protein bands with the molecular weight (MW) of around 39–50 and less than 18.3 kDa were found as the major component in the crude extract (lane 1A). It was observed that the protein pattern of the precipitate fraction (lane 4A) was present as higher band intensity than those of the crude extract and other fractions. For the first TPP containing the crude extract to t-butanol at the ratio of 1.0:0.5 with 50% (NH4 )2 SO4 , a numbers of protein band mostly presented in the bottom phase (lane 5A) than in the interphase (lane 2A). There are previous reports that the maximum proteolytic activity of C. procera protein found a protein band possessing around 30 kDa [20,21]. It was reported that the purified protease from C. procera appeared as a single band at around 29 kDa in a reducing condition of SDS-PAGE [11]. Fig. 2B shows the activity staining (zymography) of the crude extract and the fractions from TPP optimization. Clear zone on the dark background was remarked as the activity of protease against casein substrate on the gel. A protein band with the molecular weight of ∼30 kDa was clearly observed in all fractions. Depending on the intensity of the clear zone, the fraction of the interphase provided interesting protease activity (lanes 2B and 3B) as expected. The results revealed that TPP was able to partition the proteases into the interphase of system in which the salt and t-butanol content was optimized. 3.4. Overall partial purification of protease from C. procera latex by TPP The isolation of protease from C. procera latex (by the first TPP with 50% (w/v) (NH4 )2 SO4 and 1.0:0.5 ratio of the crude extract to t-butanol) gave the highest protease recovery (182%) in the interphase (Table 1). However, for further optimization of enzyme partitioning in the second TPP, the bottom phase of the system containing 30% saturated (NH4 )2 SO4 was used as it provided the highest yield among the bottom phases of all conditions tested. Performing the second TPP with 60% saturated (NH4 )2 SO4 resulted in 132% recovery in the interphase with higher purity (6.92 folds). High protease recovery obtained may probably due to the TPP led to simultaneous activation of the enzyme which results of such an apparently observed value more than 100%. Singh et al. [22] reported that the tertiary structure of serin protease (proteinase K) was altered by TPP, reflecting remarkable enhancement of the enzyme activity. Dennison and Lovrein [6] reported that Bacillus
subtilis protease, Saccharomyces cerevisiae invertase, Candida cylindracea lipase gave a yield of 300, 100 and 900%, respectively, after TPP. These unusual percentage yields were believed to be due to adopting a number of amino acid residues on the enzyme molecule. Sharma and Gupta [1,2] also reported that enzyme activation frequently observed during TPP may be a result of increased flexibility in the enzyme molecule. From the second TPP, the overall result of protease activity recovery, as well as of final purity, is considerably better than the first TPP. Roy and Gupta [3] reported that using TPP for biological activity recovery provided the best results with high recovery activity (90–98%) and high purification fold (65–100-fold). Increasing of purification fold of 18 times with 81% activity recovery was achieved with the second TPP. Saxena et al. [19] also found that by using the second TPP can increase the purification fold of amylase inhibitor and trypsin inhibitor from 8.9 and 8.65-fold to 20.1 and 16-fold purification, respectively. To compare between the first and second TPP in terms of recovery and purity of enzyme from C. procera, the first TPP possessed higher protease yield of 182% than that of the second TPP (132%). In addition, purity of enzyme was increased from 0.95 folds in the first step to 6.92 folds in the second step. This indicates that purification of enzyme was greatly obtained during operation of the second TPP. It can be pointed that only first step was sufficient when consideration of enzyme recovery. However, when regarding to the purity of the enzyme, the second step should be needed. Thus procedures utilizing the two cycles of TPP constitute a fairly simple and efficient isolation process for protease and they also yield a partial purified protein. The availability of this simple partial purification strategy should be employed in production of protease with a more efficient and economic way. 4. Conclusion The TPP was a useful technique for partial purification of protease from C. procera. It is a simple, quick and economical technique and scaling up is convenient. The TPP system consists of the ratio of crude extract to t-butanol of 1.0:0.5 and 50% (w/v) (NH4 )2 SO4 gave the highest yield but rather low purity in the interphase. Increasing the (NH4 )2 SO4 saturation of the bottom phase to the first TPP up to 60% could improve the purity of the enzyme up to 6.92 folds. Acknowledgements The authors would like to express their sincere thanks to Mae Fah Luang University, Thailand and The Thailand Research Funds under The Senior Research Scholar program for the financial support. References [1] A. Sharma, M.N. Gupta, Purification of pectinases by three-phase partitioning, Biotechnol. Lett. 23 (2001) 1625–1627. [2] A. Sharma, M.N. Gupta, Three phase partitioning as a large-scale separation method for purification of a wheat germ bifunctional protease/amylase inhibitor, Process Biochem. 37 (2001) 193–196.
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