Affinity based reverse micellar extraction and purification of bromelain from pineapple (Ananas comosus L. Merryl) waste

Process Biochemistry 46 (2011) 1216–1220

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Affinity based reverse micellar extraction and purification of bromelain from pineapple (Ananas comosus L. Merryl) waste Sunil Kumar, A.B. Hemavathi, H. Umesh Hebbar ∗ Department of Food Engineering, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Cheluvamba Mansion, Mysore 570 020, India

a r t i c l e

i n f o

Article history: Received 12 August 2010 Received in revised form 3 February 2011 Accepted 8 February 2011 Keywords: ARMES Bromelain Concanavalin A Pineapple waste CTAB Purification

a b s t r a c t Affinity based reverse micellar extraction and separation (ARMES) technique is employed for the first time to extract and purify bromelain from pineapple (Ananas comosus L. Merryl) waste. The reverse micellar system of cetyltrimethylammonium bromide (CTAB)/isooctane/butanol/hexanol and sodium bis(2-ethyl1-hexyl) sulfosuccinate (AOT)/isooctane is used for forward and reverse extractions respectively, with concanavalin A (Con A) as an affinity ligand. The effect of various process parameters like concentration of Con A during forward extraction, type of counter ligand and its concentration during back extraction, type of surfactants and its concentration, aqueous phase pH for reverse extraction on the purification and activity recovery of bromelain has been studied in detail. The optimized conditions of extraction resulted in purification of 12.32 fold with an activity recovery of 185.6%, which is higher than that reported for conventional reverse micellar extraction (RME). The forward, back, reverse and overall extraction efficiencies were found to be 49%, 44%, 48% and 14% respectively under optimized conditions. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction The recent advances in genetic engineering and fermentation process that have spawned the biotechnology industry have also stimulated new thinking and research in downstream processing. As new therapeutic products and processes are being developed, isolation and purification of such materials is becoming a challenging task. Due to the inherent complexity of biological systems, separation and purification task with very stringent quality requirements, downstream processing accounts for 60–90% of the total production cost [1], especially for products used in therapeutic and diagnostic applications. Conventional purification techniques such as precipitation, chromatography and electrophoresis pose a considerable problem for scale-up making them uneconomical unless the product is of high value [2]. Reverse micellar extraction can be easily scaled up and can be operated in continuous mode [3,4]. Separation based on physicochemical factors becomes very complex when the components have very similar properties. This demands the application of specific external agent that can identify and distinguish the target biomolecule from others, thus yielding a better resolution which otherwise would have involved a number of complex steps, ending up with low product yield. The phenomenon of biorecognition or bioaffinity is gaining importance in this regard to carry out such challenging separations [5]. Affinity based reverse micellar extraction and separation (ARMES) has the potential to

∗ Corresponding author. Tel.: +91 0821 2513910; fax: +91 0821 2517233. E-mail address: [email protected] (H.U. Hebbar). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.02.008

selectively extract biomolecules using affinity ligands. This technique involves the affinity interaction between protein and ligand coupled with incorporation and stripping of these complexes into and out of the reverse micelles (RMs), respectively [6]. Three steps are commonly involved in any affinity based separation process, namely, (i) formation of a reversible ligand–ligate complex, (ii) selective extraction of the complex into RMs and (iii) dissociation of the complex, resulting in the isolation of pure ligate. The affinity ligand thus recovered could be further used in subsequent extractions [7]. Woll et al. [8] demonstrated the practical application of reverse micellar assisted affinity extraction for the first time wherein selective extraction of Con A into AOT-isooctane RMs was achieved using n-octyl-␤-d-glucopyranoside as an affinity co-surfactant. This technique has been shown to work with the model systems [9] and with protein mixtures [10]. The salient features of ARMES are: (1) the affinity interaction is intraphasic, so the mass transfer limitation of ligand–ligate interaction is absent. Hence, the ligand utilization is very high resulting in high productivity, (2) no chemical modification of the ligand is needed, (3) ease of operation and inherent scalability of the process compared to other affinity based separations such as affinity chromatography/affinity electrophoresis/affinity precipitation and (4) dual selectivity (in binding and extraction stages) due to combination of biospecific interaction between ligand and ligate along with the driving force of RME [10]. All the studies reported so far involve the application of ARMES technique for model systems using anionic surfactant (AOT). Bromelain is a type of proteolytic enzyme present in the tissues of plant family Bromeliaceae, of which pineapple is the best

S. Kumar et al. / Process Biochemistry 46 (2011) 1216–1220

known source. Bromelain has wide range of applications such as cleansing agent, as meat tenderizer, as a digestive aid, as an anti-inflammatory agent, as a fibrinolytic agent, as an antibiotic potentiating agent, wound debridement agent, etc [11–14]. Pineapple processing industries generate nearly 35% of wastes, which are either disposed off or used in composting [15]. Bromelain is reported to be present in pineapple wastes such as core, peel and leaves [16] and hence, there is a scope for utilizing the wastes generated in the processing industry for value addition and to decrease the load on environment (faster degradation due to size reduction). Hebbar et al. [17] reported the extraction of bromelain using conventional reverse micellar systems from pineapple wastes (core, peel, crown and extended stem). In the present study bromelain is extracted and purified from pineapple core, main waste generated in pineapple processing (juice, canned slices) industry employing ARMES technique that is considered to be more selective and efficient than conventional RME method. Application of this technique for a real system using cationic surfactant CTAB has been reported for the first time. Effect of various process parameters on the extraction efficiencies at different stages of ARMES and purification of bromelain are studied in detail. 2. Materials and methods 2.1. Materials 2.1.1. Enzyme source Fresh pineapple fruits (Ananas comosus L. Merryl, cv. Kew, 7–9◦ Brix) used in the study was purchased from the local super market.

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unit of bromelain activity is defined as 1 ␮g of tyrosine released in 1 min per mL of sample when casein is hydrolyzed under the standard conditions of 37 ◦ C and pH 7.0 for 10 min. 2.2.4. Protein content Protein content was determined by measuring absorbance at 280 nm using bovine serum albumin (BSA) as standard. Protein concentration readings were taken in triplicate and an average value is used for the calculation of forward, back and reverse extraction efficiencies. The activity recovery (%), purification (fold) and extraction efficiencies (%) at different stages of ARMES were estimated using the formulae given below: activity recovery (%) = purification (fold) =

activity of bromelain after reverse extraction × 100 activity of bromelain in feed

specific activity of bromelain after reverse extraction specific activity of bromelain in feed

(1) (2)

forward extraction efficiency (%) =

protein concentration in organic phase after forward extraction × 100 protein concentration in feed

(3)

back extraction efficiency (%) =

protein concentration in back extracted aqueous phase × 100 protein concentration in organic phase after forward extraction

(4)

reverse extraction efficiency (%) =

protein concentration in reverse extracted aqueous phase × 100 protein concentration in back extracted aqueous phase

(5)

overall extraction efficiency (%) protein concentration in reverse extracted aqueous phase × 100 protein concentration in feed

2.1.2. Chemicals CTAB and AOT were obtained from Merck, Germany and BDH Laboratory Supplies, UK, respectively. Isooctane (HPLC grade) was purchased from Merck, Mumbai, India. Hexanol from SRL, Mumbai, casein (Hammerstein grade), hexanol and nbutanol from Loba chemicals, India were used. Concanavalin A from Canavalia ensiformis (Jack bean) Type IV and methyl-␣-d-mannopyranoside (MMP) were obtained from Sigma, USA. All other chemicals of AR grade were used for the experiments and analyses.

=

2.2. Methods

3. Results and discussion

2.2.1. Preparation of enzyme extract The core portion of pineapple fruit was manually separated and a known quantity was ground in a blender along with extraction buffer (0.01 M sodium phosphate buffer of pH 6.5 and containing 1% polyvinylpyrrolidone) at 1:1 ratio and then filtered (using cheese cloth). The filtrate was centrifuged at 10,000 × g for 15 min (Eltek TC 4100 D research centrifuge, India). The supernatant obtained was used for further experiments.

The selective binding of bromelain to affinity ligand Con A coupled with reverse micellar extraction of the affinity complex provides a facile biocompatible separation method. Bromelain is a glycoprotein containing a single oligosaccharide chain attached to the polypeptide, this property allows its affinity binding to Con A [20]. The affinity ligand (Con A) binds to ligate (bromelain) to form the affinity complex in the aqueous phase. The ligate–ligand complex is then extracted into the reverse micellar organic phase (RMOP) via a mechanism that is dependent upon several parameters. Various process parameters such as concentration of Con A being used, type and concentration of counter ligand, type and concentration of surfactant, pH of the aqueous phase used have been studied in order to optimize the bromelain extraction conditions. Optimised forward (feed pH 8.0, 0.1 M NaCl, 150 mM CTAB) and back extraction conditions of RME (0.5 M KBr, pH 4.2 acetate buffer) reported by Hebbar et al. [17] are used in the present study. All experiments were carried out in duplicate and independent assays were done in triplicate for each experimental condition.

2.2.2. Affinity based reverse micellar extraction and purification Forward extraction was carried out by mixing 10 mL of organic phase (150 mM CTAB/80% (v/v) isooctane/5% (v/v) hexanol/15% (v/v) butanol) with 10 mL of aqueous phase (enzyme extract/salt/concanavalin A) for 1 h on magnetic stirrer, wherein bromelain-Con A complex will be extracted into reverse micelles. Back extraction was carried out by mixing 10 mL of reverse micellar phase with 10 mL of fresh stripping solution (buffer/salt/counter ligand). In this stage bromelain-Con A complex was recovered from reverse micelles into the aqueous phase, while Con A would be detached from bromelain due to the presence of counter ligand. For the reverse extraction, pH of the aqueous phase (obtained from back extraction) was adjusted and mixed with fresh organic phase (AOT/isooctane) by magnetic stirring for 30 min. This enabled the retention of bromelain in aqueous phase and transfer of Con A into AOT reverse micellar phase. In each of the above steps, the phases were mixed thoroughly and then centrifuged at 4000 × g for 15 min (Eltek TC 4100 D research centrifuge, India) for the separation of phases. The aqueous phases after forward, back and reverse extraction were collected and aliquots of these were analyzed for enzyme activity and protein content. All the phase mixing experiments were carried out at 25 ± 2 ◦ C. 2.2.3. Enzyme assay Bromelain activity in aqueous phase was determined according to the casein digestion unit (CDU) method using Hammerstein grade casein (0.6%) as substrate in the presence of cysteine and EDTA [18]. The assays were based on proteolytic hydrolysis of the casein substrate. The absorbance of the clear filtrate (solubilized casein) was measured at 275 nm using spectrophotometer (Shimadzu UV-160, Japan). One

(6)

2.2.5. SDS–PAGE electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) with 12% gel was performed using electrophoresis unit (Genei, Bangalore, India) as per the procedure described by Laemmli [19].

3.1. Concentration of concanavalin A The efficiency of bromelain extraction was found to depend on the concentration of Con A used. It was observed that with increase in concentration of Con A up to 0.7 mg/mL, both activity recovery and purification increased. Fig. 1 shows the variations in forward extraction efficiency, activity recovery and purification with respect to variation in concentration (0–1 mg/mL) of Con

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12

10 15

120

8

6 80

4

purification (fold)

back extraction efficiency (%)

150

10

100

5

50

40 2

0

0 control

0

0 0

0.25

0.48

0.7

0.85

concentration of con A (mg/ml)

3.2. Selection of counter ligand The effect of addition of different types of counter ligand during back extraction of bromelain-Con A complex was studied. Con A has affinity for many monosaccharides [21] and presence of this can act as competitive inhibitor for bromelain, as they compete with bromelain at binding sites. The presence of counter ligand in the back extraction aqueous phase is expected to disrupt the bromelain-Con A binding due to competitive inhibition. Various types of monosaccharides such as glucose, fructose, galactose, dextrose and methyl-␣-d-mannopyranoside (MMP) were used to breakdown the complex. Concentration of 150 mM was maintained for each counter ligand. Out of all the sugars tested, methyl-␣-d-mannopyranoside resulted in maximum purification of 9.02 fold with an activity recovery of 147.2%. The inhibitory power of sugars observed was in the order of methyl␣-d-mannopyranoside > glucose > fructose > galactose > dextrose. This trend was similar to that observed in conventional Con A binding studies involving agglutination of erythrocytes reported by Paradkar and Dordick [9]. The reason for this trend could be the difference in affinity scale and structure of these counter ligands [22], which would have led to changes in configuration of enzyme and/or enzyme-Con A complex when added to the aqueous phase of back extraction. Fig. 2(A) shows the variation in purification,

12

purification (fold)

A. Maximum purification of 6.82 fold and an activity recovery of 132.3% was achieved at 0.7 mg/mL concentration. Above this concentration, forward extraction efficiency, purification as well as activity recovery decreased. At higher concentrations (above the maximum binding capacity), the unbound (excess) Con A may also get transfer/extracted resulting in lower purification of bromelain. Above 0.7 mg/mL a Con A precipitate was observed at the interface, this may be due to self aggregation of excess Con A at the interphase. A similar observation was reported at high concentrations of Con A during the extraction of glycoproteins [11]. At feed pH 8.0, bromelain is negatively charged while Con A is positively charged, so the total charge of ligand–ligate complex should be negative to extract it into cationic CTAB reverse micelles. It was suspected that, at higher concentrations, the negative charge on bromelain was shielded by Con A, which reduced the driving force for forward extraction. Paradkar and Dordick [9] used high concentration of Con A to shield negative charges on HRP (extraction pH being above the isoelectric point of HRP (horseradish peroxidase) and below the isoelectric point of Con A), in order to extract Con A-HRP complex into negatively charged AOT/isooctane reverse micelles.

fructose galactose

MMP

dextrose

14

1

Fig. 1. Effect of concanavalin A concentration on forward extraction efficiency, activity recovery and purification.

glucose

250 purification (fold) activity recovery (%) back extraction efficiency (%)

B 200

10 150

8 6

100

4

activity recovery/back extraction efficiency (%)

activity recovery/forward extraction efficiency (%)

forward extraction efficiency (%)

activity recovery/back extraction efficiency (%)

purification (fold)

activity recovery (%)

purification (fold)

160

200

A

activity recovery (%)

purification (fold)

50 2

0

0 50

100

200

300

MMP concentration (mM) Fig. 2. (A) Effect of type of counter ligand and (B) effect of MMP concentration on back extraction efficiency, activity recovery and purification.

activity recovery and back extraction efficiency with respect to various counter ligands tested. 3.3. Concentration of MMP As MMP gave better results compared to other counter ligands, its concentration was optimised in order to obtain higher efficiency. Initially as the concentration of MMP increased (50 and 100 mM), recovery and purification of bromelain also increased, which is due to the release of enzyme from the complex and effective binding of Con A with MMP. However, as the amount of MMP concentration exceeded the optimum level (100 mM), activity recovery and purification fold decreased may be due to blockage of active sites of bromelain. Back extraction efficiency increased up to 200 mM of MMP concentration and then decreases as shown in Fig. 2(B). However, the maximum purification of 10.23 fold with an activity recovery of 158.5% has been obtained at 100 mM MMP concentration. 3.4. Selection of surfactant Two surfactants (AOT and CTAB) system were studied for the reverse extraction of Con A. The overall extraction efficiency and purification were low with CTAB system. When aqueous phase pH is below 8.0, Con A will be positively charged (isoelectric point of Con A is 8.3–8.5) and hence there will be repulsion between Con A and CTAB, resulting in lower recovery of Con A. Also below pH 8.0, bromelain has the tendency of getting extracted due to its negative charge, which is evident from the decrease in activity recovery of bromelain (Table 1). Since, bromelain is not stable above pH 8.5 (where Con A is negatively charged), CTAB could not be used in this

S. Kumar et al. / Process Biochemistry 46 (2011) 1216–1220

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Table 1 Effect of surfactant type on activity recovery, purification and extraction efficiencies. Type of surfactant

Activity recovery (%)

Purification (fold)

Reverse extraction efficiency (%)

Overall extraction efficiency (%)

9.38 ± 0.6 2.31 ± 0.4

46.42 ± 1.1 60.41 ± 1.6

15.06 ± 0.86 12.46 ± 1.52

156.36 ± 2.8 34.08 ± 1.3

AOT CTAB

range. Studies with anionic surfactant AOT showed much better performance with an activity recovery of 156.36% and purification of 9.38 fold. Hence AOT is selected for further studies. 3.5. Concentration of AOT As AOT was found suitable for reverse extraction of Con A, its concentration to be used was optimised. Surfactant concentration plays a vital role in reverse micellar extraction and it was reported that extraction increases with an increase in surfactant concentration. On the other hand, an excess of surfactant molecules will lower degree of extraction due to the increased interaction and collapse of reverse micellar structure [23]. As the concentration of AOT increased, number of reverse micelles available for the uptake of Con A from the aqueous phase increases and hence higher purification is achieved. At AOT concentration higher than 150 mM, purification as well as activity recovery decreased as phase separation was hindered by the increased interaction and collapse of reverse micellar structures. Fig. 3(A) shows the variations in activity recovery, reverse extraction efficiency and purification with respect to variation in AOT concentration (50–200 mM). Maximum purification of 11.92 fold with an activity recovery of 183.33% has been achieved at 150 mM AOT concentration. 3.6. Aqueous phase pH The aqueous phase (obtained after back extraction) pH needs to be optimized for efficient reverse extraction of Con A into the AOT 14

200

purification (fold)

reverse extraction efficiency (%)

10 160 8 120

6 80 4

activity recovery/reverse extraction efficiency (%)

12

activity recovery (%)

3.7. SDS–PAGE analysis The purity of the bromelain obtained from ARMES was confirmed using SDS–PAGE and the profile is shown in Fig. 4. The aqueous phase containing bromelain obtained from reverse

240

A

purification (fold)

micellar phase. The reverse extraction of Con A helps in recovery of ligand as well as purification of ligate. The aqueous phase pH should be such that AOT selectively extracts Con A, and at the same time retains the native activity of the Con A being extracted. The aqueous phase pH was varied between 5.8 and 9.0 in order to obtain higher efficiency. It was observed in Fig. 3(B) that the maximum purification of 12.32 fold with an activity recovery of 185.6% was achieved when aqueous phase pH was adjusted to 8.0. At this pH, Con A is positively charged and is selectively reverse extracted into anionic AOT reverse micelles due to electrostatic interaction. Bromelain being negatively charged under this condition will remain in the aqueous phase. The pH of aqueous phase plays a major role in controlling electrostatic interaction between enzyme and surfactant [24–27]. The activity recovery of bromelain is higher than 100%, which may be due to the removal of enzyme activity inhibitors during purification. Many researchers have reported [28–30] enzyme activity recovery of more than 100% during liquid–liquid extractions. The purification of bromelain obtained in this study employing ARMES technique (12.32 fold) is higher than in previous reports using conventional RME (5.2–4.54 fold) [17,31], or other purification methods involving aqueous two phase extraction (4–1.2 fold) [32,33], membrane processing (1.13 fold) [34] and ion exchange chromatography (10–2.6 fold) [35,36]. However, the aqueous phase of ARMES could be subjected to further processing in order to improve the purity and to remove the residual chemicals, if any.

40

2

0

0 50

100

150

200

16

240

B

purification (fold)

purification (fold)

14

12

activity recovery

200

reverse extraction efficiency (%)

160

10 8

120

6

80

4 40

2 0

activity recovery/ reverse extraction efficiency (%)

AOT concentration (mM)

0 5.8

6.5

7

8

9

aqueous phase pH Fig. 3. (A) Effect of AOT concentration and (B) effect of aqueous phase pH on reverse extraction efficiency, activity recovery and purification.

Fig. 4. SDS–PAGE pattern of purified enzyme. Lane 1: marker; lane 2: ARMES sample; lane 3: standard bromelain from Sigma.

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extraction was dialysed overnight at 4 ◦ C and lyophilized. The concentrated sample was loaded to 12% gel along with the marker. The purified sample gave a single band for bromelain indicating reasonably good purification of the enzyme. The SDS lane patterns matched well with the sigma standard. The band obtained was found to be around 25 kDa, which lies in between the reported range of 24 and 28 kDa for bromelain [12,35,37]. 4. Conclusions Affinity based reverse micellar extraction could be successfully applied for the selective extraction and purification of bromelain from pineapple waste. ARMES technique found to be more selective and efficient compared to conventional RME technique. The processing conditions greatly affected the extraction efficiency and emphasized the need for optimizing the processing conditions for improved extraction. The outcome of the present work demonstrated that ARMES could be extended to complex mixtures of biomolecules/real system. The optimized conditions for extraction resulted in purification of 12.32 fold with an activity recovery of 185.6%. The forward, back, reverse and overall extraction efficiencies of 49%, 44%, 48% and 14%, respectively were obtained. Acknowledgements The authors thank Dr. V. Prakash, Director, CFTRI, for the encouragement and keen interest in the area of downstream processing. Authors wish to thank Dr. KSMS Raghavarao, Head, Food Engineering for the support and guidance. References [1] Lightfoot EN. Protein purification from molecular mechanisms to large scale processes. In: Ladisch MR, Willson RC, Painton CC, Builder SE, editors. Separation in biotechnology—the key role of adsorptive separations, vol. 427. ACS Publications; 1990. p. 35–51. [2] Nandini KE, Rastogi NK. Reverse micellar extraction for downstream processing of lipase: effect of various parameters on extraction. Process Biochem 2009;44:1172–8. [3] Chen YL, Su CK, Chiang BH. Optimization of reversed micellar extraction of chitosanases produced by Bacillus cereus. Process Biochem 2006;41:752–8. [4] Hebbar UH, Raghavarao KSMS. Extraction of bovine serum albumin using nanoparticulate reverse micelles. Process Biochem 2007;42:1602–8. [5] Pommerening K, Mohr P. Affinity chromatography: practical and theoretical aspects. New York: Marcel Dekker Inc; 1985. [6] Harikrishna S, Srinivas ND, Raghavarao KSMS, Karanth NG. Reverse micellar extraction for downstream processing of proteins/enzymes. Adv Biochem Eng/Biotechnol 2002;75:119–83. [7] Senstad C, Mattiasson B. Precipitation of soluble affinity complexes by a second affinity interaction: a model study. Biotechnol Appl Biochem 1989;11:41–8. [8] Woll JM, Hatton TA, Yarmush ML. Bioaffinity separations using reversed micellar extraction. Biotechnol Prog 1989;5:57–62. [9] Paradkar VM, Dordick JS. Purification of glycoproteins by selective transport using concanavalin mediated reverse micellar extraction. Biotechnol Prog 1991;7:330–4. [10] Paradkar VM, Dordick JS. Affinity based reverse micellar extraction and separation (ARMES): a facile technique for the purification of peroxidase from soybean hulls. Biotechnol Prog 1993;9:199–203.

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