- Email: [email protected]
Purification and drying of bromelain
Separation and Purification Technology 64 (2009) 259–264
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Purification and drying of bromelain R.V. Devakate, V.V. Patil, S.S. Waje ∗ , B.N. Thorat Department of Chemical Engineering, Institute of Chemical Technology, N. M. Parekh Road, Matunga (E), Mumbai 400019, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 18 June 2008 Received in revised form 6 September 2008 Accepted 28 September 2008 Keywords: Chromatography Drying Bromelain Purification Freeze drying
a b s t r a c t The present work deals with the purification and drying of bromelain, one of the vegetal proteases. The purification of the enzyme from fruit Ananas comosus was carried out by precipitation and by chromatography. Further, the drying of chromatographically purified fraction was carried out in freeze dryer and spray dryer. The activity was expressed in terms of casein digesting units (CDU). The bromelain extract of higher purity was obtained from the fruit using ion exchange chromatography. The elution efficiency in chromatographic purification was obtained in the range of 80–90%. The fold purity obtained by chromatography was 3.3 times as compared to that obtained by precipitation. Bromelain thus obtained was subjected to spray and freeze drying. It was observed that the yield obtained in spray dryer at an outlet temperature of 40–50 ◦ C was almost 50–70% and in case of freeze dryer the yield obtained was 96%. The resulting residual activity and the specific activity was compared between spray-dried and freeze-dried powder. The purity of enzyme obtained in the present study was found to be 2.8 times more than that of a commercial sample. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Bromelain (EC 3.4.22.4) is a collective name for proteolytic enzymes or proteases found in tissues including stem, fruit and leaves of the pineapple plant family Bromeliaceae [1]. The isolation of enzymes from pineapple fruit and its study have been investigated since 1894. The enzymes occurring in the stem and the fruit of Ananas comosus (Smouth cayenne) are the most studied. Unlike crude stem bromelain, which is used widely in industry, fruit bromelain is not commercially available despite the large quantities of waste pineapple fruit portions at pineapple canneries [2]. The continued interest in bromelain, for its numerous applications in the food industry as well as in medicine and pharmacology make this enzyme one of the best vegetal proteases. The potential therapeutic value of bromelain is due to its biochemical and pharmacological properties and hence, it is desired to obtain bromelain in its highest purified form. The preparation in pure form of a proteolytic enzyme has always proved difficult, and the bromelains appear to be no exception [3]. The crude commercial bromelain
Abbreviations: P, precipitate; D, dialysate; S, supernatant; CDU, casein digesting unit; db, dry basis; MWCO, molecular weight cut-off. ∗ Corresponding author. Tel.: +91 22 2414 5616x2022/2023; fax: +91 22 2414 5614. E-mail address: [email protected] (S.S. Waje). 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.09.012
used in the manufacture of pharmaceuticals is not chemically homogeneous. The main ingredient in crude bromelain is a proteolytic enzyme named glycoprotein, besides substances such as insoluble materials, e.g. colored pigments, organic acids, minerals, protease inhibitors, organic solvents and excipient used for enzyme recovery have been reported [4]. The present study examines the extract of fresh pineapple fruit for the presence of cysteine proteinases, besides protein degradation kinetics. 2. Experimental 2.1. Materials A ripe pineapple fruits were purchased from a local supermarket. l-Cysteine and casein (Hammarsten grade) were obtained from Sisco Research Laboratory (SRL, Mumbai, India). Trichloroacetic acid (TCA) was purchased from S.D. Fine Chemicals Ltd. (Mumbai, India). Bovine serum albumin (BSA) and tyrosine were purchased from Hi-Media Laboratory (India). All other chemicals used were of analytical grade and purchased from S.D. Fine Chemicals Ltd. (Mumbai, India). A purified commercial bromelain sample was obtained from Hong Mao Biochemical Co. Ltd. (Thailand) as a gift sample. Dialysis Membrane having molecular-weight-cut-off (MWCO) of 12,000 Da was also purchased from Hi-Media Laboratory (India). Ion exchange resins were made available from Resindion Sisco Research Laboratory (Italy) as a free sample.
260
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
2.2. Clarification of fruit crude juice The stalk (central core) of ripe pineapple fruit was separated from fleshy fruit. The fruit portion was then cut into small pieces and crushed in a laboratory juicer to get approximately 500 ml juice. The juice was filtered through a muslin cloth to remove the fibrous material. The filtrate was then subjected to vacuum filtration to remove ruptured plant cells. The filtered crude juice was kept for centrifugation (Remi R-24) at 10,000 × g for 20 min at 4 ◦ C to remove any insoluble materials. The clear supernatant obtained as a clarified extract was stored in aliquots at 4 ◦ C and used whenever required. The protein content in the supernatant was determined before further purification using precipitation and ion exchange chromatography. 2.3. Purification of bromelain from clarified juice Clarified pineapple juice was subjected to precipitation and ion exchange chromatography for the separation of bromelain enzyme. These methods are described briefly as follows.
Fig. 1. Schematic of spray dryer.
2.4. Dialysis 2.3.1. Precipitation The precipitation of bromelain from clarified pineapple fruit juice (20 ml) was carried out by slow addition of ammonium sulfate [(NH4 )2 SO4 ] at 4 ◦ C under constant stirring. Initially, 2.27 g (NH4 )2 SO4 was added to get 0–20% saturation. The stirring was continued for 30 min after the complete addition of salt to allow equilibration between the dissolved and aggregated proteins. The salt-enriched solution was centrifuged (Remi R-24 Research Centrifuge) at 10,000 × g for 15 min and the precipitate was collected. The supernatant was separated, its volume was noted and the amount of salt required for the next cut was calculated. The amount of (NH4 )2 SO4 added to the solution in order to increase % saturation level from S1 to S2 was calculated using following equation [5]: Amount of (NH4 )2 SO4 required (g) 533(S2 − S1 ) = × volume of solution (lit.) 100 − 0.3S2
(1)
where S1 initial saturation of solution (%) and S2 required saturation of solution (%). The equation is related by the assumption that 100% saturation corresponds to 4.05 M salt solution. Ammonium sulfate precipitate fractions and supernatants were collected at the levels of 20% saturation and assayed for protein content and specific protease activity after dialysis. 2.3.2. Ion exchange chromatography Chromatographic purification of clarified crude juice was carried out on a preparative scale. The glass column of 25 mm internal diameter and 300 mm length was packed with cation exchange resins and equilibrated with four column volumes of acetate buffer (25 mM, pH 4.0). Sufficient quantity of clarified crude juice was passed in upward direction through the packed bed of adsorbent and was kept constant at 324 ml h−1 . This was followed by a wash of acetate buffer until no proteins were detected at 280 nm using an online spectrophotometer (Model U-1100, Hitachi, India). Finally, the adsorbed enzyme (protein) was eluted using 1N sodium chloride solution at the flow rate of 324 ml h−1 . The eluting protein was determined at 280 nm on online spectrophotometer. The enzyme activity and protein content for unbound fraction, washings and elution fraction were measured to calculate the total recovery of enzyme units with respect to the bound one. The procedures for estimation of protein content and enzyme activity are mentioned below.
The salt in the purified bromelain samples as a result of above purification methods was removed by dialysis using ammonium phosphate buffer (50 mM, pH 7.0) as a process for buffer exchange. Dialysis membrane (Hi-Media Laboratory, Mumbai, India) having molecular weight cut-off of 12,000 Da was used for dialysis. The enzyme activity units and protein content after dialysis were checked for the percentage loss in the process. 2.5. Drying of purified bromelain Chromatographically purified bromelain was dried using laboratory spray dryer and vacuum freeze dryer. 2.5.1. Spray drying A laboratory bench-top spray dryer (Model: LU-22, Labultima, Mumbai, India) having 1 l/h water evaporation capacity was used for experimental purpose. Fig. 1 shows the schematic of the spray dryer. Peristaltic pump was used to feed bromelain solution to a two-fluid nozzle atomizer (inside diameter: 0.7 mm), with compressed air from a compressor (Fouji, India). The drying chamber (height: 400 mm and diameter: 100 mm) and cyclones (2 nos.) were made of thick transparent glass. Inlet drying air from HEPA filter after passing through an electric heater flows co-currently with the spray through the main chamber. The dried powder was collected in the receivers from the bottom of the cyclones. The remaining ultra-fine powder was recovered in the filter bag. The parameters such as feed flow rate, drying air temperature and compressed air pressure for atomization were set and controlled through a personal computer. In all experiments, the atomizer pressure and feed flowrate were kept constant at 2 kg cm−2 and 225 ml h−1 , respectively. Inlet air temperature was varied in the range from 100 to 170 ◦ C. Outlet air temperatures were recorded online continuously. The drying air flow rate and the feed temperature were kept at 41 m3 h−1 and 27 ◦ C, respectively. The duration of each experimental run was 90 min. 2.5.2. Vacuum freeze drying A laboratory scale vacuum freeze dryer (M/s. Ref-vac Consultancy, India) was used for this study. It consists of cylindrical stainless steel drying chamber (L = 0.4 m, D = 0.4 m) including heating plates (3 nos., total heating surface area = 0.25 m2 ), a condenser (maximum ice collection capacity = 1.8 kg, surface area = 0.25 m2 )
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
capable of operating at −35 ◦ C and a vacuum pump (M/s. Crompton Greaves, India; air displacement rate = 16 m3 h−1 ). The condenser was connected to the drying chamber by a pipe. An electronic weighing balance (M/s. Tapsons Weighing Systems, India, least count = 0.1 g) was placed inside the drying chamber for online sample weight measurement which could be observed through the transparent window. Several arrangements were made to allow comprehensive control, measurement and recording of pressure and temperature inside the drying chamber. The pressure inside the drying chamber was measured with pirani manometers and regulated with solenoid valve. The temperature of heating plates and sample were measured with PT 100 thermocouples (3 nos.) and controlled using an integrated electrical resistance heater. A purified bromelain sample (20 ml) filled in petri-dish was frozen in an external deep freezer (Blue Star, India) at −23 ◦ C for 6 h. Meanwhile, freeze dryer was started and the temperature of the condenser was allowed to reach at its minimum level of −35 ◦ C. Thereafter, the frozen bromelain sample was placed on weighing balance in the drying chamber and subsequently started the vacuum pump and the heater. The temperature of the sample and pressure inside the chamber were set at the predetermined values of 25 ◦ C and 30 Pa, respectively. The weight of the sample was recorded every 5 min time interval, till it attained a constant value. 2.6. Measurement of enzyme activity The enzyme activity assay was based on estimation of amount of small molecular weight digestion products (Trichloroacetic acid (TCA) soluble material) formed from proteins due to proteolytic action of the enzyme [6]. Proteolytic activity of bromelain was measured by the method described by Dapeau [7]. The assay consisted of 5 ml solution of 0.75% casein prepared in anhydrous disodium phosphate buffer (50 mM, pH 7). The pH was adjusted using slow addition of 0.1N HCl. The solution was brought to 37 ◦ C by preincubation for 10 min. To this substrate, a known volume of enzyme was added after diluting it to 1 ml with activating buffer. The 30 mM cysteine hydrochloride monohydrate in 6 mM disodium EDTA was used as an activating buffer. Casein proteolysis was stopped after 10 min by addition of 5 ml of 30% (w/v) TCA and was allowed to stand for 30 min at 37 ◦ C. Thereafter, the solution was cooled to room temperature and filtered twice using Whatman filter paper (No. 42). Absorbance of the filtrate was measured at 280 nm using UV–vis spectrophotometer (Chemito, UV 2500, Mumbai, India) against the tyrosine standard plot of absorbance versus tyrosine concentration (g ml−1 ). One unit of bromelain was taken as the amount of enzyme which while acting on the casein substrate under specified conditions, produces one microgram of tyrosine per minute. 2.7. Measurement of protein content Protein content was measured spectrophotometrically by using the Bicinchoninic method of protein estimation [8]. Sigma Bicinchoninic acid protein assay kit was used. 0.2 ml Bicinchoninic acid was added to 0.025 ml of suitably diluted protein and the mixture was vortexed for about 30 s. The color developed was measured at 565 nm using microplate reader (Model 680, Bio-Rad California) after 2 h. Bovine serum albumin was used as the standard for protein assay. A stock solution of 0.1 mg ml−1 BSA was prepared in distilled water. Solutions of different concentrations of BSA were prepared by diluting the stock solution with distilled water. The absorbance of these dilutions was measured at 565 nm and a plot of absorbance versus BSA protein concentrations (g ml−1 ) was used as a standard.
261
2.8. Differential scanning calorimetry (DSC) Thermal analysis of freeze-dried powder was performed using differential scanning calorimeter (DSC) (Perkin Elmer Pyris-6, Germany). Before analysis, the dried samples were transferred to vacuum desiccator and equilibrated for 3 days over P2 O5 for ‘zero’ moisture content. Approximately 5 mg powder was used for analysis. The sample was sealed in an aluminium pan and an empty pan was used as a reference. Heating rate of 10 ◦ C min−1 was used. The glass transition temperature was reported as the midpoint temperature of heat capacity step. 2.9. Fourier transform infrared (FTIR) spectroscopy Fourier transform infrared (FTIR) spectra were measured using a Perkin Elmer FTIR spectrometer (Series 1600) and analyzed using PE-GRAMS/32 1600 software, as described by Liao et al. [9]. Briefly, a dry protein sample (approximately 0.5 mg) was mixed with 300 mg ground potassium bromide and compressed to get a pellet. The spectra were smoothed with a nine-point Savitsky–Golay function to remove any possible white-noise. The baseline of the spectrum in the amide I region was leveled and zeroed, then the spectrum of the sample was normalized for area in the region and the intensity of ␣-helical band was recorded. 3. Results and discussion 3.1. Purification using precipitation From Table 1, it is clear that the fractions collected at 40–60% and 60–80% (w/v) ammonium sulfate saturation were found to have the higher specific protease activity and protein content as compared to other saturation levels. The enzyme recovery and protein content were found to be about 80% and 34% of total enzyme and protein contents, respectively. Further increase in the percentage saturation level from 80% to 100% causes the denaturation of the proteins and enzymes which also tends to reduce the specific activity. In the fractions obtained using lower (NH4 )2 SO4 saturation (<40% saturation), the specific enzyme activity was found to be very less (<79 Units per mg protein). This means that proteases are soluble in a solution having (NH4 )2 SO4 saturation less than 40%. Proteins other than proteases which are not soluble in the supernatant were precipitated when (NH4 )2 SO4 saturation is less than 40%. To get the optimum range of (NH4 )2 SO4 saturation, the 40–80% (w/v) saturation interval was further divided into smaller fractions (Table 2) and it was observed that 40–70% (w/v) (NH4 )2 SO4 fraction contained 68% of the protease activity and approximately 3-fold increase in specific activity compared to the crude fruit juice. To more precisely obtain, the precipitation kinetics in the saturation range of 40–60%, the saturation range was increased by 2%, and for every 2% increase in saturation, the precipitate and supernatant were collected and analyzed for bromelain activity. A second preliminary experiment shows that an increase in solid-phase activity could be correlated with a fall in the supernatant activity. The kinetics of bromelain precipitation in the saturation range of 40–60% is as shown in Fig. 2. 3.2. Purification using ion exchange chromatography The bromelain was found as much as 10-fold pure with 84.5% enzyme recovery using cation exchange chromatography technique (Table 3). No bromelain activity was detected in the washing flow. The elution efficiency of 97.6% was obtained. The cation exchange chromatography of bromelain resulted in one prominent peak at
262
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
Table 1 Effect of percent saturation on bromelain and total protein recovery on precipitation. Saturation level
Units (CDU)
Protein content (mg)
Specific activity (Units per mg protein)
Fold purity
% yield
Crude 0–20% (D) 20–40% (D) 40–60% (D) 60–80% (D) 80–100% (D) 100%–+(D)
160721 7393.2 27804.7 66056.3 61556.1 6589.6 5786.0
2733.6 476.2 350.9 401.0 451.1 401.0 601.5
58.8 15.5 79.2 164.7 136.5 16.4 9.6
1.0 0.26 1.35 2.81 2.33 0.28 0.16
100 4.6 17.3 41.1 38.3 4.1 3.6
Table 2 Effect of percent saturation on bromelain and total protein recovery (precipitation range of 40–80%). Saturation level
Units (CDU)
Protein content (mg)
Specific activity (Units per mg protein)
Fold purity
% yield
20–40% (S) 40–70% (P) 70–80% (P)
124458.6 86772.4 5351.7
1783.2 418.2 1042.7
69.8 207.5 5.1
1.00 2.97 0.07
100 69.7 4.3
Fig. 2. Precipitation kinetics of bromelain in the range of 40–60% saturation.
the fifth fraction as shown in Fig. 3. The specific activity of the crude juice and elute were found to be 58.8 and 590.9 Units per mg protein, respectively. The unbound fraction mainly contains sugars which can be further processed for concentration of juice or sugar recovery. 3.3. Effect of drying methods The evaporation rate in spray drying was found to be 210 ml h−1 . Fig. 4 shows the moisture content profile of sample with respect to time during freeze drying. The final moisture content in case of freeze drying was found to be 4.7% wet basis. Table 4 shows the higher retention of the residual activity of freeze-dried bromelain powder as compared to spray-dried powder. The residual activity retention of bromelain in freeze drying was found to be 95 ± 2% as against 78 ± 2% in spray drying. The residual protein content of freeze-dried bromelain (87 ± 2%) was also found to be higher than
Fig. 3. Chromatographic purification profile for the bromelain from pineapple fruit juice.
that of spray-dried bromelain (73 ± 2%). The reason attributed to these findings is the desired low temperature of the material during freeze drying which maintains its structure and the morphology during the process of dewatering. In case of spray drying, the effect of drying air temperature on the residual enzyme activity was also studied. Fig. 5 shows that with an increase in inlet air temperature the residual enzyme activity decreases for a given feed rate condition. At higher inlet air temperature, the material is exposed to higher outlet air temperature which causes the denaturation of the proteins. Therefore, it is desirable to operate the spray dryer at lower air temperature to get the maximum enzyme activity reten-
Table 3 Results of chromatographic purification. Parameter
Observation
Ion exchanger Resin volume (ml) Enzyme recovery (%) Elution efficiency (%) Protein recovery (%) Specific activity of crude (Units per mg protein) Specific activity of elute (Units per mg protein) Purification factor
Cation 100 84.5 97.6 8.7 58.8 590.9 10
Fig. 4. Plot of moisture content (db) vs. time during freeze drying of bromelain.
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
Fig. 5. Effect of inlet air temperature on the residual enzyme activity (%) (data: inlet air flow rate, 45 m3 h−1 ; feed rate, 225 ml h−1 ).
tion. However, the moisture content was found to vary in between 5.2% and 6% on wet basis for different inlet air temperatures. The powder deposited in spray drying chamber showed a negligible activity as it is exposed to a higher temperature for longer period of time. The inactivation temperature of the enzyme was reported in the range of 65–70 ◦ C. It shows almost zero activity at outlet air temperature of 65 ◦ C. Similar results have been reported by Samborska et al. [10] in case of spray drying of ␣-amylase, which is a major class of proteases. The yield of bromelain powder obtained in a spray dryer was observed to be less as compared to the freeze dryer. The probable reason might be an inefficient powder collection system in spray drying which leads to carry over of fine particles along with exhaust air. The powder deposition in spray drying chamber also contributes to lower yield values. The specific activity of freezedried protein was observed to be 482.3 Units per mg protein and that of standard commercial powder was 171 Units per mg protein, i.e. 1 mg protein contains 482.3 casein digesting units (CDU). This proves that the enzyme produced in the present study is almost 2.8fold more pure than the commercially available bromelain (Table 4). 3.4. Inactivation kinetics of bromelain in an aqueous solution Fig. 6 shows the profile of bromelain inactivation with respect to time in aqueous solution at various temperatures. The solid lines shown in the figure were calculated from the parameters obtained by nonlinear regression analysis according to a first-order kinetic expression. The kinetics of inactivation can be described by firstorder rate equation as dA/dt = −kA. Where A is the relative activity of enzyme and k is the rate constant (min−1 ). The thermal inactivation is not at all a physical destruction of the materials, but the whole process is obviously a set of elementary reactions, which altogether represent the mechanism of the overall reaction. Approximating the experimental data, the temperature effect on inactivation rate may be described similarly to the kinetics of chemical reactions by the Arrhenius type of equation: k = k0 exp(−E/RT), where k0 is the
Fig. 6. Profiles of bromelain inactivation in pH 7.4 aqueous solution (♦: 45 ◦ C, : 50 ◦ C, : 55 ◦ C, : 60 ◦ C).
Fig. 7. Arrhenius plots of apparent first-order rate constants obtained for bromelain inactivation.
numerical index representing the nature of the substance nature and characterizes the state of an object i.e. the presence of labile sites and bonds as well as their heat tolerance; E is the inactivation energy (kcal mol−1 ), namely, the minimum energy that causes an irreversible change in the substance exposed to the temperature [11]; R is a universal gas constant (kcal mol−1 K−1 ) and T is temperature (K).
Table 4 Effect of drying methods on product characteristics. Parameter
Freeze drying
Spray drying
Residual activity (%) Residual proteins (%) Specific activity (CDU per mg protein) Yield (%)
95 ± 2 87 ± 3 482.3 96 ± 1
78 ± 2 73 ± 3 462.5 73 ± 2
Note: commercial sample specific activity, 171 CDU per mg protein.
263
Fig. 8. DSC thermogram for freeze-dried bromelain powder.
264
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
any structural changes and protein denaturation in powder the wet bulb temperature of outlet drying air should be less than 61 ◦ C. 3.6. Fourier transform infrared spectroscopy FTIR spectrum of freeze-dried bromelain is as shown in Fig. 9. The characteristic C–N stretch vibration frequencies of monoalkyl guanidinium are assigned to observed IR bands at 1655–1685, 1615–1635 and 1170–1180 cm−1 . The band at 1630–1860 cm−1 shows presence of >C O stretching groups (amides at ∼1650 cm−1 ). It confirms the presence of amino acids which may contain amide group as their side chain, i.e. aspargine and glutamine. The spectra at 3280–3340 cm−1 shows the presence of >CH– stretching vibrations. The experimental spectra of phenylalanine, proline, valine, leucine and isoleucine are in the range of 900–3700 cm−1 . 3.7. Storage and thermal stability Fig. 9. FTIR spectra of freeze-dried bromelain powder.
Fig. 10 shows the storage of bromelain powder at different temperatures over the period of 4 days. The powder samples stored at 4, 30 and 60 ◦ C shows the loss in the activity by 4%, 50% and 90%, respectively, at the end of 4 days. Thus, the inactivation rate was found to be a strong function of storage temperature and time. 4. Conclusions
Fig. 10. Time and temperature stability of freeze-dried bromelain ( : 60 ◦ C).
: 4 ◦ C, : 27 ◦ C,
The inactivation energy was calculated as 21.4 kcal mol−1 by assuming the linearity of the plot. Fig. 7 shows the Arrhenius plot of apparent first-order rate constant obtained for bromelain inactivation. The information available on the inactivation energy of protein degradation is very limited in the literature [12]. The rate constants obtained by nonlinear regression analysis thus provides a reasonable Arrhenius relationship and useful information. The results obtained suggest that the inactivation of enzymes in aqueous solution can be modeled even if the inactivation profile is complicated and the dependence of kinetic parameters on temperature can be thus determined. 3.5. Differential scanning calorimetry From the DSC thermogram of bromelain powder as shown in Fig. 8, the glass transition temperature was found to be 61 ◦ C. The thermogram is an endotherm indicating that the powder is amorphous. From DSC study, it can be concluded that in order to avoid
Bromelain obtained using ion exchange chromatography was found to be about 3 times more pure as compared to the ammonium sulfate precipitation method. Because of lower drying temperature, freeze drying was found to give powder with higher enzyme activity and protein content. Bromelain produced in the present study using ion exchange chromatography followed by freeze drying shows almost 2.8 times more purity as compared to commercial sample. The yield of powder in spray drying can be increased by using properly designed polycyclones and by avoiding powder deposition in spray chamber. The proteins denaturation during spray drying can be avoided by adjusting wet bulb temperature of the outlet air below 61 ◦ C. References [1] M.B. Doko, V. Bassani, J. Casadebaig, L. Cavailles, M. Jacob, Int. J. Pharm. 76 (1991) 199–206. [2] J.C. Caygill, Enzyme Microb. Technol. 1 (1979) 233–242. [3] S. Ota, S. Moore, W.H. Stein, Biochemistry 3 (1964) 180–185. [4] T. Murachi, M. Yasui, Y. Yasuda, Biochemistry 3 (1964) 48–55. [5] R.K. Scopes, in: R.K. Scopes (Ed.), Protein Purification: Principles and Practice, second ed., Springer-Verlag, New York, 1982, pp. 41–65. [6] R. Arnon, E. Shapira, Biochemistry 6 (1967) 3942–3950. [7] G.R. Dapeau, Methods Enzymol. 45 (1976) 471. [8] P.K. Smith, P.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, O.C. Klenk, Anal. Biochem. 150 (1985) 76–85. [9] Y. Liao, M.B. Brown, G.P. Martin, Eur. J. Pharm. Biopharm. 58 (2004) 15–24. [10] K. Samborska, D. Witrowa-Rajchert, A. Gonclaves, Dry. Technol. 23 (2005) 941–953. [11] P.S. Kuts, E.G. Tutova, Dry. Technol. 2 (1983-84) 171–201. [12] T. Tsuda, M. Uchiyama, T. Sato, H. Yoshino, Y. Tsuchiya, S. Ishikawa, M. Ohmae, S. Watanabe, Y. Miyake, J. Pharm. Sci. 79 (1990) 223–227.
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Purification and drying of bromelain R.V. Devakate, V.V. Patil, S.S. Waje ∗ , B.N. Thorat Department of Chemical Engineering, Institute of Chemical Technology, N. M. Parekh Road, Matunga (E), Mumbai 400019, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 18 June 2008 Received in revised form 6 September 2008 Accepted 28 September 2008 Keywords: Chromatography Drying Bromelain Purification Freeze drying
a b s t r a c t The present work deals with the purification and drying of bromelain, one of the vegetal proteases. The purification of the enzyme from fruit Ananas comosus was carried out by precipitation and by chromatography. Further, the drying of chromatographically purified fraction was carried out in freeze dryer and spray dryer. The activity was expressed in terms of casein digesting units (CDU). The bromelain extract of higher purity was obtained from the fruit using ion exchange chromatography. The elution efficiency in chromatographic purification was obtained in the range of 80–90%. The fold purity obtained by chromatography was 3.3 times as compared to that obtained by precipitation. Bromelain thus obtained was subjected to spray and freeze drying. It was observed that the yield obtained in spray dryer at an outlet temperature of 40–50 ◦ C was almost 50–70% and in case of freeze dryer the yield obtained was 96%. The resulting residual activity and the specific activity was compared between spray-dried and freeze-dried powder. The purity of enzyme obtained in the present study was found to be 2.8 times more than that of a commercial sample. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Bromelain (EC 3.4.22.4) is a collective name for proteolytic enzymes or proteases found in tissues including stem, fruit and leaves of the pineapple plant family Bromeliaceae [1]. The isolation of enzymes from pineapple fruit and its study have been investigated since 1894. The enzymes occurring in the stem and the fruit of Ananas comosus (Smouth cayenne) are the most studied. Unlike crude stem bromelain, which is used widely in industry, fruit bromelain is not commercially available despite the large quantities of waste pineapple fruit portions at pineapple canneries [2]. The continued interest in bromelain, for its numerous applications in the food industry as well as in medicine and pharmacology make this enzyme one of the best vegetal proteases. The potential therapeutic value of bromelain is due to its biochemical and pharmacological properties and hence, it is desired to obtain bromelain in its highest purified form. The preparation in pure form of a proteolytic enzyme has always proved difficult, and the bromelains appear to be no exception [3]. The crude commercial bromelain
Abbreviations: P, precipitate; D, dialysate; S, supernatant; CDU, casein digesting unit; db, dry basis; MWCO, molecular weight cut-off. ∗ Corresponding author. Tel.: +91 22 2414 5616x2022/2023; fax: +91 22 2414 5614. E-mail address: [email protected] (S.S. Waje). 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.09.012
used in the manufacture of pharmaceuticals is not chemically homogeneous. The main ingredient in crude bromelain is a proteolytic enzyme named glycoprotein, besides substances such as insoluble materials, e.g. colored pigments, organic acids, minerals, protease inhibitors, organic solvents and excipient used for enzyme recovery have been reported [4]. The present study examines the extract of fresh pineapple fruit for the presence of cysteine proteinases, besides protein degradation kinetics. 2. Experimental 2.1. Materials A ripe pineapple fruits were purchased from a local supermarket. l-Cysteine and casein (Hammarsten grade) were obtained from Sisco Research Laboratory (SRL, Mumbai, India). Trichloroacetic acid (TCA) was purchased from S.D. Fine Chemicals Ltd. (Mumbai, India). Bovine serum albumin (BSA) and tyrosine were purchased from Hi-Media Laboratory (India). All other chemicals used were of analytical grade and purchased from S.D. Fine Chemicals Ltd. (Mumbai, India). A purified commercial bromelain sample was obtained from Hong Mao Biochemical Co. Ltd. (Thailand) as a gift sample. Dialysis Membrane having molecular-weight-cut-off (MWCO) of 12,000 Da was also purchased from Hi-Media Laboratory (India). Ion exchange resins were made available from Resindion Sisco Research Laboratory (Italy) as a free sample.
260
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
2.2. Clarification of fruit crude juice The stalk (central core) of ripe pineapple fruit was separated from fleshy fruit. The fruit portion was then cut into small pieces and crushed in a laboratory juicer to get approximately 500 ml juice. The juice was filtered through a muslin cloth to remove the fibrous material. The filtrate was then subjected to vacuum filtration to remove ruptured plant cells. The filtered crude juice was kept for centrifugation (Remi R-24) at 10,000 × g for 20 min at 4 ◦ C to remove any insoluble materials. The clear supernatant obtained as a clarified extract was stored in aliquots at 4 ◦ C and used whenever required. The protein content in the supernatant was determined before further purification using precipitation and ion exchange chromatography. 2.3. Purification of bromelain from clarified juice Clarified pineapple juice was subjected to precipitation and ion exchange chromatography for the separation of bromelain enzyme. These methods are described briefly as follows.
Fig. 1. Schematic of spray dryer.
2.4. Dialysis 2.3.1. Precipitation The precipitation of bromelain from clarified pineapple fruit juice (20 ml) was carried out by slow addition of ammonium sulfate [(NH4 )2 SO4 ] at 4 ◦ C under constant stirring. Initially, 2.27 g (NH4 )2 SO4 was added to get 0–20% saturation. The stirring was continued for 30 min after the complete addition of salt to allow equilibration between the dissolved and aggregated proteins. The salt-enriched solution was centrifuged (Remi R-24 Research Centrifuge) at 10,000 × g for 15 min and the precipitate was collected. The supernatant was separated, its volume was noted and the amount of salt required for the next cut was calculated. The amount of (NH4 )2 SO4 added to the solution in order to increase % saturation level from S1 to S2 was calculated using following equation [5]: Amount of (NH4 )2 SO4 required (g) 533(S2 − S1 ) = × volume of solution (lit.) 100 − 0.3S2
(1)
where S1 initial saturation of solution (%) and S2 required saturation of solution (%). The equation is related by the assumption that 100% saturation corresponds to 4.05 M salt solution. Ammonium sulfate precipitate fractions and supernatants were collected at the levels of 20% saturation and assayed for protein content and specific protease activity after dialysis. 2.3.2. Ion exchange chromatography Chromatographic purification of clarified crude juice was carried out on a preparative scale. The glass column of 25 mm internal diameter and 300 mm length was packed with cation exchange resins and equilibrated with four column volumes of acetate buffer (25 mM, pH 4.0). Sufficient quantity of clarified crude juice was passed in upward direction through the packed bed of adsorbent and was kept constant at 324 ml h−1 . This was followed by a wash of acetate buffer until no proteins were detected at 280 nm using an online spectrophotometer (Model U-1100, Hitachi, India). Finally, the adsorbed enzyme (protein) was eluted using 1N sodium chloride solution at the flow rate of 324 ml h−1 . The eluting protein was determined at 280 nm on online spectrophotometer. The enzyme activity and protein content for unbound fraction, washings and elution fraction were measured to calculate the total recovery of enzyme units with respect to the bound one. The procedures for estimation of protein content and enzyme activity are mentioned below.
The salt in the purified bromelain samples as a result of above purification methods was removed by dialysis using ammonium phosphate buffer (50 mM, pH 7.0) as a process for buffer exchange. Dialysis membrane (Hi-Media Laboratory, Mumbai, India) having molecular weight cut-off of 12,000 Da was used for dialysis. The enzyme activity units and protein content after dialysis were checked for the percentage loss in the process. 2.5. Drying of purified bromelain Chromatographically purified bromelain was dried using laboratory spray dryer and vacuum freeze dryer. 2.5.1. Spray drying A laboratory bench-top spray dryer (Model: LU-22, Labultima, Mumbai, India) having 1 l/h water evaporation capacity was used for experimental purpose. Fig. 1 shows the schematic of the spray dryer. Peristaltic pump was used to feed bromelain solution to a two-fluid nozzle atomizer (inside diameter: 0.7 mm), with compressed air from a compressor (Fouji, India). The drying chamber (height: 400 mm and diameter: 100 mm) and cyclones (2 nos.) were made of thick transparent glass. Inlet drying air from HEPA filter after passing through an electric heater flows co-currently with the spray through the main chamber. The dried powder was collected in the receivers from the bottom of the cyclones. The remaining ultra-fine powder was recovered in the filter bag. The parameters such as feed flow rate, drying air temperature and compressed air pressure for atomization were set and controlled through a personal computer. In all experiments, the atomizer pressure and feed flowrate were kept constant at 2 kg cm−2 and 225 ml h−1 , respectively. Inlet air temperature was varied in the range from 100 to 170 ◦ C. Outlet air temperatures were recorded online continuously. The drying air flow rate and the feed temperature were kept at 41 m3 h−1 and 27 ◦ C, respectively. The duration of each experimental run was 90 min. 2.5.2. Vacuum freeze drying A laboratory scale vacuum freeze dryer (M/s. Ref-vac Consultancy, India) was used for this study. It consists of cylindrical stainless steel drying chamber (L = 0.4 m, D = 0.4 m) including heating plates (3 nos., total heating surface area = 0.25 m2 ), a condenser (maximum ice collection capacity = 1.8 kg, surface area = 0.25 m2 )
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
capable of operating at −35 ◦ C and a vacuum pump (M/s. Crompton Greaves, India; air displacement rate = 16 m3 h−1 ). The condenser was connected to the drying chamber by a pipe. An electronic weighing balance (M/s. Tapsons Weighing Systems, India, least count = 0.1 g) was placed inside the drying chamber for online sample weight measurement which could be observed through the transparent window. Several arrangements were made to allow comprehensive control, measurement and recording of pressure and temperature inside the drying chamber. The pressure inside the drying chamber was measured with pirani manometers and regulated with solenoid valve. The temperature of heating plates and sample were measured with PT 100 thermocouples (3 nos.) and controlled using an integrated electrical resistance heater. A purified bromelain sample (20 ml) filled in petri-dish was frozen in an external deep freezer (Blue Star, India) at −23 ◦ C for 6 h. Meanwhile, freeze dryer was started and the temperature of the condenser was allowed to reach at its minimum level of −35 ◦ C. Thereafter, the frozen bromelain sample was placed on weighing balance in the drying chamber and subsequently started the vacuum pump and the heater. The temperature of the sample and pressure inside the chamber were set at the predetermined values of 25 ◦ C and 30 Pa, respectively. The weight of the sample was recorded every 5 min time interval, till it attained a constant value. 2.6. Measurement of enzyme activity The enzyme activity assay was based on estimation of amount of small molecular weight digestion products (Trichloroacetic acid (TCA) soluble material) formed from proteins due to proteolytic action of the enzyme [6]. Proteolytic activity of bromelain was measured by the method described by Dapeau [7]. The assay consisted of 5 ml solution of 0.75% casein prepared in anhydrous disodium phosphate buffer (50 mM, pH 7). The pH was adjusted using slow addition of 0.1N HCl. The solution was brought to 37 ◦ C by preincubation for 10 min. To this substrate, a known volume of enzyme was added after diluting it to 1 ml with activating buffer. The 30 mM cysteine hydrochloride monohydrate in 6 mM disodium EDTA was used as an activating buffer. Casein proteolysis was stopped after 10 min by addition of 5 ml of 30% (w/v) TCA and was allowed to stand for 30 min at 37 ◦ C. Thereafter, the solution was cooled to room temperature and filtered twice using Whatman filter paper (No. 42). Absorbance of the filtrate was measured at 280 nm using UV–vis spectrophotometer (Chemito, UV 2500, Mumbai, India) against the tyrosine standard plot of absorbance versus tyrosine concentration (g ml−1 ). One unit of bromelain was taken as the amount of enzyme which while acting on the casein substrate under specified conditions, produces one microgram of tyrosine per minute. 2.7. Measurement of protein content Protein content was measured spectrophotometrically by using the Bicinchoninic method of protein estimation [8]. Sigma Bicinchoninic acid protein assay kit was used. 0.2 ml Bicinchoninic acid was added to 0.025 ml of suitably diluted protein and the mixture was vortexed for about 30 s. The color developed was measured at 565 nm using microplate reader (Model 680, Bio-Rad California) after 2 h. Bovine serum albumin was used as the standard for protein assay. A stock solution of 0.1 mg ml−1 BSA was prepared in distilled water. Solutions of different concentrations of BSA were prepared by diluting the stock solution with distilled water. The absorbance of these dilutions was measured at 565 nm and a plot of absorbance versus BSA protein concentrations (g ml−1 ) was used as a standard.
261
2.8. Differential scanning calorimetry (DSC) Thermal analysis of freeze-dried powder was performed using differential scanning calorimeter (DSC) (Perkin Elmer Pyris-6, Germany). Before analysis, the dried samples were transferred to vacuum desiccator and equilibrated for 3 days over P2 O5 for ‘zero’ moisture content. Approximately 5 mg powder was used for analysis. The sample was sealed in an aluminium pan and an empty pan was used as a reference. Heating rate of 10 ◦ C min−1 was used. The glass transition temperature was reported as the midpoint temperature of heat capacity step. 2.9. Fourier transform infrared (FTIR) spectroscopy Fourier transform infrared (FTIR) spectra were measured using a Perkin Elmer FTIR spectrometer (Series 1600) and analyzed using PE-GRAMS/32 1600 software, as described by Liao et al. [9]. Briefly, a dry protein sample (approximately 0.5 mg) was mixed with 300 mg ground potassium bromide and compressed to get a pellet. The spectra were smoothed with a nine-point Savitsky–Golay function to remove any possible white-noise. The baseline of the spectrum in the amide I region was leveled and zeroed, then the spectrum of the sample was normalized for area in the region and the intensity of ␣-helical band was recorded. 3. Results and discussion 3.1. Purification using precipitation From Table 1, it is clear that the fractions collected at 40–60% and 60–80% (w/v) ammonium sulfate saturation were found to have the higher specific protease activity and protein content as compared to other saturation levels. The enzyme recovery and protein content were found to be about 80% and 34% of total enzyme and protein contents, respectively. Further increase in the percentage saturation level from 80% to 100% causes the denaturation of the proteins and enzymes which also tends to reduce the specific activity. In the fractions obtained using lower (NH4 )2 SO4 saturation (<40% saturation), the specific enzyme activity was found to be very less (<79 Units per mg protein). This means that proteases are soluble in a solution having (NH4 )2 SO4 saturation less than 40%. Proteins other than proteases which are not soluble in the supernatant were precipitated when (NH4 )2 SO4 saturation is less than 40%. To get the optimum range of (NH4 )2 SO4 saturation, the 40–80% (w/v) saturation interval was further divided into smaller fractions (Table 2) and it was observed that 40–70% (w/v) (NH4 )2 SO4 fraction contained 68% of the protease activity and approximately 3-fold increase in specific activity compared to the crude fruit juice. To more precisely obtain, the precipitation kinetics in the saturation range of 40–60%, the saturation range was increased by 2%, and for every 2% increase in saturation, the precipitate and supernatant were collected and analyzed for bromelain activity. A second preliminary experiment shows that an increase in solid-phase activity could be correlated with a fall in the supernatant activity. The kinetics of bromelain precipitation in the saturation range of 40–60% is as shown in Fig. 2. 3.2. Purification using ion exchange chromatography The bromelain was found as much as 10-fold pure with 84.5% enzyme recovery using cation exchange chromatography technique (Table 3). No bromelain activity was detected in the washing flow. The elution efficiency of 97.6% was obtained. The cation exchange chromatography of bromelain resulted in one prominent peak at
262
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
Table 1 Effect of percent saturation on bromelain and total protein recovery on precipitation. Saturation level
Units (CDU)
Protein content (mg)
Specific activity (Units per mg protein)
Fold purity
% yield
Crude 0–20% (D) 20–40% (D) 40–60% (D) 60–80% (D) 80–100% (D) 100%–+(D)
160721 7393.2 27804.7 66056.3 61556.1 6589.6 5786.0
2733.6 476.2 350.9 401.0 451.1 401.0 601.5
58.8 15.5 79.2 164.7 136.5 16.4 9.6
1.0 0.26 1.35 2.81 2.33 0.28 0.16
100 4.6 17.3 41.1 38.3 4.1 3.6
Table 2 Effect of percent saturation on bromelain and total protein recovery (precipitation range of 40–80%). Saturation level
Units (CDU)
Protein content (mg)
Specific activity (Units per mg protein)
Fold purity
% yield
20–40% (S) 40–70% (P) 70–80% (P)
124458.6 86772.4 5351.7
1783.2 418.2 1042.7
69.8 207.5 5.1
1.00 2.97 0.07
100 69.7 4.3
Fig. 2. Precipitation kinetics of bromelain in the range of 40–60% saturation.
the fifth fraction as shown in Fig. 3. The specific activity of the crude juice and elute were found to be 58.8 and 590.9 Units per mg protein, respectively. The unbound fraction mainly contains sugars which can be further processed for concentration of juice or sugar recovery. 3.3. Effect of drying methods The evaporation rate in spray drying was found to be 210 ml h−1 . Fig. 4 shows the moisture content profile of sample with respect to time during freeze drying. The final moisture content in case of freeze drying was found to be 4.7% wet basis. Table 4 shows the higher retention of the residual activity of freeze-dried bromelain powder as compared to spray-dried powder. The residual activity retention of bromelain in freeze drying was found to be 95 ± 2% as against 78 ± 2% in spray drying. The residual protein content of freeze-dried bromelain (87 ± 2%) was also found to be higher than
Fig. 3. Chromatographic purification profile for the bromelain from pineapple fruit juice.
that of spray-dried bromelain (73 ± 2%). The reason attributed to these findings is the desired low temperature of the material during freeze drying which maintains its structure and the morphology during the process of dewatering. In case of spray drying, the effect of drying air temperature on the residual enzyme activity was also studied. Fig. 5 shows that with an increase in inlet air temperature the residual enzyme activity decreases for a given feed rate condition. At higher inlet air temperature, the material is exposed to higher outlet air temperature which causes the denaturation of the proteins. Therefore, it is desirable to operate the spray dryer at lower air temperature to get the maximum enzyme activity reten-
Table 3 Results of chromatographic purification. Parameter
Observation
Ion exchanger Resin volume (ml) Enzyme recovery (%) Elution efficiency (%) Protein recovery (%) Specific activity of crude (Units per mg protein) Specific activity of elute (Units per mg protein) Purification factor
Cation 100 84.5 97.6 8.7 58.8 590.9 10
Fig. 4. Plot of moisture content (db) vs. time during freeze drying of bromelain.
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
Fig. 5. Effect of inlet air temperature on the residual enzyme activity (%) (data: inlet air flow rate, 45 m3 h−1 ; feed rate, 225 ml h−1 ).
tion. However, the moisture content was found to vary in between 5.2% and 6% on wet basis for different inlet air temperatures. The powder deposited in spray drying chamber showed a negligible activity as it is exposed to a higher temperature for longer period of time. The inactivation temperature of the enzyme was reported in the range of 65–70 ◦ C. It shows almost zero activity at outlet air temperature of 65 ◦ C. Similar results have been reported by Samborska et al. [10] in case of spray drying of ␣-amylase, which is a major class of proteases. The yield of bromelain powder obtained in a spray dryer was observed to be less as compared to the freeze dryer. The probable reason might be an inefficient powder collection system in spray drying which leads to carry over of fine particles along with exhaust air. The powder deposition in spray drying chamber also contributes to lower yield values. The specific activity of freezedried protein was observed to be 482.3 Units per mg protein and that of standard commercial powder was 171 Units per mg protein, i.e. 1 mg protein contains 482.3 casein digesting units (CDU). This proves that the enzyme produced in the present study is almost 2.8fold more pure than the commercially available bromelain (Table 4). 3.4. Inactivation kinetics of bromelain in an aqueous solution Fig. 6 shows the profile of bromelain inactivation with respect to time in aqueous solution at various temperatures. The solid lines shown in the figure were calculated from the parameters obtained by nonlinear regression analysis according to a first-order kinetic expression. The kinetics of inactivation can be described by firstorder rate equation as dA/dt = −kA. Where A is the relative activity of enzyme and k is the rate constant (min−1 ). The thermal inactivation is not at all a physical destruction of the materials, but the whole process is obviously a set of elementary reactions, which altogether represent the mechanism of the overall reaction. Approximating the experimental data, the temperature effect on inactivation rate may be described similarly to the kinetics of chemical reactions by the Arrhenius type of equation: k = k0 exp(−E/RT), where k0 is the
Fig. 6. Profiles of bromelain inactivation in pH 7.4 aqueous solution (♦: 45 ◦ C, : 50 ◦ C, : 55 ◦ C, : 60 ◦ C).
Fig. 7. Arrhenius plots of apparent first-order rate constants obtained for bromelain inactivation.
numerical index representing the nature of the substance nature and characterizes the state of an object i.e. the presence of labile sites and bonds as well as their heat tolerance; E is the inactivation energy (kcal mol−1 ), namely, the minimum energy that causes an irreversible change in the substance exposed to the temperature [11]; R is a universal gas constant (kcal mol−1 K−1 ) and T is temperature (K).
Table 4 Effect of drying methods on product characteristics. Parameter
Freeze drying
Spray drying
Residual activity (%) Residual proteins (%) Specific activity (CDU per mg protein) Yield (%)
95 ± 2 87 ± 3 482.3 96 ± 1
78 ± 2 73 ± 3 462.5 73 ± 2
Note: commercial sample specific activity, 171 CDU per mg protein.
263
Fig. 8. DSC thermogram for freeze-dried bromelain powder.
264
R.V. Devakate et al. / Separation and Purification Technology 64 (2009) 259–264
any structural changes and protein denaturation in powder the wet bulb temperature of outlet drying air should be less than 61 ◦ C. 3.6. Fourier transform infrared spectroscopy FTIR spectrum of freeze-dried bromelain is as shown in Fig. 9. The characteristic C–N stretch vibration frequencies of monoalkyl guanidinium are assigned to observed IR bands at 1655–1685, 1615–1635 and 1170–1180 cm−1 . The band at 1630–1860 cm−1 shows presence of >C O stretching groups (amides at ∼1650 cm−1 ). It confirms the presence of amino acids which may contain amide group as their side chain, i.e. aspargine and glutamine. The spectra at 3280–3340 cm−1 shows the presence of >CH– stretching vibrations. The experimental spectra of phenylalanine, proline, valine, leucine and isoleucine are in the range of 900–3700 cm−1 . 3.7. Storage and thermal stability Fig. 9. FTIR spectra of freeze-dried bromelain powder.
Fig. 10 shows the storage of bromelain powder at different temperatures over the period of 4 days. The powder samples stored at 4, 30 and 60 ◦ C shows the loss in the activity by 4%, 50% and 90%, respectively, at the end of 4 days. Thus, the inactivation rate was found to be a strong function of storage temperature and time. 4. Conclusions
Fig. 10. Time and temperature stability of freeze-dried bromelain ( : 60 ◦ C).
: 4 ◦ C, : 27 ◦ C,
The inactivation energy was calculated as 21.4 kcal mol−1 by assuming the linearity of the plot. Fig. 7 shows the Arrhenius plot of apparent first-order rate constant obtained for bromelain inactivation. The information available on the inactivation energy of protein degradation is very limited in the literature [12]. The rate constants obtained by nonlinear regression analysis thus provides a reasonable Arrhenius relationship and useful information. The results obtained suggest that the inactivation of enzymes in aqueous solution can be modeled even if the inactivation profile is complicated and the dependence of kinetic parameters on temperature can be thus determined. 3.5. Differential scanning calorimetry From the DSC thermogram of bromelain powder as shown in Fig. 8, the glass transition temperature was found to be 61 ◦ C. The thermogram is an endotherm indicating that the powder is amorphous. From DSC study, it can be concluded that in order to avoid
Bromelain obtained using ion exchange chromatography was found to be about 3 times more pure as compared to the ammonium sulfate precipitation method. Because of lower drying temperature, freeze drying was found to give powder with higher enzyme activity and protein content. Bromelain produced in the present study using ion exchange chromatography followed by freeze drying shows almost 2.8 times more purity as compared to commercial sample. The yield of powder in spray drying can be increased by using properly designed polycyclones and by avoiding powder deposition in spray chamber. The proteins denaturation during spray drying can be avoided by adjusting wet bulb temperature of the outlet air below 61 ◦ C. References [1] M.B. Doko, V. Bassani, J. Casadebaig, L. Cavailles, M. Jacob, Int. J. Pharm. 76 (1991) 199–206. [2] J.C. Caygill, Enzyme Microb. Technol. 1 (1979) 233–242. [3] S. Ota, S. Moore, W.H. Stein, Biochemistry 3 (1964) 180–185. [4] T. Murachi, M. Yasui, Y. Yasuda, Biochemistry 3 (1964) 48–55. [5] R.K. Scopes, in: R.K. Scopes (Ed.), Protein Purification: Principles and Practice, second ed., Springer-Verlag, New York, 1982, pp. 41–65. [6] R. Arnon, E. Shapira, Biochemistry 6 (1967) 3942–3950. [7] G.R. Dapeau, Methods Enzymol. 45 (1976) 471. [8] P.K. Smith, P.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, O.C. Klenk, Anal. Biochem. 150 (1985) 76–85. [9] Y. Liao, M.B. Brown, G.P. Martin, Eur. J. Pharm. Biopharm. 58 (2004) 15–24. [10] K. Samborska, D. Witrowa-Rajchert, A. Gonclaves, Dry. Technol. 23 (2005) 941–953. [11] P.S. Kuts, E.G. Tutova, Dry. Technol. 2 (1983-84) 171–201. [12] T. Tsuda, M. Uchiyama, T. Sato, H. Yoshino, Y. Tsuchiya, S. Ishikawa, M. Ohmae, S. Watanabe, Y. Miyake, J. Pharm. Sci. 79 (1990) 223–227.