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International Food Research Journal 19(2): 727-731 (2012

)

Differential scanning calorimetry as tool in observing thermal and
storage stability of recombinant bromelain
Nurul, A. I. and *Azura, A.

Biomolecular Engineering Research Group, Department of Biotechnology
Engineering, Kuliyyah of Engineering, International Islamic University Malaysia,
P.O. Box. 10, 50728 Kuala Lumpur

Abstract: Knowledge about the thermal and storage behavior of produced protein is important for the purpose
of storage, transport and shelve life during industrial application. Recombinant bromelain thermal and storage
stability were measured and compared to the commercial bromelain using Differential Scanning Calorimetry
(DSC). Recombinant bromelain is more stable than commercial bromelain at higher temperature but the stability
was reduced after 7 days of storage at 4oC. Higher energy is needed to break the bond between amino acid
chains in recombinant bromelain as shown by the enthalpy obtained, suggesting that recombinant bromelain has
good protein structure and conformation compared to commercial.

Keywords: DSC, thermal stability, storage stability, recombinant bromelain

Introduction in solution and the stability of this respiratory protein
was obtained (Cueto et al., 2003). DSC is also used to
The mechanisms of protein degradation by detect the glass transition temperature, Tg in protein
chemical or physical means have been extensively solutions and lyophilized products. The determination
reviewed (Cueto et al., 2003; Gao et al., 2005). of Tg is necessary in defining a freeze-drying cycle
Protein structures are stabilized by non-covalent and storage temperature for the lyophilized products.
intramolecular interactions between amino acid The peak transition or denaturation temperature, Td,
side chains. The higher levels of protein structure is a measure of thermal stability, while the enthalpy
are maintained by relatively weak non-covalent change (DH), measured as area under the endothermic
interactions. Disruption of these weak interactions peak, represents the proportion of undenatured protein
can be caused by factors such as temperature (heat or in a sample, or extent of ordered structure (Charrier
cold), pressure, salt, pH, shear, surface interactions, et al., 2006). The sharpness of the transition peak,
and freeze-drying application. Thermal analysis is measured as width at half peak height (DT1/2), is
useful for detecting the effects of these factors on an index of the cooperatively of the transition from
proteins or protein products. native to denatured state (Amako and Xiong, 2001;
Differential scanning calorimetry (DSC) has Tang, 2007). Therefore, the aims of this study are to
been widely used in studying the thermal stability of examine and characterize the thermal and storage
proteins and as one of the most sensitive technique for stability of recombinant bromelain by DSC and
measuring the thermodynamic parameters of thermal compare it with the commercial bromelain.
protein unfolding (Chen and Oakley, 1995; Cordella et
al., 2003). DSC determines the calorimetric changes Material and Methods
in proteins as a function of temperature. The thermal
denaturation of proteins is attributed to the rupture Vector construction
of intramolecular hydrogen bonds. The denaturation Bromelain gene was isolated and purified from
temperatures are measures of the thermal stability Ananas comosus stem. The cDNA was synthesis from
of proteins. Their determination under controlled total RNA and cloned into the entry vector. E. coli
conditions can provide direct comparison of the BL21-AI was used as expression system as described
thermal stability of the different proteins. previously (Ismail and Amid, 2008a; 2008b; Amid et
Equilibrium analysis of DSC thermograms al., 2011).
corresponding to reversible unfolding of proteins
provides information about the thermodynamics and Purification of recombinant bromelain
mechanisms of the reversible unfolding. A kinetic Purification of the recombinant bromelain
model for the irreversible denaturation was applied was carried out following the instruction of the
to analyze heat capacity curves that are dependent on manufacturer (Qiagen, Germany). The expressed
the DSC scan rate. Thus, information on the structure recombinant bromelain was purified by affinity

*Corresponding author.
Email: azuraamid@iium.edu.my © All Rights Reserved
Tel: +603-61964429; Fax: +603-61964442

A. Up to 15 mL of cell lysate was measures the thermal stability and unfolding of a loaded onto the Ni-NTA column at a flow rate of 1 ml/ protein. and Azura.0). Millipore. aluminium pans was pierced just before analysis. folded protein which stabilizing the native states or allowing desorbed water to leave the pan. Units/mL enzyme = Figure 1. The recombinant protein was eluted by elution buffer (50 mM NaH2PO4.53oC) at day 1 and day 7 (Figures (DSC) (Mettler Toledo. were heated from 29°C to 150°C at heating rate of 10ºC min-1. Figures 1 and 2 show the glass transition temperature. The freezed recombinant the product stability to high temperature while the bromelain was plug into a freeze dryer (Labconco. Figure 2.87oC) showed higher Thermal analysis of freeze-dried powder was thermal stability compared to commercial bromelain performed using differential scanning calorimetry (90. I. Tg for recombinant and commercial Freeze drying bromelain for day 1 and after 7 days of storage in The purified recombinant bromelain was 4oC. Approximately.6212 = milimolar extinction coefficient of p. All biological processes depend on proteins being stable and in the appropriate folded conformation.nitrophenol at 340 nm 10 = volume (in microliters) of enzyme used Results and Discussion A stable and correctly folded protein is an absolute requirement for a successful biotherapeutic. The Tg is defined as the temperature at which an concentrated by spinning it at 7400 rpm. 30 min at amorphous system changes from a hard or glass to the 4oC using (Centrifugal Filter Unit. The lid of the liganded protein. low Tg value indicate the vulnerability of the product USA) and left for 4 days until powderized.6. DSC directly (Ni-NTA) agarose. The enzyme activity was calculated by applying equation (1). 5 1 and 2). (a) Recombinant bromelain (b) Commercial bromelain (ΔA340nm/min Test -ΔA340nm/minBlank)(280)(df) (0.. (a) Recombinant bromelain (b) Commercial bromelain The thermal and storage behavior of recombinant International Food Research Journal 19(2): 727-731 . DSC thermograms show Transition midpoint at day 1. min. The higher Tg of recombinant Differential scanning calorimetry bromelain (98. Switzerland) for the first five min. USA) rubbery state. DSC822e). A ligand will bind to a protein only if mg sample was sealed in an aluminum pan and an the resulting complex is more stable than the non- empty pan was used as a reference. DSC thermograms show Transition midpoint after 7 days of storage at 4oC.61oC and 90.19oC and 94. Binding can occur to the native.6212)(10) (1) where : 280 = volume (in microliters) of assay df = dilution factor 0. during processing. 2006).728 Nurul. handling and storage in response to temperature. A. The assay was conducted in triplicate to confirm the precision of the measurement. The it can destabilize the native protein by binding to the powdered recombinant and commercial bromelain denatured protein (Charrier et al. The reaction was measured spectrophotometrically at 340 nm (Tecan. The high Tg value indicate the greater and kept at -80oC overnight. Enzymatic assay of bromelain The protease activity towards Nα-CBZ-L- Lysine p-Nitrophenyl Ester (LNPE) was studied at 45oC and pH 4. chromatography using a nickelnitrilotriacetic acid bromelain was investigated by DSC. 300 mM NaCl and 250 mM Temperature stability imidazole at pH 8.

recombinant bromelain can maintained its structure Bromelain contains a single oligosaccharide moiety and stably folded although exposing to higher per molecule which interacts largely with the sugar. 2008). 2001). Thermal and storage stability of recombinant bromelain 729 techniques it can be expected that the presence of The use of enzyme in industrial processes may stabilizers such as certain sugars is necessary for require reaction to be conducted at high temperature successful drying and storage stability of the dried in order to improve productivity and the the results proteins (Wang. (2001) shows that most protein are stabilized in could be considered as potential candidate for various the presence of sugar such as trehalose and sucrose. exhibited higher storage stability compared to From the existing knowledge from other drying recombinant when the Tg is maintained at 90oC after International Food Research Journal 19(2): 727-731 . 2000).19 90.03 J/g were needed and therapeutically important enzymes without fail. 2000. Bromelain (Merck. had a strong at a constant rate and the heat change associated impact on the particle morphology and showed with thermal denaturation is detectable.81 for its destabilization... stable glass The enthalpy change was measured when all transition temperature. Melo absorbs energy from the surrounding in the form of et al. This showed higher amount of heat needed to break the could also make trehalose an unstable matrix and bond between the amino acid chains in recombinant changes of the formulation matrix which influence bromelain compared to commercial.02 J/g and 94. activity of the enzymes are glycerol and sorbitol USA) USA) Tm (Li et al. was observed after 7 days storage of the enzyme. The availability of sugar such as (transition midpoint) 98. Crystallinity property is related to enthalpy due to Meanwhile.06 78.02 90.15 Activity Storage stability In terms of storage stability study. to denature the protein structure and conformation However Habib et al. Summary of DSC results for recombinant and commercial The binding of sugars to the folded enzyme (native) bromelain at day 1 and after 7 days of storage is expected to be more than to the unfolded ones Duration Time Day 1 A week (Day 7) (denatured). This content or in the presence of crystalline phase. the protein is heated Remarkably.53 sucrose in unpurified commercial bromelain results (oC) Crystallinity 53. temperature. Enthalpy and thermodynamic measurements on the stability changes observed in Table 1 may be associated with of bromelain under thermal stress revealed that this molecular changes as a result of protein unfolding..39 93.87oC after 7 days storage in 40C more rapidly when the protein absorbs more energy (Figures 1 and 2). higher percentage of was reduced from 98. 2008). The protein will crystallize reduced to 94. Other stabilizers which exhibited a Recombinant Commercial Bromelain Recombinant Commercial Bromelain higher stabilizing effect and preserved 75% of enzyme Proteins Bromelain (Merck. The Tg of recombinant bromelain (Li et al. (2007) reported that both kinetic at day 1 and day 7 respectively (Table 1).03 59.99 in osmolyte exclusion and preferentially hydration of (%) Enthalpy the denatured bromelain and appears to be the reason (ΔH) (J/g) – 100% 123. such as disruption of hydrogen bonds crystalline phase compared to trehalose which forms and exothermic reactions. 2000). Previous studied by Melo et obtained suggested that recombinant bromelain al.17 85. explained that the protein conformation (Allison et al. the Tg of recombinant bromelain is unfolding of the protein. Therefore. commercial bromelain the protein is crystallized (100% crystallization). sugar is a universal protein stabilizer. 1995). such as disruption of a heterogeneous system. The bromelain more than sucrose at temperature changes are due to a combination of endothermic 60oC. Tg. Table 1. The Kaushik and Bhat (2003) reported on the thermal amount of heat absorbed is measured by DSC in the stabilization of five proteins differing in their various form of unfolding enthalpy due to heat denaturation physico-chemical properties with sugar.19oC to 94. Commercial bromelain (Table 1).59 89. and that (Chen and Oakley. Enzyme 57. 2008). The process distinct physical characteristics (Wang.61 94. It appears that 1 M trehalose inactivated of the ordered secondary structure of a protein.. industrial applications. In DSC.61 47..87 90.67 94.87oC after 7 days of crystallinity was observed when higher amount of storage simultaneously indicated by the reduction in enthalpy change absorbed for the protein to denature the enzyme activity (Table 1). heat which describes the endothermic process. enzyme is less stable in the presence of sucrose or The enthalpy value is correlated with the net content trehalose. sucrose and trehalose. locally differing in water hydrophobic interactions (Li et al.62 41. and can be Higher enthalpy was observed by recombinant used to increase the stability of many industrially bromelain where 123. Sucrose showed a stable system during the reactions.

S.. 2001. Davis. and Hua. and Llabrés. J-P.V. C. M. E. Thus.M. A. quality. I. and time. A. combinations of disaccharides and dextran. manufactured and marketing in all over the world.. Randolph. Journal of Pharmacology Science 89: 199– 214. 2006.C. Optimization on the storage conditions such as water activity.A. and Younus. Charsley. P. N. previous study (Hale et al. and Sbirrazzuoli.. Yusof. concluded that in order to avoid any structural Cueto..3oC. The powder samples chicken thigh and breast muscles. the recombinant bromelain exhibited higher thermal Devakate..V. 1995.A. Thermochimica Acta 435: 38-43.. and Griffiths. M. The The ability of DSC to detect differences in Tg in Protein Journal 26(2): 117-124. R.G. dichroism experiment (59. H.K. Markhama. Biochimica et fruit is higher than stem bromelain. A. Laye.E. S. Conclusions Habib.. Determination of whereas recombinant bromelain is purely enzyme the temperature and enthalpy of the solid-solid phase extracted from pineapple stem. This is due in protein characterization and formation screening.. B. Li et al. H.730 Nurul. Cabrol-Bass. stability and lower storage stability compared to 2009. the inactivation rate was Stem Bromelain from Ananas comosus. 2005. Arroyo-Reyna. T. Xu. The stability of protein is strongly associated with Allison. T..L. Thus. M.P. From DSC study. which depends K. our study shows that 252: 159-166. Faucon. Thermal destabilization of stem bromelain by trehalose. transition of caesium nitrate by differential scanning Devakate et al. is due to the combination of fruit and stem bromelain Berger. Tang. E. G-Y. Hence. Dorta. 2007.. F. Effects of to characterize the properties. New approach to stability assessment of bromelain lypholization especially in spray drying protein solution formulation by differential scanning process.S. The 2005) shown that proteinase activity of pineapple thermal denaturation of stem bromelain is consistent with an irreversible two-state model. D. Process found to be strong function of storage temperature Biochemistry. P. This would Biophysics Acta 1248: 123-128.. these components addition may Acknowledgment decrease the bond between amino acid chains in commercial bromelain and easily break and denatured We would like to thank the Ministry of Ministry of at lower temperature compared to recombinant Higher Education. O. N. changes and protein denaturation in recombinant 2003. 2006. 7 days of storage (Devakate et al. J.. and Thorat. Y. 1991). Malaysia (MOHE) for providing a explained the lower thermal stability of commercial FRGS grant no FRGS0409-112 for this project.5% and 4. complex protein formulations is extremely valuable Hale. Thermochimica Acta 445: 36-39. Zhou. L. and Oakley. stability and carrageenan on thermal stability of proteins from safety of the food systems. 1995. Thermochimica recorded the maximum of the heat capacity curve Acta 248: 229-244.. Purification and drying of bromelain. it can be 290.. Thermal analysis of whereas Arroyo-Reyna and Hernandez-Arana (1995) proteins of pharmaceutical interest.7% Purification and Characterization of a recombinant respectively (Table 1). 2005..N. the bulb temperature of outlet drying air calorimetry. Charrier et al.. temperature of freeze-dried bromelain is 61oC. Munguia. occurred at 59. Chen.F. H.W. 1995. D. (2009) found the glass transition calorimetry.S. International Journal of Pharmaceutics should be less than 100oC. suggest that higher activity of commercial bromelain Charrier.N. D. and James. C. V. Expression. Mohd.L. at 4oC shows the loss in activities by recombinant Amid. Patil. Waje.6oC) performed under Journal of Thermal Analysis and Calorimetry 71: 279- 1oC/min heating rate. to the addition of stabilizer and other components It can be used to evaluate protein stability and in in commercial bromelain since it is commercially process development is gaining recognition. J. C. Separation and Purification Technology 64: 259-264. and its aqueous solution. Y. and Carpenter.L..M. B... Tm value observed in circular species characterization and adulteration detection.. S. commercial. References 2008).. 2009). A. Manning. T. Ismail. Trinh. and Hernandez-Arana. (2011) 46 (12): 2232-2239. This is in agreement with the Cordella.T. T-C. bromelain (Arroyo-Reyna and Hernandez-Arana. C.T. 2000. Unfortunately. and commercial bromelain are 27..D. the Tg can be set as a reference parameter Amako. A. Food Research of recombinant and commercial bromelain stored International 34: 247-253. of storage stability of lyophilized actin using humidity and temperature (Ross and Karel. 2003. Proteinase activity and stability of natural bromelain International Food Research Journal 19(2): 727-731 . M. A.. Middleton.. and Azura. Application of DSC as a tool for honey floral transition midpoint. M. 2007. Glass bromelain formulation are needed to improve the transition and enthalpy relaxation of ethylene glycol storage stability of the enzyme. the glass transition temperature (Tg). Khan. Future studies on the recombinant Gao. and Xiong.. Greer. Besides that.

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