Ravi Thesis
Ravi Thesis
Ravi Thesis
A THESIS
SUBMITTED TO THE
UNIVERSITY OF MUMBAI
FOR THE
MASTER OF TECHNOLOGY DEGREE
IN
BIOPROCESS TECHNOLOGY
(PARTLY BY PAPERS PARTLY BY RESEARCH)
SUBMITTED BY
RAVIKANT VITHALRAO DEVAKATE
The work described in this thesis has been carried out by Mr. Ravikant Vithalrao
Devakate under my supervision at the Chemical Engineering Department,
Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai –
400 019. I certify that it is his bonafide work. The work described is original and
has not been submitted for any other degree of this or any other university.
Professor B. N. Thorat
Research Supervisor
Professor of Chemical Engineering
Chemical Engineering Department,
Institute of Chemical Technology,
University of Mumbai, Date: 30 June 2006
Matunga, Mumbai-400 019 Place: Mumbai
STATEMENT BY THE CANDIDATE
As required by the University Regulation No. R 2304, I wish to state that the
work embodied in this thesis titled “Crystallization and Drying studies of
Biomaterials” forms my own contribution to the research work carried out
under the guidance of Professor B. N. Thorat at the Chemical Engineering
Department, Institute of Chemical Technology, University of Mumbai, Matunga,
Mumbai – 400 019. This work has not been submitted for any other degree for
this or any other University. Whenever references have been made to previous
work of others, it has been clearly indicated as such and included in
bibliography.
Professor B. N. Thorat
Research Supervisor
Professor of Chemical Engineering
Chemical Engineering Department,
Institute of Chemical Technology,
University of Mumbai,
Matunga, Mumbai – 400 019
This thesis is the end of my journey in obtaining my degree at UDCT. I have not traveled
in a vacuum in this journey. There are some people who made this journey easier with
words of encouragement and more intellectually satisfying by offering different places to
look to expand my theories and ideas. I would like to express my gratitude to all those
who gave me the possibility to complete this thesis.
………..Ravikant
...dedicated to
My Parents
Index
1. INTRODUCTION
1.1.1. Enzymes 1
1.5. References 17
2.5. Bromelains 28
2.5.2.1. Stability 32
2.6. References 42
3.3.5.1.Freeze Dryer 58
3.3.5.2.Spray Dryer 59
3.4. References 64
4.5.4. Optimum pH 87
4.6. References 91
5. CONCLUSION 93
SYNOPSIS
List of Tables
No.
press)
proteinases
recovery in precipitation
bromelain
List of Figures
No.
Introduction
Introduction
organism, its vital functions and the activities. Most frequently, it is a single cell or a system
of cells which form a living organism. However, this can be a structural part of a cell or an
organism which constitutes a compact entity; a component built into various structural parts
of processes in which living organisms are involved (Kudra and Strumillo, 1998).
In the literature the term ‘bio – product’ is regarded as equivalent to a ‘bio – material’ and
Biopolymers are related to bio – material, bio – product and biotechnology. Biopolymer
1.1.1. Enzymes
Enzymes are proteins with catalytic activity allowing chemical reactions in a living cell to
energy of substrates, i.e. the energy necessary to complete a given chemical reaction. Thus,
they enhance reaction equilibrium. Besides the active form, the enzymes may be inactive
and can be activated when needed. The structure of enzymes is similar to the structure of
amino acids and proteins. The central region directly engaged in a biochemical reaction is
called the active centre. This region includes atoms or groups of atoms which control
enzymatic activity, e.g. ions of metals, lipids, hydrocarbons, etc. The enzymatic reaction
rate is affected by the factors including duration of the reaction, temperature, pH, enzyme
stability, substrate concentration and presence of activators. Because enzymes are proteins,
their structures change easily under the action of the above mentioned factors which may
isomerases and ligases. Due to enzymatic treatment, the source materials are used
economically, processes are more efficient, production cycles are shortened, product’s
quality and durability are improved, and reaction rates can be easily controlled. For these
biotechnology.
conditions of the solute in the solvent, as compared to precipitation due to chemical reaction.
A solution system remains at equilibrium until the point is reached where there is
insufficient solvent to maintain full hydration of the solute molecules. If more solute is
added to the solution, the system is no longer at equilibrium under this so-called
equilibrium corresponding free energy minimum by forming a solid phase. The solid phase
similar for small molecules (e.g., salts and small organic compounds) and macromolecules
(e.g., proteins, DNA, and RNA). Three stages are common to all crystallizations: nucleation,
aggregates (dimers, trimers, etc.) produce a stable aggregate with a repeating structure. The
nucleus must first exceed a specific size, called the critical size, before it is capable of
further growth. However, the formation of crystal nuclei from supersaturated solution does
crystal growth can occur for many reasons. The most obvious reason is that the equilibrium
The solubility of a protein can be described by a phase diagram as shown in Figure 1.1.
The solubility curve, S, divides unsaturated from supersaturated areas. On the curve proteins
are in equilibrium between solution and crystal, they will not crystallize beneath S. The
Zone 1 (Metastable zone): The solution may not nucleate for a long time but this zone will
Zone 3 (Precipitation zone): Proteins do not nucleate but precipitate out of solution.
The crystallization of proteins is usually more difficult than small molecule crystallization.
This is probably a result of the difficulty to obtain a high quality protein sample for
crystallization. Because precise protein–protein contacts are needed for the crystallization of
proteins, any factor that introduces heterogeneity into the protein sample may have
interfering effects on the crystallization. There are numerous factors that can introduce
molecules, different conformations of the protein can be present, contamination in the form
The basic strategy to crystallize proteins is to bring the system into a state of limited degree
of supersaturation. This can be done, for example, by removing the solvent, adding a
there are dozens of other parameters that are involved in protein crystallization.
multifactorial process.
1. Purity of proteins: Proteins need not be absolutely pure to give crystals, crystals from
impure solutions, however, frequently give poor diffraction data. To get good quality
crystals, proteins need not only to be reasonable free of contaminating proteins but
“conformationally pure”. Denatured proteins are more disruptive to crystal growth than
unrelated proteins. An important purity issue is that all traces of protease must be
enzyme substrates. These stabilizes the quaternary structure of the protein and promote
lattice packing.
3. pH: The net charge on the surface of a protein is an important determinant of lattice
6. Nature of crystallizing agent (Precipitant): The most widely used precipitants are
ammonium sulphate and potassium phosphate. Other common ones are polyethylene
glycol (PEG) 6000, acetone and ethanol. The nature of the precipitant affects the kinetics
of approach to supersaturation. This approach is usually slower with PEG than with salts,
for instance.
• Structure determination
where large, high quality crystals are needed. Obtaining high quality diffractive crystals is
protein crystals are most often crystallized by small-scale microdiffusion methods. Very
The structural analysis is based on the principle that in a perfect crystal all the molecules
have the same conformation and orientation. When X-rays are focused through protein (or
any other pure substance) crystal, individual atoms diffract the rays. The number of
electrons in each atom determines the intensity of the scattering of X-rays and thus, the X-
rays striking particular atoms will all be diffracted in the same way. A diffraction pattern is
generated by the interference of individual X-rays. A series of diffraction patterns are taken
from several angles and these patterns represent the way atoms are arranged in the molecule
and can be used to determine the structure of the protein. There are currently more than
18000 protein structures in the Protein Data Bank (PDB) (Berman et al., 2000), which have
• Protein purification
crystallization is relatively rare even though the quality of the crystals is not crucial.
although the latter was commonly used earlier. However, protein purification by
crystallization has many advantages: high yield, high purity in one step, unlimited scale up
possibilities, and the product is highly concentrated protein crystal slurry ready for further
formulation. Some industrial proteins, like xylose isomerase (EC 5.3.1.5) (Visuri, 1987),
cellulase (EC 3.2.1.4) (Nilsson et al., 1998) and protease (Gros and Cunefare, 2001) have
• Medical applications
A majority of small molecular weight drugs are produced in crystalline form because of the
high storage stability, purity and reproducibility of the drug properties (Hancock and
Zografi, 1997). There are hundreds of macromolecular therapeutic agents used in clinical
crystallization as presented above; b) high stability of the protein product compared with
soluble or amorphous forms; c) crystals are the most concentrated form of proteins, which is
a benefit for storage, formulation and for drugs that are needed in high doses (e.g.,
antibiotics); d) the rate of crystal dissolution depends on the morphology, size and additives;
In many applications crystallized proteins are not suitable for use as such, as a result of their
fragility and solubility. In order to produce a crystalline protein matrix that is insoluble also
in other conditions than those used in crystallization, the crystals have to be chemically
protein crystals creates an active and microporous protein matrix that can be used in
Enzymes are obtained from plants, animals and from microorganisms as a result of
fermentation processes (Poutanen, 1997). In the presence of water proteins can undergo a
variety of chemical and physical degradation reactions (Manning et al., 1989) and one of the
ways to achieve long term stability is to dry protein formulations (Pikal, 1990). The final
in solution.
Since most enzymes are not stable in solution, drying is often used to improve the stability
during storage. Drying of enzymes may be performed according to the following methods
(Strumillo et al., 1991): spray drying, vacuum drying, heat pump drying and freeze drying.
The choice of drying method depends on the quality parameters of enzyme, production
output, end applications and the cost of drying. Although freeze drying is usually favoured
for drying of high – valued and thermally labile enzymes in small quantities, spray drying is
largely applied in large scale production of enzymes because of its much lower cost
(Yamamoto and Sano, 1992). The design of a proper drying process should guarantee a high
level retention of enzyme activity. Generally, enzyme activity after drying is a function of
the composition of the initial liquid to be dehydrated, the process parameters and the
physicochemical characteristics of the enzyme. So, the drying of each enzyme should be
industries for preparation and storage of many therapeutic proteins, as well as labile
The process consists of two major steps: freezing of a protein solution and drying of the
frozen solid under vacuum. The drying step is further divided into two phases: primary and
secondary drying. The primary drying removes the frozen water and the secondary drying
A glassy material is formed following crystallization of ice during the freeing process. As
water becomes ice, freeze concentration takes place and the uncrystallized mixture gets
concentrated and changes from a ‘viscous liquid’ to a ‘brittle glassy’ structure, i.e. the
material undergoes the so called “glass transition”. The temperature where this transition
occurs in the maximally freeze – concentrated mixture is known as Tg, termed as the ‘glass
transition of the maximally freeze concentrated solute’ (Levine and Slade, 1988). Thus, non
– crystallizing solutes, such as proteins, remain with glassy matrix and become kinetically
frozen in.
increased viscosity that helps to reduce the degree of conformational distortion due to
drying (Pikal, 1990). Also, the lower initial temperature keeps unwanted reactions between
Spray drying is a convective drying technique that uses hot air to transfer heat and remove
the water evaporated. It is a short time process in the range of few seconds and if process
conditions are optimized and stabilizers are added, it can be suitable even for heat sensitive
The process may be summarized in three phases: spray formation, drying and powder
separation. The well mixed co-current system with the air disperser is preferred to avoid
Design and choice of operating conditions for spray drying of heat – sensitive products, are
still largely empirical. Therefore, the prediction of activity retention via simulation is of
considerable importance for the drying of enzymes. To predict the degree of enzymes
inactivation during drying, the thermal inactivation kinetics determined at different water
content must be integrated with drying models (Banga and Singh, 1994; Liou et al., 1985).
Generally the inactivation process of enzymes is assumed to be a first order reaction (Luyben
et al., 1982; Yamamoto and Sano, 1992) and the dependence of rate constants on temperature
is described by the Arrhenius equation (Meerdink and Van’t Riet, 1991; Sadykov et al.,
1997).
Several authors have studied the simulation of enzymes degradation during drying.
A theoretical description of inactivation of enzymes during spray drying has been done based
on literature data concerning inactivation behaviour of several enzymes during the spray
single suspended droplet could be predicted on the basis of a model calculation which
included the inactivation rate constants, the water diffusivity and water activity as a function
of water content and temperature (Yamamoto and Sano, 1992). The model calculations and
the experimental results showed that lower temperature and small droplets allow higher
enzyme retention.
In a collaborative European research program, a model solution with sensitive tracers was
established to characterize and compare different spray driers with respect to their effect on
enzyme activity retention. Results indicated that α – amylase and peroxidase were stable
during spray drying while tyrosinase was significantly affected. Low outlet temperature was
Alkaline phosphatase activity, moisture content and bubble volume in spray dried condensed
milk were measured experimentally and compared with predictions from a mathematical
model, including bubble formation during spray – drying. Enzyme activity decreased with
• handle heat sensitive, non – heat sensitive and heat – resistant pumpable fluids as feed
• produce dry material of controllable particle size, shape, form, moisture content and
• handle wide range of production rates i.e. any individual capacity requirements can be
• provide extensive flexibility in spray dryer design, such as drying of aqueous feed stocks,
• product deposit on the drying chamber may lead to degraded product or even fire hazard.
Table 1.1 gives the summary of comparison among the spray drying and freeze drying
operations.
Table 1.1: Comparison among spray drying and freeze drying (Filkova et al., 2004)
Drying systems incorporating a dehumidification cycle, called heat pump dryer (HPD), have
been developed that not only accelerate the drying process and preserve the quality of the
product by drying at low temperature but also conserve energy of the drying processes. The
heat pump recovers the sensible and latent heats by condensing moisture from the drying air.
Consequently, partial vapour pressure in the drying air decreases which increases the driving
potential of evaporated moisture. The recovered heat is recycled back to the dryer by heating
There are several advantages as well as limitations associated with heat pump – assisted
dryers. Some of these can be offset by using hybrid drying technologies. Following are some
• Heat pump drying (HPD) offers one of the highest Specific Moisture Extraction Rate
(SMER) often in the range of 1.0 to 4.0, since heat can be recovered from the moisture –
laden air.
• Heat pump dryers can significantly improve product quality by drying at low
temperatures. At low temperatures, the drying potential of the air can be maintained by
auxiliary heating) and relative humidity 15 to 80% (with humidification system) can be
generated.
• Excellent control of environment for high value products and reduced electrical energy
• Higher initial capital cost and maintenance cost due to need to maintain compressor,
• Leakage of refrigerant.
crystallization or drying of plant protease named, Bromelain, from the fruit, stem and leaf
• To purify bromelain from fruit portion of pineapple plant (Ananas comosus) using
• To obtain purified bromelain in dry powder form using different drying techniques
• To see the effect of drying temperature on retention of enzyme activity and protein
1.5 REFERENCES
Arakawa, T., Prestrelski, S.J., Kenney, W.C., Carpenter, J.F. (1993). Factors affecting
short – term and long – term stabilities of proteins. Advance Drug Delivery Reviews, 10,
1 – 28.
Banga, J.R., Singh, R.P. (1994). Optimization of air drying of foods. Journal of Food
Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H.,
Shindyalov, I.N., Bourne, P.E. (2000). The protein data bank. Nucleic Acids Research,
Carpenter, J.F., Crowe, W.H. (1989). An infrared spectroscopic study of the interactions
Etzel, M.R., Suen, S.Y., Halverson, S.L., Budijono, S. (1996). Enzyme inactivation in a
droplet forming a bubble during drying. Journal of Food Engineering, 27, 17 – 34.
Gros, E.H., Cunefare, J.L. (2001). Crystalline protease and method for producing same,
US Patent 6207437.
Fikova, I., Huang, L.X., Mujumdar. A.S. (2004). Heat Pump – Assisted Drying, pp.215
– 256. In: Handbook of Industrial drying (Second Edition), A.S. Mujumdar (Ed.),
Kiang, C.S., Jon. C.K. (2004). Heat Pump Drying Systems. pp. 1103 – 1132. In:
Handbook of Industrial drying (Second Edition), A.S. Mujumdar (Ed.), Mercel Dekker
Jen, A., Merkle, H.P. (2001). Diamonds in the rough: protein crystals from a
Letters, 9, 21 – 63.
Liapis, A.I., Bruttini, R. (1995). Freeze – drying, pp.309 – 343. In: Handbook of
Industrial drying (Second Edition), A.S. Mujumdar (Ed.), Mercel Dekker Inc., New
109 – 116.
Luyben, K.Ch.A.M., Liou, J.K., Bruin, S. (1982). Enzyme degradation during drying.
Manning, M.C., Patel, K., Borchardt, R.T. (1989). Stability of protein pharmaceuticals.
Patent 5728559.
Pikal, M.J. (1990). Freeze – drying of proteins. Part I. Process design. BioPharm, 3 (8),
18–27.
Roy, J.J., Abraham, T.E. (2006). Preparation and characterization of cross – linked
biotechnological products, pp. 36 – 55. In: Drying 91, A.S. Mujumdar and I. Filkova
Visuri, K. (1987). Stable glucose isomerase concentrate and a process for the thereof.
US Patent 4699882.
Heinemann, USA.
Yamamoto, S., Sano, Y. (1992). Drying of enzymes: Enzyme retention during drying of
Bromelain – A
Literature Overview
Bromelain – A literature overview
The commercial exploitation of enzymes ranges from very high volume of low cost
enzymes for the industrial purpose such as detergent enzymes to highly purified low
volume high cost enzymes for medicinal and therapeutic purposes. The market is
generally divided as industrial enzymes, enzymes for medicinal use and enzymes for
analytical and diagnostic purposes. Although about 3000 enzymes have been isolated and
characterized and only 300 enzymes are commercially available. The vast majority of
enzymes used industrially are hydrolases. They constitute 85% of total enzymes,
whereas, remaining 15% being divided between oxidoreductases and isomerases. Of the
hydrolyze lipids (Fowler, 1988). The industrial enzyme market consists of various
Among the hydrolases, proteases are the single class of enzymes which occupy a pivotal
position with respect to their applications in both physiological and commercial fields.
Proteolytic enzymes catalyze the cleavage of peptide bonds in other proteins. Proteases
are degradative enzymes which catalyze the total hydrolysis of proteins. Advances in
analytical techniques have demonstrated that proteases conduct highly specific and
selective modifications of proteins such as blood clotting and lysis of fibrin clots and
Glucoamylase 8-10
Pectinase 4-5
Lipase 2-3
Their involvement in the life cycle of disease-causing organisms has led them to become
a potential target for developing therapeutic agents against fatal diseases such as cancer
and AIDS. Proteases have a long history of application in the food and detergent
industries. Their application in the leather industry for dehairing and bating of hides to
substitute currently used toxic chemicals is a relatively new development and has
to the specificity of their action, has attracted worldwide attention in attempts to exploit
proteases has increased with the realization that they play key roles in rheumatoid
Since proteases are physiologically necessary for living organisms, they are ubiquitous,
being found in a wide diversity of sources such as plants, animals, and microorganisms.
The most familiar proteases of animal origin are pancreatic trypsin, chymotrypsin, pepsin
and rennin. The inability of the plant and animal proteases to meet current world
represent an excellent source of enzymes owing to their broad biochemical diversity and
approximately 40% of the total worldwide enzyme sales. Proteases from microbial
sources are preferred to the enzymes from plant and animal sources since they possess
almost all the characteristics desired for their biotechnological applications. Most
commercial proteases, mainly neutral and alkaline are produced by organisms belonging
The use of plants as a source of proteases is governed by several factors such as the
availability of land for cultivation and the suitability of climatic conditions for growth.
bromelain, keratinases and ficin represent some of the well-known proteases of plant
origin. Among the plant enzymes, papain is the single industrial product available in
large quantity.
and Molecular Biology, proteases are grossly subdivided into two major groups, i.e.,
cleave the peptide bond proximal to the amino or carboxy termini of the substrate,
whereas endopeptidases cleave peptide bonds distant from the termini of the substrate.
Based on the functional group present at the active site, proteases are further classified
into four prominent groups, i.e., Serine proteases, Aspartic proteases, Cysteine proteases,
and Metalloproteases.
As the present work deals with one of the medicinally as well as industrially important
plant cysteine protease named as bromelain (from the pineapple fruit), the further
cysteine proteases have been recognized. Distribution of cysteine proteases is very wide
in both plant and animal kingdoms, ranging from the bacteria (peptidase-B of
Clostridium) through many higher plant families: genera and species of these include
Carica papaya, the tropical paw-paw, source of papain; Bromelia penguin, source of
penguinain; Asclepia, milkweeds containing asclapain in stem and roots; Ficus, tropical
“fig” trees of several species, the source of ficins and Ananas comosus (Pineapple the
proteinase from bacteria are reasonably similar. Each of these several proteases differ in
degree relative to composition and activity: examples are the bromelains, papain and the
ficins, which contrast greatly in origin but nevertheless shows remarkable homology in
the amino acid sequences around the reactive, essential cysteine site of each (Murachi,
1964). It should be noted, however, that not all plant proteases are sulfhydryl enzymes.
Thus, solanain (from the berries of the horsenettle), arachain (from the cotyledon and
embryo of the peanut) do not require activation by sulfhydryl reagents and are not
affected by mild oxidizing agents. Several mammalian lysosomal cathepsins, and the
Trypanosoma, Schistosoma) also belong to this class (Drenth et al., 1971). The activity of
all cysteine proteases depends on a catalytic dyad consisting of cysteine and histidine.
The order of Cys and His (Cys-His or His-Cys) residues differ among the families.
Generally, cysteine proteases are active only in the presence of reducing agents such as
protease. Cysteine proteases have neutral pH optima, although a few of them, e.g.,
lysosomal proteases, are maximally active at acidic pH. They are susceptible to
sulfhydryl agents such as Para Chloro Mercury Benzoate (PCMB) but are unaffected by
(Di isopropyl Fluoro Phosphate (DFP) and metal – chelating agents. Papain was the first
recognized member of the class of proteolytic enzymes (cysteine proteases) that need free
sulfhydryl group for activity. These enzymes usually need an activator for activity which
has a function of releasing the blocked SH groups (Drenth et al., 1971). Like the serine
involves a cysteine and a histidine residue. The essential Cys25 and His159 (papain
numbering) play the same role as Ser195 and His57 respectively. The detail mechanism
of action for cysteine proteases is described below (Rao et al., 1998). All these plant
proteases have broader specificities than trypsin but peptides of positively charged amino
The mechanism of action of proteases has been a subject of great interest to researchers.
well documented.
similar to that of serine proteases. A striking similarity is also observed in the reaction
mechanism for several peptidases of different evolutionary origins. The plant peptidase
papain can be considered the archetype of cysteine peptidases and constitutes a good
model for this family of enzymes. They catalyze the hydrolysis of peptide, amide ester,
thiol ester, and thiono ester bonds. The initial step in the catalytic process (Figure 1)
involves the noncovalent binding of the free enzyme and the substrate to form the
complex. This is followed by the acylation of the enzyme, with the formation and release
of the first product, the amine. In the next deacylation step, the acyl-enzyme reacts with a
water molecule to release the second product, with the regeneration of free enzyme. The
2.5. BROMELAINS
The bromelains are proteases occurring as glycoproteins from the pineapple plant,
a perennial herb native to tropical America. Principal sources today include Hawaii,
Japan and Taiwan. The existence of bromelain was probably first established in 1891 by
Chittenden (Chittenden, 1891) who salted it out of the juice and studied its action in
considerable detail. Later on Heinicke and Gortner (1957) showed that the juice of the
stem of the pineapple plant is a rich source of proteolytic enzymes. The properties of the
bromelain lead to conclusion that it is similar to papain (Balls et al., 1941). Bromelain is
(Murachi et al., 1964). In 1950, the Pineapple Research Institute of Hawaii began a study
of the proteases of the pineapple plant. They found that not only all varieties of
commercial pineapples contain proteases, but that probably all species of genera of the
Furthermore, they found that the proteases of the fruit, the leaves, and the stems
represented different mixtures of proteases. To avoid coining, several hundred new plant
protease names based on species name, Heinicke and Gortner (1957) suggested that the
name bromelin replace by bromelain and be the generic name for any protease obtained
from any member of the family Bromeliaceae. They further suggested that the individual
preparations be distinguished by prefixing the binomial latin name of the plant source and
the organ yielding the enzyme. Thus the name for the proteases from the fruit of
commercial pineapple plant would be as Ananas comosus (L.) Merr. Var. Cayenne fruit
bromelain. Similarly the enzyme from stem part is Ananas comosus (L.) Merr. Var.
The Pernambuco variety of pineapple appears to be richer in enzyme than the Cayenne,
but the latter is the only variety in industrial use. The bromelain content of pineapple
plants is as shown in Table 2.2. Bromelain is well distributed all over in the pineapple
plant. The enzyme bromelain is present in the leaves and stalks as well as in the fruit. The
enzyme from fruit part was first described as bromelain and now called as Fruit
Bromelain (EC 3.4.22.33) and that from stalk (stem) part is designated as Stem
Bromelain (EC 3.4.22.32). It is evident that the bromelain follows the juice, not the solid
matter. The enzyme is remarkably stable toward heat, as shown by the fact that the press
juice heated to 600C (less stable than papain) and screened thereafter still contained a
large proportion of the original enzyme in the active state (Balls et al., 1941).
The juice of the pineapple contains various amounts of polysaccharides and polyuronides.
These may range from 30 – 70%. The polyuronides are partially responsible for the high
viscosity of fruit juice. Furthermore, when present in the enzyme preparations, they
account for some of the strong binding of inorganic cations (Collins, 1960).
Table 2.2: Bromelain content of pineapple plants (press juice from hand press)
(Balls et al., 1941)
Milk units/ml
Variety Material % juice Milk units/ml
juice(not
obtained juice(activated)
activated)
Cayenne
Pernambuco
Different enzymes from the pineapple juice have been reported. About half of the protein
in pineapple flesh is accounted by the protease bromelain. The enzyme is not present at
all during the early stages of fruit development and then increases very rapidly and stays
Pineapple is unique among fruits in having high concentration of protease in the ripe
fruit. Unlike papain, bromelain doesn’t disappears as fruit ripens (Table 2.2). Papain has
high levels of enzyme in the green stage, but become completely inactive when the fruit
Other non proteolytic enzymes reported from pineapple fruit are IAA oxidase,
peroxidase, phosphatase, catalase, and cellulase (Dull, 1971). The other proteolytic
The economic advantages in the manufacture of bromelain over papain are apparent.
Since, the fruit part is the more valuable part of plant, the enzyme is produced from the
parts of the plant unusable for food and thus, enzyme is usually a byproduct from
pineapple industry. The waste parts, leaves, cores (stems) and the skin of the fruit are
another industry and always obtained from a single variety of the plant, it would therefore
be unusually uniform. The quantity made could be easily adjusted to suit the market, and
manufacture would require very little addition to the existing facilities for pineapple
papain. The material used must be factory waste, since other pineapple products are more
valuable. The amount of enzyme present is small, and it would not pay to destroy the
sugar or citric acid even in the waste in order to get the bromelain (Balls et al., 1941).
Two kinds of bromelain are available commercially, stem bromelain and fruit bromelain.
Commercial bromelains are slightly soluble in water and glycerol but insoluble in most
depending upon the protein it is acting upon. It is most active at temperature range of 50-
2.5.2.1. Stability:
The enzyme retains full activity against casein when kept at 50C for 24 hours over a range
of pH from 4 to 10 (Inagami and Murachi, 1963). The enzyme is stable in 25% (v/v)
methanol at 250C for 20 minutes, while it looses 33% caseinolytic activity in 20% (v/v)
ethanol at 370C for 20 minutes. A 50% loss of the activity is caused by heating the
enzyme solution at 550C for 20 minutes at pH 6.1 (El-Gharbawi and Whitaker, 1963).
There is still uncertainty about the exact molecular weight of stem bromelain, as it was
found from literature that different author has given a different molecular weight for their
molecular weight has been done by polyacrylamide gel electrophoresis in the presence of
(1973). Measured values range from 25,600 to 28,100 Dalton and in practice it is
Physical properties of bromelain protein reported by Yamada et al. (1976) are listed in
Table 2.3.
of 18,000 as determined by Sephadex G-75 gel filtration was reported by Ota et al.
(1972) and 31,000 by polyacrylamide gel electrophoresis in the presence of SDS and by
Murachi et al. (1964) have found the isoelectric point of their bromelain preparation to be
at about pH 9.5 by isoelectric focusing technique. Wharton (1974) also gave an evidence
of the alkaline isoelectric point of stem bromelain. At pH values of 4.0, 7.0, and 9.0,
migrations takes place towards cathode and at pH 9.5, it was slightly towards anode
lower. Yamada et al. (1976) reported an isoelectric point of pH 4.6 for their purified fruit
In Table 2.3, the amino acid composition of stem bromelain reported by different
investigators (Feinstein and Whitaker, 1964; Murachi, 1964; Husain and Lowe, 1968) is
shown. The stem enzyme is basic and amino acid analysis shows a relatively high content
of lysine and arginine as compared to amino acid composition of fruit bromelain (Ota et
al., 1964). The principal amino terminal residue is valine (Takahashi et al., 1973) and the
Stem bromelain contains four methionine residues per molecule; whereas, no methionine
is present in papain (Murachi, 1964). The abundance of basic amino acids is more
apparent in stem bromelain than papain. This is in accord with the finding that stem
bromelain has an isoelectric point higher than that for papain, pH 8.75 (Smith et al.,
1954). Stem bromelain has only one cysteinyl and histidyl residue per molecule whereas
1 2 3 4 5 6
Lysine 14 17 17 12 5 8
Histidine 1 1 1 1 1 1
Arginine 8 8 9 6 5 9
Aspartic acid 23 23 23 16 17 32
Threonine 10 10 11 8 8 14
Serine 21 21 22 16 18 30
Glutamic acid 19 17 18 12 13 25
Proline 11 13 11 8 7 10
Glycine 27 24 26 19 18 35
Alanine 30 26 28 20 14 25
Half-cystine 9 9 7 5 6 9
Valine 16 16 18 12 11 20
Methionine 3 3 4 2 3 5
Isoleucine 17 17 18 12 9 10
Leucine 7 8 8 5 4 10
Tyrosine 14 16 16 11 13 16
Phenylalanine 8 8 7 5 4 8
Tryptophan 7 7 6 5 3 7
Total (245) (244) (250) (179) (161) (282)
Ammonia (amide) -- 21 28 19 24 --
Glucosamine 2 2 2 4 <0.2 0
Carbohydrate (%) 2.1 2.1 0.9 2.0 3.2 0
Column 1: (Takahashi et al., 1973). Nearest integral number of residues per mole of
28,000 for component SB 1. Column 2: (Murachi, 1964) Number of residues per mole of
MW 33,000 reported earlier for step 6 preparation has been recalculated on the basis of
MW of 28,000. Column 3: (Ota et al., 1972). Number of residues per mole of 28,000 for
component I-1. Column 4: (Feinstein and Whitaker, 1964) for component II, taken
methionine as two residues per molecule. Column 5: (Ota et al., 1972). Number of
residues per mole of 18,000 for component A. Column 6: (Yamada et al., 1976). Nearest
The amino acid composition of fruit enzyme is similar to stem enzyme with the notable
exception that it contains much less lysine, and smaller alanine content relative to glycine
(Yamada et al. 1976). This difference is reflected by in the isoelectric points of the two
proteins (Ota, 1966). The amino terminal residue is alanine (Yamada et al., 1976).
Three separate laboratories studied that the stem bromelain contained a small amount of
carbohydrate. Murachi et al. (1964) were first to observe that stem enzyme appears to be
whereas Ota et al. (1964) reported 1.46% carbohydrate. Feinstein and Whitaker (1964)
moles of carbohydrate per mole of purified enzyme. Scocca and Lee (1969) have reported
the same oligosaccharide content of 2:1:1:2 of mannose, fucose, xylose and glucosamine
respectively for their purified fractions bromelain II and bromelain III which are
while among the proteolytic enzymes from animal origin plasmin and enterokinase have
been reported to contain 1.5% hexose and 41.1% carbohydrate, respectively (Murachi,
bromelain with Pronase, contained mannose, fucose, xylose, and glucosamine in the ratio
3:1:1:4.
al. (1976) reported that fruit bromelain (purified by them called FA2) contained neither
amino sugar nor neutral carbohydrates determined by four different methods and stated
that FA2 is not a glycoprotein. While, Ota et al. (1964) reported that their purified fruit
The crude bromelain enzyme shows about 25% of their maximum activity against casein
without any addition of activating agent (Ota et al., 1964). The enzyme can be fully
(Murachi, 1970). Ota et al. (1964) suggested the use of mercaptoethanol or cysteine plus
EDTA for the maximum proteolytic activity. But, Murachi and Neurath (1960) reported
that the addition of EDTA besides cysteine was not essential for full activity. Cysteine
and cyanide activate the enzyme to about the same degree, while H2S and Na2S produce a
much lower degree of activation. The reason behind the low activation by sulfides is
might be that the oxidized sulfhydryl groups of bromelain are not readily accessible by
Stem bromelain as well as fruit bromelain are reversibly inhibited by inorganic mercuric
ion, organic mercurials, and tetrathionate. Murachi and Neurath (1960) gave evidence
that the inhibition by mercuric ion was instantaneous and could be completely reversed
by the addition of excess cysteine, suggesting that the sulfhydryl groups of the enzyme
protein are essential for its catalytic activity. Irreversible inactivation occurs when stem
alkylate the essential sulfhydryl group of the enzyme protein. Chloromethyl ketone
heptanone (TLCK) also alkylate the –SH group, resulting in inactivation of the enzyme.
activity (Murachi, 1970). Murachi and Neurath. (1960) also reported that 0.001M DFP in
propanol neither inhibits the enzyme nor does it change its activation by cysteine
suggesting that, in contrast to other endopeptidases, no reaction with active site had
occurred. The results of them are in agreement with those reported by Heinicke, who
The specificity of the stem bromelain has been examined on a number of substrates. It
hydrolyses proteins like casein and haemoglobin at high rates, while a synthetic
substrates of smaller molecular size, like Benzoyl Arginine Ethyl Ester (BAEE) has also
shown to be hydrolyzed at moderate rate (Inagami and Murachi, 1963). Although the
differences have also been observed. In comparison with papain, the pineapple enzyme
hydrolyzed casein equally well; haemoglobin is hydrolyzed four times faster and BAEE
and Benzoyl Argininamide (BAA) much more slowly (Murachi and Neurath, 1960;
Basic amino acyl residues are preferred, but the preference is less strict than in the case of
papain (Murachi, 1970). A preliminary experiment carried out by the Inagami and
Murachi (1963) indicates that arginine derivatives like BAEE and BAA are the best
substrates among various amino acid esters and amides, respectively (Table 2.4). Among
the three substrates (esters of the L-arginine derivatives) examined by Inagami and
Murachi (1963).
BAA 0.040 10
The fruit enzyme is more active against BAA and BAEE than the stem enzyme (Ota et
al., 1964). The pH optima for casein, denatured hemoglobin are at 8.3 and pH 8.0,
respectively.
Bromelain is said to possess wide variety of uses both in industrial purpose as well as
medicinal purpose. Industrial uses include as detergents; for dehairing and tanning in
leather industries; cleaning agent; for processing of raw silk and spot remover in textile
Proteases from plant origin such as papain and bromelain are used frequently
Bromelain has been proved to be having wide range of biological activities, including
actions. Evidence indicates that bromelain's action is in part a result of inhibiting the
generation of bradykinin at the inflammatory site via depletion of the plasma kallikrein
percent of the high molecular weight substances detected in the blood after oral
administration. The highest concentration of bromelain is found in the blood one hour
Bromelain is generally considered safe, even at high doses. Bromelain can cause an
allergic reaction (red or itchy eyes, sneezing, running nose, irritated throat) in people who
Food Uses
commodity
Beer Chill-proof; improve flavor; retain foam
Bread dough Reduce kneading time; baked goods; increase fluidity of glutin
gels
Eggs yolks Partial digestion to prevent gel formation in frozen state
Fats Deodorize
Fish Solubilize the protein
Meat pickling Improved corned meat products
Meat smoking Release of casings after processing
Meat Antimortem injections
tenderizing
2.6 REFERENCES
Balls, A.K., Thompson, R.R., Kies, M.W. (1941). Bromelain: Properties and Commercial
Chittenden, R.H. (1891). On the proteolytic action of bromelin, the ferment of pineapple
Collins, J.L. (1960). “The Pineapple”, Interscience Publishers Inc., New York, p. 253 –
254.
Drenth, J.N. Jansonius, J.N. Koekoek, R., Wolthers, D.G. (1971). “Papain, X-ray
Structure”, in The Enzymes, Ed.-P.D. Boyer, Edition-III, Vol.3, Academic Press, New
Dull, G.G. (1971). “The Pineapple: General”, in The Biochemistry of Fruits and their
El-Gharbawi, M., Whitaker, J.R. (1963). Fractionation and partial characterization of the
Feinstein, G., Whitaker, J.R. (1964). Molecular weights of the proteolytic enzymes of
Felton, G.E. (1971). “Pineapple Juice”, in Fruit and vegetable juice processing
technology, Edition-II, Eds-Tressler, D.K. and Joslyn, M.A., The Avi Publishing
Heinicke, R.M., Gortner, W.A. (1957). Stem Bromelain – A new protease preparation
Hussain, S.S., Lowe, G. (1968). Amino acid sequence around the active site cysteine and
M.S. and Johnson, A.H., The Avi Publishing Company Inc., Westport Connecticut, p. 94
– 97.
Murachi, T. (1964). Amino acid composition of stem bromelain. Biochemistry, 3 (7), 932
– 934.
G.E., and Lovand, L., Vol. 19, Academic Press, New York, p. 273 – 285.
G.E. And Lorand, L., Vol. 45, Academic Press, New York, p. 475 – 485.
Murachi, T., Neurath, H. (1960). Fractionation and specificity studies on stem bromelain.
Murachi, T., Suzuki, A., Takahashi, N. (1967). Evidence for glycoprotein nature of stem
Murachi, T., Yasui, M., Yasuda, Y. (1964). Purification and physical characterization of
Ota, S., Horie, K., Hagino, F., Hashimoto, C., Date, H. (1972). Fractionation and some
properties of the proteolytically active components of bromelains in the stem and the fruit
Ota, S., Moore, S., Stein, W. (1964). Preparation and chemical properties of purified stem
Rao, M.B., Tanksale, A.M., Ghatge, M.S., Deshpande, V.V. (1998). Molecular and
Reed, G. (1966). “Enzymes in Food Processing”, Academic Press New York, p. 128 –
130.
Scocca, J., Lee, Y.C. (1969). The composition and structure of the carbohydrate of
Smith, E.L., Kimmel, J.R. and Brown, D.M. (1954). “Crystalline Papain II”, Journal of
Takahashi, N., Yasuda, Y., Goto, K., Miyake, T., Murachi, T. (1973). Multiple molecular
Wharton, C.W. (1974). The structure and mechanism of stem bromelain. Biochemistry
Fresh pineapple fruits were purchased from a local supermarket (Mumbai, India). L –
Cysteine for biochemistry and Casein (acc. to Hammarsten) for biochemistry were obtained
from Sisco Research Laboratories (SRL) Pvt. Ltd., India. Trichloroacetic acid (TCA) was
purchased from S.D. Fine Chemicals Ltd. Mumbai. All other chemicals used were of
analytical grade and purchased from S.D. Fine Chemicals Ltd., Mumbai. Standard purified
bromelain sample was obtained from Hong Mao Biochemical Co. Ltd. Thailand. Bovine
Serum Albumin (BSA) and Tyrosine were purchased from Hi – Media Laboratory.
Dialysis Membrane having molecular weight cut off (MWCO) 12000 was also purchased
from Hi – Media Laboratory. Ion exchange resins were made available from Resindion
Bromelain when assayed for proteolytic activity against casein shows specificity similar to
that of papain. The assay was based on the estimation of the amount of small molecular
weight digestion products (Trichloroacetic acid (TCA) soluble material) formed from
proteins due to proteolytic action of the enzyme (Arnon and Shapira, 1967).
A stock solution of 1 mg/ml in dilute HCl of Tyrosine was prepared. Serial dilutions of this
stock solution were prepared by using dilute HCl so as to obtain the solutions of different
concentrations of tyrosine ranging from 31.5 µgm/ml to 125 µgm/ml. The absorbance of
this prepared solution was measured at 280 nm using Chemito 2500 UV-VIS
(μgm/ml) was plotted and is shown in Figure 3.1, which is used as a standard.
0.8
Absorbance y = 0.0061x
0.6
R 2 = 0.9996
0.4
0.2
0
0 50 100 150 200
Conc. of Tyrosine (ugm/ml)
Figure 3.1 Standard plot for enzyme assay using tyrosine as a standard.
(1976).
Phosphate buffer and the pH was adjusted to 7 using 0.1 N HCl (slow addition) to avoid
destruction of matrix and was brought to 37 ºC by pre incubation for 10 min. To this
substrate a known volume of enzyme was added after diluting it to 1 ml with activating
used as activating buffer. Casein proteolysis was stopped after 10 min by addition of 5 ml
of TCA (Trichloroacetic acid) stock solution. 0.11 M Tri Chloro acetic acid and 0.22 M
sodium acetate prepared in 0.33 M acetic acid was used as a TCA stock solution. The
mixture was allowed to stand for 30 min at 37 ºC after addition of TCA. After cooling to
room temperature, the solution was filtered twice through Whatman No. 42 filter paper.
Spectrophotometer.
“One unit of bromelain was taken as the amount of enzyme which while acting on the
casein substrate under specified conditions, produced one microgram of tyrosine per
of protein estimation (Smith et al., 1985). Sigma Bicinchoninic Acid Protein Assay Kit was
used for assay purpose. The principle of the bicinchoninic acid (BCA) assay is similar to
the Lowry (Lowry et al., 1951) procedure, in that both rely on the formation of a Cu2+
protein complex under alkaline conditions, followed by reduction of the Cu2+ to Cu1+. The
complex with Cu1+ in alkaline environments, thus providing a basis to monitor the
reduction of alkaline Cu2+ by proteins. To determine the protein concentration, 200 µl BCA
working Reagent was added to 25 µl of suitably diluted protein. The protein assay
containers were sealed with film and incubated at room temperature for 2 hours. The color
developed was measured at 565 nm using micro plate reader (Model 680, BioRad) after 2
hours. Bovine serum albumin (BSA) was used as the standard for protein assay. A stock
solution of 0.1 mg/ml BSA was prepared. Solutions of different concentrations were
prepared by diluting it with distilled water from this stock solution. The assay of these
prepared samples were carried out by the method mentioned above. A plot of absorbance at
565 nm versus BSA protein concentrations in micrograms per ml was plotted and used as a
0.8
0.7
Absorbance 0.6
0.5
0.4 y = 0.0007x
R2 = 0.9943
0.3
0.2
0.1
0
0 200 400 600 800 1000 1200
Conc (ugm/ml)
Figure 3.2 Standard plot for Protein estimation using BSA as a standard.
Crude juice, chromatographic fractions and dried samples were assayed for reducing sugars
by DNSA (Dinitrosalicylic acid) method (Miller, 1959). To determine the total reducing
sugar, 1 ml of the diluted sample was treated with 1 ml of DNSA reagent and the mixture
was kept for 10 minutes in boiling water bath. The resultant mixture was then cooled and
final volume was made up to 12 ml with the help of distilled water. The absorbance of this
Glucose was used as a standard for reducing sugar assay. Solutions of different
concentrations were prepared by diluting it with distilled water from this stock solution. A
plot of absorbance at 540 nm versus glucose concentrations (mg/ml) was plotted and used
0.4
0.35
0.3
Absorbance
0.25
0.2
0.15 y = 0.3503x
R2 = 0.9996
0.1
0.05
0
0 0.2 0.4 0.6 0.8 1 1.2
Figure 3.3 Standard plot for sugar estimation using glucose as a standard.
The Freeze dried, spray dried, heat pump dried and crystallized samples were subjected to
analyze water activity in a water activity meter (M/S Aqua Lab) in order to estimate free
moisture. The water activity was calculated at room temperature. The effect of temperature
p …………….(3.1)
a =
w p
w
Where,
Moisture contents of crude juice, unbound fraction, washout and elute were analyzed using
loss on drying (LOD) method. 10 ml of sample in petri plates was subjected to vacuum
drying at a temperature of 50 ºC. Drying was carried out for sufficient time (approximately
Residual moisture content of dried formulations was measured with the Karl-Fisher method
(MacLeod, 1991) on Microcontroller based Digital Karl Fischer (Model No. MI 453, M/s
Polmon Instruments Pvt. Ltd., India). At least 100 mg of powder was mixed with dry
methanol and titrated with Karl Fisher reagent untill the end point was reached. The
samples were dispersed in methanol and the water content was determined. Methanol was
used as blank. The water content of every formulation was given as the average of
Thermal analysis of freeze dried and spray dried powders was performed using differential
scanning calorimeter (DSC) (Perkin Elmer Pyris – 6) Prior to measurement, the dried
samples were transferred into vacuum desiccators and equilibrated for 3 – 4 days over P2O5
for ‘zero’ moisture content. Approximately 2 – 10 mg of powder was used. The sample
was sealed in an aluminium pan and an empty pan was used as a reference. Dried sample
were heated in the range of 40 to 300 ºC. Heating rate of 10 ºC/min was used. All glass
transition temperatures were reported as the midpoint temperature of the heat capacity step
Fourier transform infrared (FTIR) spectra were measured utilizing a Perkin Elmer 1600
series FTIR, and analyzed using a PE-GRAMS/32 1600 software, as described previously
(Liao et al., 2004). Briefly, a dry protein sample (approximately 0.5 mg protein) was mixed
with about 300 mg ground potassium bromide and compressed into 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 the α –
The stalk (central core) of the pineapple fruit and the leaves were separated from fleshy
fruit part of pineapple. The fruit portion was then cut into small pieces and blended in
mixer. Approximately 400 – 500 ml of juice was prepared from one pineapple fruit. This
juice was filtered through a muslin cloth to remove the fibrous material. The resultant juice
still contained ruptured plant cells so in order to remove that it was subjected to filtration
under vacuum. The clarified crude juice was also subjected to centrifugation on a Remi R –
24 research centrifuge for twenty minutes at low temperature and 10,000×g. The clear
supernatant obtained as a clarified extract was stored in aliquots at 4 °C and used for
crystallization study. Each batch of bromelain extract from the pineapple gave different
bromelain content in the range of 1000 – 1500 casein hydrolyzing units per ml or 70,000 to
Crystallization of clarified juice was carried out by using different precipitants such as
The following is a preliminary description of the procedure which was found to yield crude
crystalline material.
A cryostat bath (M/s Asha Scientific Company, Mumbai) containing water was maintained
at 4 ºC and the sample undergoing fractionation was chilled upto 4 ºC. The protein
concentration was in the range of 15 – 20 mg/ml. Slow addition of precipitating agent with
efficient cooling and under constant stirring was carried out. After the last bit of salt was
dissolved, stirring was continued for 10 – 30 min to allow complete equilibration between
dissolved and aggregated proteins. Then, the solution was centrifuged (Remi R – 24
research centrifuge) at 10000×g for 10 to 15 min and the precipitate was collected. The
supernatant was decanted, its volume noted and the amount of salt required for the next cut
was calculated. The precipitate was dissolved in minimum quantity of suitable buffer and
For ammonium sulfate precipitation, the amount of ammonium sulfate added to the
533 (S − S )
g= 2 1 …………….(3.2)
100 − 0.3 S
2
Where,
Crystallization using acetone as a precipitating agent was carried out using a method
A 20 g of fresh tissue (callas or leaves) was homogenized in a mixer with 150 ml of 0.05 M
potassium phosphate buffer (pH 6.1). The suspension was further homogenized using a
homogenizer to ensure maximum cell disruption. The suspension obtained from leaves was
passed through a muslin cloth prior to the second homogenization to eliminate fibres. The
homogenate was centrifuged at 10000 rpm for 15 min in a Remi R – 24 research centrifuge
using SS – 34 rotor at 0 – 5 °C. The supernatant was taken for further purification.
80% fraction gives maximum enzyme activity. The supernatant was brought to 40%
saturation by the addition of pre – chilled acetone at 4 °C. The precipitate isolated by
centrifugation at 10000 rpm for 10 min was discarded. The supernatant was brought to
80% saturation and the precipitate obtained after centrifugation as above was collected and
A formula for calculating the amount of organic solvent to be added is given as:
v =1000 (y − x) …………….(3.3)
100 − y
Where,
x = initial % saturation,
y = final % saturation.
content was studied. Dialysis of the precipitate was also carried out.
bromelain with ammonium sulfate and sodium chloride (Murachi and Neurath, 1960).
50 ml of Stem bromelain juice after adjusting to pH 7.5 with 1 N NaOH was centrifuged
for 30 minutes at 14,000 rpm. To the supernatant solution (50 ml), 12.5 g of ammonium
sulfate of pH 7.5 and room temperature was added and after 1 hour the mixture was
centrifuged. The precipitate was washed with a solution of 6.25 g of ammonium sulfate in
25 ml of water and the washed precipitate was dissolved in water to make a solution of 25
ml. At pH 7.5, 2.25 g of ammonium sulfate was added and the precipitate was collected by
30 g of NaCl, washed with saturated NaCl solution, and then dissolved in 400 ml of 80%
saturated sodium chloride solution by adjusting to pH 9.0 with 1N NaOH. When 0.1 N
acetic acid was added slowly to lower the pH to approximately 8, a faint turbidity due to
crystalline material appeared which did not increase substantially by standing but could be
increased by further cautious addition of acetic acid to a yield of about 5% of the starting
material. A similar type of crude crystalline material was also obtained when stem
bromelain was treated in the same manner except that 0.01 M cysteine was employed
throughout the procedure instead of converting the enzyme into the mercury derivative.
To remove the salt from the precipitated sample it was further dialyzed against 50 mM
ammonium sulfate or phosphate buffer, pH 7.0 as a process for buffer exchange. The
dialysis was carried out by using the dialysis membrane available from Hi-Media
(Mumbai, India) Laboratory having MWCO (Molecular Weight Cut Off) of 12,000. The
enzyme activity units and protein content after dialysis were checked for the percentage
column dimensions were 30 cm (L) x 2.5 cm (D). Acetate buffer was used as an
equilibrating buffer and for removal of unbound or weakly bound protein, sodium chloride
(1N) solution was used as elution buffer. The column was regenerated using 0.5N NaOH
solution.
Ion exchange resins (cation) were packed in a glass column of 25 mm internal diameter 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. This was followed by the wash of equilibration buffer till no protein was
India). Finally the bound enzyme (protein) was eluted using 1N sodium chloride solution.
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
carried out by using the methods mentioned in previous section to calculate the total
The purified sample was subjected to drying. Three different types of dryers were used.
A laboratory freeze dryer (M/s Ref-vac Consultancy, India) having condenser ice collection
capacity of 1.8 kg and temperature of –35 ºC was used. The drying chamber (0.4 m (L) ×
0.4 m (D)) was equipped with heating plates. 20 ml of purified Bromelain sample was
filled manually into the petri plate. An electronic weighing balance was kept inside the
drying chamber for online sample LOD measurement in order to study the drying kinetics.
The freezing step was performed by placing the petri plate in a deep freezer for 6 hr. at -23
ºC. Then, the petri plate was transferred into the drying chamber which was kept at room
temperature. The product temperature was recorded using thermocouple, which was placed
on the surface of the product. Pressure inside the chamber was recorded at different time
intervals. The shelf temperature was kept at 30 ºC and pressure inside the chamber was
kept at 24 Pa.
The powder obtained was dissolved in distilled water at proper dilution and was further
analyzed for activity retention and residual protein content. The activity retention and
protein retention were calculated on the basis of solid content. Activity of the freeze dried
samples was then expressed as a percentage of the activity of the initial sample.
T1 From Atm.
HOT AIR H1
F
To Atm
P
COMP V
AIR
ASP
T2
H2 DC
SC
CY-1 CY-2
C1
FEED C2 C3
AG
SC: Scrubber
ASP: Aspirator
F: Filter
P: Pressure regulator
V: Vacuum gauge
AG: Agitator
F: Feed Pump
A laboratory spray dryer (LU-222, M/s Labultima, India) of 1 lit/hr evaporation capacity
was used. The dimensions of the drying chamber were 0.4 m (L) × 0.1 m (D). The spray
dryer operates in a co-current manner and has a two fluid spray nozzle with an orifice of
0.7 mm in diameter. The spray dryer was equipped with two cyclone collector for
collection of product. The air used for the heating purpose was passes through HEPA
filters.
In the present work, two different set of operating parameters were used.
• In order to study the effect of air inlet temperature on the enzyme activity retention, the
inlet air temperature was varied in the range of 150 – 200 ºC while the outlet air
temperature was maintained at 40 ºC by varying the feed flow rate. The flow rate of
• In the second set of operating parameters, the effect of outlet air temperature on
enzyme activity retention was studied. In this case, inlet air temperature was kept at 160
ºC and the feed flow rate was varied in such a way that the outlet temperature was
varied in the range of 35 – 65 ºC. The flow rate of drying air was kept at 41.3 Nm3/h.
Effect of inlet and outlet air temperature on enzyme inactivation kinetics was studied.
Every experiment was carried out in duplicates, without any additive and in second,
Maltodextrin was used as an additive. However, the powder obtained was hygroscopic and
activity loss was high and hence, no further experiments were carried out with
Maltodextrin.
Drying Ratio: Drying ratio was equal to powder solid content divided by feed solid
content.
Productivity: Productivity (kg/h) was equal to feed rate divided by drying ratio.
Drying Rate: Drying rate (kg/h) was equal to productivity subtracted from feed flow rate.
Figure 3.6 shows an open loop heat pump drying system (HPD) used in the present study.
R134a was used as a refrigerant. The system used for the study consisted of heat bypass
coil for improving the performance of heat pump and hence, the specific moisture removal
rate in HPD. The heat pump system comprised of a 1.5 kWh heat load providing the
dehumidified air of 100 – 175 Nm3/hr. The temperature range obtained from this HP unit
was in between 30 – 45 °C and the corresponding relative humidity of air was in the range
of 10 – 20%.
The 20 ml of purified bromelain sample was dried in HPD for a period of 5 – 7 hrs. The
samples were collected after every 30 min time interval for determining its weight loss, in
Desuperheater Internal
Dryer Reboiler
Condenser
Compressor
Expansion Valve
External
Condenser
Precooler Evaporator
Ambient air
3.4. REFERENCES
Apte, P.V., Kaklij, G.S., Heble, M.R. (1979). Proteolytic enzymes (bromelains) in tissue
ASTM (1991). Standard test method for glass transition temperatures by differential
Dapeau, G.R. Methods in Enzymology, vol. XLV (Lorand, L., ed.) pg. 471, Academic
Liao, Y., Brown, M. B., Martin G. P. (2004). Investigation of the stabilisation of freeze-
dried lysozyme and the physical properties of the formulations. European Journal of
Lowry, O.H., Rosebrough, N.J., Lewis Farr, A., Randall, A. (1951). Protein measurement
with the Folin phenol reagent. Journal of Biological Chemistry 193, 265 – 275.
Miller, G.L. (1959). Use of dinitro salicylic acid reagent for determination of reducing
Murachi, T., Neurath, H. (1960). Fractionation and specificity studies on stem bromelain.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D.,
Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, O.C. (1985). Measurement of protein
Ammonium sulfate precipitation was performed as the first step of bromelain purification.
Ammonium sulfate fractions i.e. supernatant and precipitate were collected at levels of
saturation of 20% (w/v) and assayed for specific protease activity before and after dialysis.
Fractions collected at 40 – 60% and 60 – 80% ammonium sulfate were found to have the
highest percent of protease activity and specific protease activity, with a recovery of 79.4%,
and accounted for 34% of the total protein concentration. The enzyme activity and specific
enzyme activity were found to be very less in the fractions obtained at lower saturation that
means proteases were soluble in 35% of saturation of ammonium sulfate, but not in 60% of
saturation of ammonium sulfate. Many other proteins in the supernatant were not soluble in
35% of saturation of ammonium sulfate and they were precipitated out of solution.
To more precisely define the ammonium sulfate fraction containing the highest amount of
specific protease activity, smaller ammonium sulfate fractions were collected over the
range of 40 – 80%. It was observed that the 40 – 70% ammonium sulfate fraction contained
68% of the protease activity whereas the 70 – 80% fraction contained < 5% of the protease
Fractions of saturation 40 – 60% and 60 – 80% were showing highest activity and hence a
saturation of 40 – 70% was obtained and was found to contain most of the bromelain and
To more precisely define, the precipitation kinetics in the saturation range of 40 – 60%, the
saturation range is increased by 2%, and for every 2% increase in saturation, the precipitate
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
20000
16000
12000
CDU
8000
4000
0
35 40 45 50 55 60 65
% Saturation
Supernatant Precipitate
Table 4.1 shows the percent recovery of bromelain and protein from crude fruit juice of
pineapple during each stage of supersaturation and Table 4.2 shows the percent recovery of
Table 4.1 Effect of percent saturation on bromelain and total Protein recovery in
precipitation
Table 4.2: Effect of percent saturation on activity retention and protein content in
ammonium sulfate precipitation
For further study, precipitates of 40 – 60% and 60 – 80% were mixed and were subjected
The advantages in using Ammonium sulfate as a precipitant over other precipitating agents
were that the enzyme obtained using Ammonium sulfate was much more stable and the
Acetone precipitation was also performed as the first step of bromelain purification. The
supernatant and precipitate were collected at levels of saturation of 20% (w/v) and assayed
for specific protease activity before and after dialysis. Fractions collected at 40 – 80%
saturation contained the highest percent of protease activity and specific protease activity,
with a recovery of 42%, and accounted for 9.8% of the total protein concentration.
Table 4.3 gives the percent recovery of bromelain and protein from leaves of pineapple
during 40 – 80 % of supersaturation
Acetone fractionation of tissue extracts eliminates most of the pigmented matter at 40%
saturation. Proteins in this fraction showed a negligible proteolytic activity. Most of the
enzyme activity was recovered in 40 – 80% fraction. The total proteolytic activity of the
matured leaves of the plantlets was comparable, but much less than that observed in the
At 60 % saturation using acetone, the recovery obtained was 42%, which was very less as
fractionation when coupled with ammonium sulfate precipitation or gel filtration may give
These results indicate that cold acetone is probably a suitable effective agent for the initial
step of protease purification. The effectiveness of cold acetone as a purification agent for
proteolytic enzymes was also reported by Popova and Pishtiyski (2001). Michail et al.
(2006) also reported that cold acetone was a much better purification agent than other
Crude crystalline enzyme was obtained by fractionation with ammonium sulfate and
The specific activity of the crude crystalline product thus obtained was the same as, or only
slightly higher than, that of the starting material as assayed toward casein in the presence of
0.005 M cysteine, which was in good agreement with the observations of Murachi and
Neurath (1960).
The nature of the salt is known to have a strong influence on protein solubility.
Experimental results show that the salting – out effect of salts mainly depends on the nature
of the anions. Salts exert their effect by dehydrating proteins through competition for water
molecules (Melander and Horvarth, 1977). Their ability to dehydrate depends primarily on
the square of the valence of the anion of the salt. Thus, salts with polyvalent anions are
more effective at salting-out than those containing univalent anions. Comparison of salting-
out ability of salts should be made on the basis of salt concentration rather than ionic
which is consistent with the Hofmeister series and for cation the sequence would be,
NH4+>K+>Na+
Of the common inexpensive salts that are effective in causing precipitation, sodium,
potassium and ammonium sulfates, phosphates are attractive candidates (Arakawa and
Timasheff, 1984).
Since the fruit bromelain was found to possess more activity than the one found in stem or
Enzyme recovery with respect to load was found to be 84.54% and elution efficiency was
observed to be 97.56%. The specific activity of the crude juice was 58.79 Units/mg Protein
and that of Elute was 590.92 Units/ mg Protein. Thus the fold purity obtained was 10.05.
The unbound fraction mainly contained sugars which could have been further processed for
The profile of Moisture content (db) with respect to time in freeze dryer is as shown in
Figure 4.2.
18
16
14
Moisture content (db)
12
10
8
6
4
2
0
0 100 200 300 400 500 600
Time (min)
Where,
When the water molecules sublime and enter the vapour phase, they also keep with them a
significant amount of the latent heat of sublimation (2840 kJ/kg ice) and thus the
The effect of freeze drying on product quality parameters such as residual activity, protein
Table 4.5 Effect of freeze drying on product quality parameters (Ts = 30ºC)
Parameter Value
Yield 95 – 97%
The stability of freeze dried product can be attributed to its ability to go through the drying
The effect of spray drying on product quality parameters such as residual activity, protein
The yield obtained was less in case of spray dryer as compared to that obtained in freeze
dryer. This may be due to poor collection efficiency. Carry over of fine particles along with
the air or the product deposition on the drying chamber May leads to lowder yield.
Table 4.6 Effect of spray drying on product quality parameters (To = 40 ± 2ºC)
Parameter Value
Yield 70 – 75%
The less enzyme activity retention may be due to the degradation of the product because of
heating, or structural changes that are occurring during drying. Also, the powder obtained
from chamber showed negligible activity. The reason behind this may be that the deposited
powder in the drying chamber faced higher temperature for longer period of time, which
The outlet temperature of the spray dryer was determined solely by the main effects of the
inlet temperature, feed flow rate and air flow rate. The outlet temperature of the spray dyer
is considered to be the most important factor in determining the residual activity of spray
Figure 4.3 shows the effect of inlet air temperature on activity retention of bromelain.
0.9
0.8
% activity retention
0.7
0.6 y = 0.7932
0.5
0.4
0.3
0.2
0.1
130 140 150 160 170 180 190 200 210
Inlet Tem perature (ºC)
Inlet temperature was having negligible effect on enzyme activity retention. The enzyme
activity retention was found to be in the range of 70 – 75%. The inlet temperature was
varied in the range of 140 – 200 ºC and the outlet temperature was maintained constant by
adjusting feed flow rate and hence the temperature attained by the dry powder was not
more than 40 ºC in any run. Thus, the enzyme activity retention was more or less remains
constant. However, the particle size obtained was different, since feed flow rate affects the
particle size of product. These results are in good agreement with those reported by
Figure 4.4 shows effect of outlet air temperature on activity retention of bromelain.
0.6
0.4
0.2
0
30 40 50 60 70
Outlet Temp (deg C)
The residual activity of spray dried bromelain was found to vary between 77% – 1% at
different outlet temperatures. At constant inlet air temperature when lower feed flow was
used, higher outlet temperature was achieved and hence, the enzyme activity was found to
decreased for each drying air temperature. At an outlet temperature of 65 ºC, the enzyme
almost showed zero activity. The reason behind this may be that the inactivation
temperature of the enzyme was reported to be 65 – 70 ºC. Feed flow rate has a remarkable
Some authors reported that proteins are more resistant to thermal denaturation at lower
water content conditions. It is generally known that when protein substance is dried, its
2005). It means that higher evaporation rate leads to better enzyme activity preservation,
The final relative bromelain activity after drying can be strongly correlated to outlet
temperature and moisture content in powders. In order to maintain the relative activity of
dried enzyme on the acceptable level, the outlet temperature should be kept below 45 ºC.
Average drying ratio, productivity and drying rate are shown in Table 4.7.
Drying ratio was found to increase from 13.42 to 13.72 with a decrease in feed rate. It can
be seen that Productivity and drying rates were increased from 5.79 to 14.21 kg/hr
The effect of heat pump drying on product quality parameters such as residual activity,
Table 4.8 Effect of heat pump drying on product quality parameters (T = 38ºC)
Parameter Value
Yield 75 – 80%
The less yield obtained in Heat pump dryer may be attributed to loss of fine powder which
was carried away along with the air during drying. Heat pump drying results in the
crystallization of bromelain powder. Though the powder was having comparable activity,
its stability was very poor and also, the moisture content was observed to be more. The
crystalline powder of bromelain looses its 50% of activity after 4 days, when it was kept at
4 ºC. The preparation of such enzymes in the crystalline state does not necessarily
The results are in good agreement with the findings of Pikal and Rigsbee (1997). They
have found that the insulin in the amorphous state is more stable than in crystalline form
during storage.
The powder obtained by different drying techniques was analyzed for different product
quality parameters and these parameters were compared with that of standard commercial
powder sample obtained from Hong Mao Biochemical Co. Ltd. The results of which are as
The enzyme activity of freeze dried powder was 9.39 CDU/mg powder, which was slightly
more than that obtained by spray and heat pump drying. But the enzyme activity of
commercial powder was 132.5 CDU/mg powder, which was very high as compared with
that obtained by freeze drying. The reason behind this may be that the enzyme subjected to
freeze drying was chromatographically purified fraction containing NaCl. NaCl was not
removed before freeze drying. Due to this reason the protein concentration in the powder
The specific activity of freeze dried protein was observed to be 482.31 Units/mg protein
and that of standard commercial powder was 170.96 Units/mg protein i.e. 1 mg protein
contains 482.31 casein digesting units (CDU). This proves that our enzyme is having much
The enzyme activity can be increased by subjecting the eluted sample to dialysis as a
concentration prior to drying so as to remove the salt and concentrate the protein sample.
However, the selection of buffer in dialysis should be checked for pH and stability. The
The water activity of both spray dried and freeze dried powder was well below 0.8 which
The reducing sugar content of standard commercial powder was 0.317 mg/mg powder
which means that after concentration sugars were added so as to increase glass transition
Figure 4.5 shows the profile of bromelain inactivation in aqueous solution at various
temperatures. The activity ratio to the initial value is plotted against time.
0.8
Relative Activity
0.6
0.4
0.2
0
0 50 100 150 200
Time (min)
The solid lines shown in the figures were calculated from the parameters obtained by
energy calculated by assuming the linearity of the plot was 21.4 kcal/mol.
Figure 4.6 shows Arrhenius plot of apparent first order rate constant obtained for
bromelain inactivation.
1000/T (ºC-1)
-2
ln K
-4
y = -21.27x + 61.699
R2 = 0.999
-6
Figure 4.6: Arrhenius plots of apparent first-order rate constants obtained for bromelain
inactivation (pH 7.4).
most of the studies published to date (Tsuda et.al., 1990). In this study, bromelain was
degradation is very limited. The kinetics of enzyme inactivation in aqueous solution was
studied for bromelain. Inactivation of bromelain was found to follow simple first-order
kinetics and the rate constant obtained appeared to confirm to the Arrhenius relationship,
suggesting that the inactivation rate can be predicted by interpolating the relationship. The
relationship. The results obtained suggest that inactivation of enzymes in aqueous solution
can be modeled even if the profile is complicated and the dependence of kinetic parameters
Figure 4.7: Fourier Transform IR spectra of the 4000 – 500 cm-1 region of freeze dried
bromelain
observed IR bands at 1655-1685 cm-1, 1615-1635 cm-1 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 which contains
The spectra at 3280 – 3340 cm-1 show presence of CH stretching vibrations. The
experimental spectra of phenylalanine, proline, valine, leucine and isoleucine are in the
Figure 4.8: DSC thermogram of freeze dried bromelain obtained at a heating rate of
10ºC/min
The freeze dried enzyme powder containing NaCl was subjected to Differential Scanning
Calorimetry (DSC) and it was found that the glass transition temperature of the powder is
61.16 0C. The thermogram is an endotherm indicating that the powder is amorphous and
the curve obtained is that of the glass transition temperature and the melting temperature is
104.83 ºC.
From DSC study, we can say that in order to avoid structural changes from plastic to
rubbery viscous state, the drying temperature should not be more than 60 ºC and hence, all
4.5.4. Optimum pH
The pH profile of protease activity of fruit portion of bromelain is as shown in Figure 4.9.
0.6
0.4
0.2
0
4 6 8 10 12
pH
Figure 4.9: The pH dependence of the proteinase activity of purified fruit bromelin.
Casein was used as substrate
The casein was not soluble below pH 6.0 and hence all assays were performed above pH
6.0. The optimum pH for fruit bromelain was found to be around 8.0 when casein was used
as substrate. This implies that the fruit portion contained protease with optimum pH on the
basic side, and hence, all the assays were carried out at pH of 8.0.
However, the optimum pH may vary depending on the substrate and the buffer in which the
cystein activators and then was subjected to assay. The temperature profile of protease
From Figure 4.10 it is clear that the enzyme is most active at a temperature of 60 ºC. This
was in good agreement with the findings of Liang et al. (1999). They have stated that the
optimum temperature of bromelain was in the range of 50 – 60 ºC and above 65 ºC, it gets
inactivated.
0.5
0.4
Absorbance (A at 280 nm)
0.3
0.2
0.1
0
0 20 40 60 80 100
Temp (deg C)
Figure 4.10: Temperature dependence of the protease activity of purified fruit bromelain.
Casein was used as substrate.
The results are in good agreement with the observations of Chittenden, 1891. He has
reported that neutralized pineapple juice exerts its maximum digestive power at about 60º
C.
From Figure 4.10, one may conclude that the enzyme should be stored at 60 ºC, but the
fact is that though the enzyme is having maximum activity at 60 ºC, it remains active at
that temperature for a short period of time and thereafter, it gets deactivated.
The enzyme sample activated by Cystein HCl was analyzed for its activity. The enzyme
was mixed with casein substrate and was allowed to react for different time intervals. The
reaction was stopped by adding TCA at different time intervals. The effect of reaction time
250
200
150
CDU/ml
100
50
0
0 5 10 15 20 25 30 35
Time (min)
The Casein degradation rate was very fast during the initial period of reaction. It increased
exponentially till 15 minutes and thereafter it was found to be almost constant. So, in order
to give sufficient time for completion of reaction, it was decided to keep reaction time
equal to 30 minutes.
The time and temperature stability of freeze dried bromelain is as shown in Figure 4.12.
100
% Activity Retention
80
60
40
20
0
0 24 48 96
Time (hrs)
4 deg C Room Temperature 60 deg C
The powder which was stored at 4 ºC showed only 4 – 5% loss of activity at the end of 4
days; whereas, the powder stored at room temperature showed 50 – 60% activity loss at the
end of 4 days and the powder which was stored at 60 ºC looses its activity by 90% at a
Thus, the inactivation rate was found to be a strong function of temperature of dry powder
4.6 REFERENCES
Apte, P.V., Kaklij, G.S., Heble, M.R. (1979). Proteolytic enzymes (bromelains) in tissue
divalent cation salts: balance between hydration and salt binding. Biochemistry, 23, 5912 –
5923.
Chittenden, R.H. (1893). On the Proteolytic action of Bromelin, the ferment of pineapple
Liang, H.H., Huang, H.H., Kwok, K.C. (1999). Properties of tea – polyphenol – complexed
Michail, M., Vasiliadou, M., Zotos, A. (2006). Partial purification and comparison of
precipitation techniques of proteolytic enzymes from trout (Salmo gairdnerii) heads. Food
Murachi, T., Neurath, H. (1960). Fractionation and Specificity studies on stem bromelain.
Pikal, M.J., Rigsbee, D.R. (1997). The stability of insulin in crystalline and amorphous
solids: observation of greater stability for the amorphous form. Pharmaceutical Research,
amylase – The effect of process variables on the enzyme inactivation. Drying Technology,
Tsuda, T., Uchiyama. M., Sato, T., Yoshino, H., Tsuchiya, Y., Ishikawa, S., Ohmae, M.,
Conclusion
Conclusion
• Dry solid formulation provides acceptable enzyme shelf life. Dried bromelain
powder results in more activity retention and longer shelf life than the crystallized
bromelain.
• The glass transition temperature of the studied enzyme, bromelain in its native
form, is about 60 ºC. Hence, to avoid structural changes drying was carried out
• In spray drying of bromelain, the yield obtained was very less; however, this
problem can be overcome by adding some bulking agents. Outlet temperature has
• Freeze dried bromelain has more enzyme activity and shelf life as compared
to the spray dried bromelain. However, the mode of drying and purification
should be decided by the end application of the product. If the end application of
digestive aid or anti swelling agent, etc.), then in such cases chromatography
final step as it provides longer shelf life and high activity retention for bromelain.
The stability study of the bromelain during dialysis in buffers having different pH
can also be studied. Their final examination can be considered as the next step of this
project work; since dialysis results in removal of salt which gives concentrated protein,
The powder properties such as particle size, porosity and generation of adsorption
temperatures for bromelain are very important parameters and have a scope for further
study. In freeze drying of the said enzyme, secondary drying was not considered, i.e.
moisture was removed by means of sublimation only. However, the enzyme can be
bromelain, to remove unfrozen moisture which will have some effect on drying time and
drying kinetics.
Some physical properties of the enzyme need to be examined. Further work can
be done for thorough examination of Michelis Menton constant, which will explain
UNIVERSITY OF MUMBAI
MASTER OF TECHNOLOGY
IN
BIOPROCESS TECHNOLOGY
Registration Number
Objectives of work:
The present work deals with the purification followed by crystallization or drying
of plant protease named, Bromelain, from the fruit portion of pineapple plant (Ananas
comosus, Family: - Bromeliaceae).
• To purify bromelain from fruit portion of pineapple plant (Ananas comosus) using
ion exchange chromatography.
• To crystallize crude bromelain from pineapple extract using different precipitants.
• To obtain purified bromelain in dry powder form using different drying
techniques such as freeze drying, spray drying and heat pump drying.
• To see the effect of drying temperature on retention of enzyme activity and
protein content in all the three drying operations.
• To find the optimum pH and temperature for bromelain.
• To study the water activity of enzyme as a function of temperature.
• To carry out FTIR study and DSC study of bromelain powder.
Experimental:
Column mode Chromatography experiments:
Experiments were performed by using 2.5 cm id glass columns. The sample was
loaded through the column containing ion exchange resins (SP). The column was then
washed with equilibration buffer (sodium acetate) and the bound protein was then eluted
with the proper buffer (sodium chloride) at desired flow rate (5-6 ml/min). The crude,
unbound, washing and elution fractions were then assayed for the presence of proteolytic
activity.