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Bromelain: An overview of industrial

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Appl Microbiol Biotechnol (2014) 98:72837297

DOI 10.1007/s00253-014-5889-y

MINI-REVIEW

Bromelain: an overview of industrial application

and purification strategies
Zatul Iffah Mohd Arshad & Azura Amid & Faridah Yusof &
Irwandi Jaswir & Kausar Ahmad & Show Pau Loke

Received: 19 March 2014 / Revised: 8 June 2014 / Accepted: 10 June 2014 / Published online: 26 June 2014
# Springer-Verlag Berlin Heidelberg 2014

Abstract This review highlights the use of bromelain in

various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel,
core, crown, and leaves. Bromelain, a cysteine protease, has
been exploited commercially in many applications in the food,
beverage, tenderization, cosmetic, pharmaceutical, and textile
industries. Researchers worldwide have been directing their
interest to purification strategies by applying conventional and
modern approaches, such as manipulating the pH, affinity,
hydrophobicity, and temperature conditions in accord with
the unique properties of bromelain. The amount of downstream processing will depend on its intended application in
industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of
recombinant bromelain for novel applications in the future.
Keywords Bromelain . Cysteine protease . Purification .
Recombinant bromelain
Z. I. M. Arshad : A. Amid (*) : F. Yusof : I. Jaswir
Bioprocess and Molecular Engineering Research Unit, Department
of Biotechnology Engineering, Faculty of Engineering, International
Islamic University Malaysia, P.O. Box 10, 50728,
Kuala Lumpur, Malaysia
e-mail: [email protected]
Z. I. M. Arshad
Faculty of Chemical and Natural Resources Engineering, Universiti
Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan,
Pahang, Malaysia
K. Ahmad
Kuliyyah of Pharmacy, International Islamic University Malaysia,
P.O. Box 10, 50728 Kuala Lumpur, Malaysia
S. P. Loke
Manufacturing and Industrial Processes Division, Faculty of
Engineering, Centre for Food and Bioproduct Processing,
University of Nottingham Malaysia Campus, Jalan Broga,
Semenyih, 43500, Selangor Darul Ehsan, Malaysia

Introduction
Modern technological advancements have raised the protease
production industrially worldwide. Today, proteases dominate
with approximately 60 % market share of the total enzyme
market worldwide where the major producers are Novo
Industries, Gist-Brocades, Genencor International, and Miles
Laboratories (Feijoo-Siota and Villa 2011). This growth is
entrenched with the rising general awareness of the need to
protect the environment from the impact of chemical industrialization. Recently, bromelain has drawn attention in diverse industrial applications owing to its properties and higher
commercial values (Heinicke and Gortner 1957). Bromelain is
a protease derived from the stem and fruit of pineapples
(Ananas comosus). Stem bromelain (EC 3.4.22.32), ananain
(EC 3.4.22.31), and comosain are extracted from pineapple
stems, while fruit bromelain (EC 3.4.22.33) is mainly from
fruit juice (Rowan et al. 1990). Similar proteases are also
present in pineapple peel, core, crown, and leaves, with the
highest proteolytic activity and protein contents detected in
the extract of pineapple crown (Ketnawa et al. 2012).
Stem bromelain can be isolated from stem juices by means
of precipitation and centrifugation (Devakate et al. 2009;
Heinicke and Gortner 1957). Moreover, using chromatographic methods, other basic proteolytic (ananain, comosain,
F4, F5, and F9) and acidic components of stem bromelain
fraction A (SBA/a) and fraction B (SBA/b) have been partially
and fully purified (Feijoo-Siota and Villa 2011; Harrach et al.
1995, 1998; Murachi et al. 1964; Napper et al. 1994; Rowan
et al. 1990; Wharton 1974). The molecular weight of purified
stem bromelain is 23.4035.73 kDa, fruit bromelain
31.00 kDa, ananain 23.4323.42 kDa, and comosain 23.56
24.51 kDa. The isoelectric point of stem and fruit bromelain is
at pH 9.55 and 4.6, respectively (Murachi et al. 1964; Yamada
et al. 1976). There is minimal difference in the amino acid
composition between stem bromelain, ananain, and comosain

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Appl Microbiol Biotechnol (2014) 98:72837297

as shown in Table 1. Stem bromelain contains 285 amino

acids where the most abundant amino acids are alanine and
glycine, while histidine and methionine are present in the
lowest amounts (Murachi 1964; Ota et al. 1964). It appeared
to differ significantly in the number of lysine, arginine, and
isoleucine compared to ananain and comosain (Napper et al.
1994). Ananain possesses more hydrophobicity in the region
near histidine-157 and sequence insert between 170 and 174
residues which cannot be found in stem bromelain (Lee et al.
1997). Stem bromelain also contains four hexosamines, and
2.1 % carbohydrate, meaning it is a glycoprotein (Murachi
1964; Murachi et al. 1964). This observation was corroborated
by the findings of Ota et al. (1964), who detected 1.5 %
carbohydrate, including six glucosamines, in purified stem
bromelain. Later, Murachi et al. (1967) isolated 30 mg of
glycopeptides from 1 g of stem bromelain using gel filtration
in an attempt to confirm the presence of a glycoprotein.
Murachi et al. (1967) and Yasuda et al. (1970) reported that
the carbohydrate composition in stem bromelain consists of
mannose, fucose, xylose, and glucosamine in the ratio of
3:1:1:4 using gas chromatography analysis, whereas Scocca
and Lee (1969) obtained the same oligosaccharide group with
a different ratio of 2:1:1:2 using automated borate
chromatography.
Stem bromelain is an endoprotease that breaks peptide
bonds within the protein molecule. Stem bromelain is under
the papain family which resembles papain unequivocally in
the alignment of amino acid sequence. A prominent similarity
is observed in their mechanism of action as shown in Fig. 1.
Initially, noncovalent bonding involves the free enzyme
(structure a) and substrate to form the complex (structure b).
Table 1 Amino acid composition of stem bromelain, ananain, and
comosain (Napper et al. 1994)
Amino acid

Stem bromelain

Ananain

Comosain

Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
IIe
Leu
Tyr
Phe
His
Lys
Arg
Cys

18
9
17
16
22
25
14
3
17
6
14
6
1
15
6
7

19
8
18
13
24
20
14
2
14
9
12
5
2
11
10
7

18
7
17
13
25
20
13
3
12
9
12
7
2
10
11
7

Fig. 1 Catalytic mechanism of cysteine proteinase action. Im and +HIm

referred to imidazole and protonated imidazole (Rao et al. 1998)

Then, the next step is the acylation of the enzyme (structure c)

which produces amine, with R-NH2 as the first product.
Finally, the deacylation reaction step takes place between the
acyl-enzyme and water to release the carboxylic acid,
RCOOH, and free enzyme (Rao et al. 1998). The presence
of asparagine-175 hydrogen-bonded with histidine-159 favors
the catalytic function in papain. Stem bromelain is distinguished from papain by its lower value of second-order rate
constant of inactivation by iodoacetate and iodoacetamide due

Appl Microbiol Biotechnol (2014) 98:72837297

7285

to the absence of asparagine-175 and two adjacent residues as

well as the mutation of serine-176 to lysine in its structure
(Ritonja et al. 1989). It is noteworthy that stem bromelain
exhibits a weak inhibition by L -3-carboxy-2,3-transepoxypropionyl-leucylamido (4-guanidino) butane (E64)
about 678 M1 s1 in contrast to papain which is about
638,000 M1 s1 (Barrett et al. 1982; Rowan et al. 1990).
Specificity of stem bromelain was markedly examined
using different substrates, such as gelatin (gelatin digestion
unit), casein (casein digestion unit), azocasein and
azoalbumin, hemoglobin, sodium caseinate, Z-Arg-Arg-NHMec, and Z-Arg-Arg-Na (Corzo et al. 2012; Gautam et al.
2010; Iversen and Jrgensen 1995; Napper et al. 1994; Rowan
et al. 1990). Its proteolytic ability efficiently had hydrolyzed
glucagon at arginine-alanine and alanine-glutamic acid bonds
where it shows preference for glutamic acid, aspartic acid,
lysine, or arginine in the P1 site yet leaves intact the argininearginine and lysine-tyrosine bonds. The release of DNP (2,4dinitrophenyl)-alanine and DNP-glutamic acid after FDB
(fluorodinitrobenzene) treatment becomes an evidence of the
successful cleavage at its preference bonds (Murachi and
Neurath 1960; Napper et al. 1994). Profiling of substrate
specificity for stem bromelain using positional scanning synthetic combinatorial libraries (PS-SCLs) had displayed a
cleavage site at arginine (Choe et al. 2006). Generally, stem
bromelain is in full activity under the presence of ethylenediaminetetraacetic acid (EDTA) and cysteine, and the addition
of mercury (II) chloride (HgCI2) causes inhibition of its activity (Murachi and Neurath 1960). The optimum pH and temperature for stem bromelain activity are in the range of
pH 6.58.0 and 5560 C, respectively, for most of the substrates which are presented in Table 2. Studies of the stability
of pineapple fruit in storage without preservative at 4 C
indicate that bromelain retains 755 % of its activity, and no
microbial growth is detected after 180 days (Bhattacharya and
Bhattacharyya 2009). The bromelain degradation profile in
aqueous solution at various temperatures follows first-order

kinetics, and its activation energy is 41.7 kcal/mol (Yoshioka

et al. 1991).
While the biochemical properties such as molecular
weight, optimum pH and temperature as well as carbohydrate
composition in this enzyme display varying degrees of homology from various researchers, judgment on the variation of
bromelain resource and purification process has to be scrutinized extensively before awaiting for further industrial development. Thanks to the major breakthrough of modern biotechnology, new analytical technologies to characterize and
ensure the safety of the source and end product application
have been developed (Staub et al. 2011). Today, escalating
growth of commercial manufacturers of stem bromelain (retrieved from http://www.chemicalbook.com) worldwide has
swiftly found usage in a wide array of industrial applications
such as food, textile, brewing, cosmetic, and dairy products
(Polaina and MacCabe 2007). Thus, this review will start with
an overview on various applications of bromelain in tenderization, baking industry, protein hydrolysate, enzymatic browning inhibition agent, animal feed, textile industry, tooth whitening as well as cosmetic industry. Next, an in-depth summary
on recent purification strategies of bromelain in the last
10 years which include ultrafiltration, precipitation, aqueous
two-phase system (ATPS), adsorption, reverse micelle extraction (RME), and chromatography will be provided.

Industrial applications of bromelain

Bromelain is a plant protease isolated from pineapple. Its
strong proteolytic activity has created a wide interest in numerous applications, mainly in tenderization, foods, detergents, and the textile industry. Bromelain has also been envisaged to have extensive applications as an active ingredient in
tooth-whitening dentifrices and skin products. In this part of
the review, the major applications of bromelain are summarized (see Table 3).

Table 2 Optimum pH and temperature for bromelain with different substrates

Type of bromelain

Optimum pH

Optimum temperature (C)

Substrates

References

Fruit

55
55
59
59
37
60
45

Azocasein
Azoalbumin
Casein
Sodium caseinate
Hemoglobin
Casein
N-CBZ-L-lysine p-nitrophenyl ester (LNPE)

Corzo et al. (2012), Bhattacharya

and Bhattacharyya (2009)

Peel
Recombinant

6.5
7.5
7.7
6.5
2.9
8
4.6

Stem

40

Casein

Khan et al. (2003)

Bz-Phe-Val-Arg-pNA

Benucci et al. (2011)

Ketnawa and Rawdkuen (2011)

Amid et al. (2011)

7286

Appl Microbiol Biotechnol (2014) 98:72837297

Table 3 Industrial applications of bromelain

Applications

Reasons

Baking industry

Improve dough relaxation and allow the dough to rise evenly Kong et al. (2007), Tanabe et al. (1996), Watanabe et al. (2000)
Produce hypoallergenic flour
Hydrolyze meat myofibril proteins
Hage et al. (2012), Sullivan and Calkins (2010), Ketnawa and
Hydrolyzing agent for meat, oyster, chicken, and squid
Rawdkuen (2011), Chuapoehuk and Raksakulthai (1992)

Tenderization

References

Fish protein
Hydrolyze fish protein to generate fish protein hydrolysate
hydrolysate
Antibrowning agent Inhibit browning of fruits and phenol oxidation
Alcohol production Enhance protein stability of beers
Prevent haze formation
Animal feed
Estimate protein degradation in ruminant feed
Textile industry
Minimize softening time in cocoon cooking
Remove scale and impurities of wool and silk fibers
Enhance dyeing properties of protein fibers
Tooth whitening
Remove stains, plaque, and food debris on the outer
surface of teeth
Cosmetic industry Treat acne, wrinkles, and dry skin
Reduce post-injection bruising and swelling

Tenderization
Tenderness is an important characteristic of meat. The overwhelming demand for guaranteed tender meat has attracted
players in the meat industry to provide an acceptable quality of
product. In the USA, certified Angus beef was introduced in
the late 1970s. Consumers have found it consistently flavorful, juicy, and tender (Koohmaraie and Geesink 2006). Many
approaches have been employed to improve postmortem tenderness, including blade tenderization (Pietrasik et al. 2010;
Pietrasik and Shand 2011), moisture enhancement technology
(Streiter et al. 2012), and enzymatic treatment (Gerelt et al.
2000; Pietrasik and Shand 2011). In a traditional tenderization
method, meat is kept cool for up to 10 days to allow postmortem proteolysis by proteolytic enzymes: cathepsins and
calpains (Nowak 2011).
The potential of proteolytic enzymes such as bromelain,
papain, and ficin has been recognized, as shown by their long
history of use in meat tenderization. Compared with papain
and ficin, bromelain is commercially available in the market
under name brands such as McCormick and Knorr.
Commercial papain, bromelain, and zingibain efficiently hydrolyze a few meat myofibril proteins, such as actomysin,
titin, and nebulin, as revealed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) (Hage et al.
2012). Sullivan and Calkins (2010) reported that bromelain
can improve the sensory evaluation rating and tenderness of
meat comparable to other exogenous enzymes. The addition
of bromelain can also produce a tenderizing effect on myosin
and other myofibrillar proteins of coarse dry sausage. A
number of methods of tenderization using bromelain have
been reported. Lizuka and Aishima (1999) successfully

Elavarasan et al. (2013), Ren et al. (2008), Tanuja et al. (2012)

Lozano-De-Gonzalez et al. (1993), Srinath et al. (2012)
Benucci et al. (2011)
Tomnkov and Kopen (1995)
Koh et al. (2006), Devi (2012), Singh et al. (2003)

Chakravarthy and Acharya (2012), Kalyana et al. (2011)

Ozlen et al. (1995), Levy and Emer (2012)

applied continuous monitoring using attenuated total reflectance infrared spectroscopy (ATR-IR) to observe the structural
changes in meat protein after treatment with pineapple juice.
Melendo et al. (1997) studied the effect of pH, temperature,
cutting method, and cooking time during marinating with
bromelain. Ketnawa and Rawdkuen (2011) demonstrated that
treatment of bromelain at up to 20 % (w/w) leads to extensive
proteolysis on beef, chicken, and squid. Besides, bromelain
can be a good substitute for hydrochloric acid as a hydrolysis
agent for oyster meat and, at the same time, receives higher
acceptability scores in sensory evaluation in oyster sauce
production (Chuapoehuk and Raksakulthai 1992).

Baking industry
Gluten is a functional component of wheat food products,
such as flour. It consists of two major proteins, gliadin and
glutenin. Gluten becomes insoluble and forms lattice-like
structures when it is hydrated. Therefore, gluten must be
degraded to avoid resistance to dough stretching (Walsh
2002). The use of proteolytic enzymes such as bromelain
can improve dough relaxation, enhance solubility, and prevent
dough shrinkage. This will allow the dough to rise evenly
during the baking process (Kong et al. 2007; Polaina and
MacCabe 2007). Bromelain also has been used to produce
hypoallergenic flour that is suitable for wheat-allergic patients.
The immunoglobulin E (IgE)-binding epitope, Gln-Gln-GlnPro-Pro, is a major allergen in flour. Thus, the addition of
bromelain can help to hydrolyze peptide bonds near proline
(Pro) residues, which degrades the epitope structure (Tanabe
et al. 1996; Watanabe et al. 2000).

Appl Microbiol Biotechnol (2014) 98:72837297

Protein hydrolysate
Protein hydrolysate has a long history of use as a nitrogen
source for growth media for microbial, plant, and animal cell
culture at the laboratory and industrial scales. It is widely used
as a nutritional supplement, pharmaceutical ingredient, and
flavor enhancer and in cosmetics and beverages. Current
practice in the manufacturing of protein hydrolysate involves
acid, alkali, and enzyme hydrolysis. However, enzymatic
hydrolysis is preferable due to the ease in controlling the
degree of hydrolysis as well as shortening the hydrolysis time.
The significant parameters for enzymatic hydrolysis are temperature, hydrolysis time, pH, and degree of hydrolysis (DH)
(Pasupuleti and Braun 2010). The addition of proteolytic
enzymes can make the enzymatic hydrolysis process efficient
and reproducible (Chalamaiah et al. 2012). There has been
considerable research on the flavor profile (sweet, salty, sour,
bitter, bouillon, and umami taste) and functional properties
(hypoantigenic, antioxidant, and antimicrobial) of protein hydrolysate isolated from fish and plant sources using commercial bromelain.
Protein hydrolysate derived from food processing byproducts can be used as precursors in the production of meat,
savory food, and chicken flavor substitutes. For example, the
hydrolysis of mungbean protein by bromelain has released 16
free amino acids which give a meaty flavor characteristic such
as bouillon, sweet smell, green, and fatty (Sonklin et al. 2011).
Besides, after bromelain hydrolysis, several amino acids such
arginine, lysine, and leucine were found from the protein byproduct hydrolysis of Gracilaria fisheri (red seaweeds) which
characterize seafood flavoring (Laohakunjit et al. 2014).
Sangjindavong et al. (2009) reported on the production of fish
sauce from the fermentation of surimi waste accelerated with
pineapple core and peel.
In spite of the fact that protein hydrolysate can be used as a
flavor agent, the hydrolysis process is often accompanied by
bitterness effect due to the reaction of endoprotease with the
protein substrate during the hydrolysis. The bitter intensity
can be expressed as the taste dilution (TD) factors. Partial
hydrolyzation of soy protein isolate (SPI) by bromelain
showed lower (TD) factors of 4 and 16 in 10 and 15 % DH,
respectively, compared to alcalase and neutrase. This phenomenon could be due to the remaining nonpolar amino acid
residues at the C-terminus of the peptide after bromelain
hydrolyzes the hydrophobic amino acid residues (Seo et al.
2008). Cheung and Li-Chan (2014) found that after 8 h of
hydrolysis, shrimp hydrolysate by bromelain demonstrated
less bitter and high umami taste in the instrumental taste
sensing system rather than alcalase and protamex hydrolysate.
The bitterness characteristic results from enzymatic hydrolysis
and is due to the presence of N-terminal peptide residues
which are glycine, leucine, isoleucine, phenylalanine, and
valine (Hevia and Olcott 1977).

7287

Recent studies have demonstrated that protein hydrolysates

from fish source possess antioxidant activities after enzymatic
hydrolysis using various proteolytic enzymes such as bromelain, papain, flavorzyme, protamex, neutrase, and alcalase
(Lamsal et al. 2007; Ren et al. 2008; Tanuja et al. 2012).
Among these enzymes, bromelain hydrolysate from freshwater carp (Catla catla) is appearing to give high disappearance
rates of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH)
(Elavarasan et al. 2013). Antimicrobial peptides from natural
sources can be promising candidates to replace synthetic
antimicrobial agents in the treatment of a wide spectrum of
bacterial infections. Thus, Ghanbari et al. (2012) explored the
application of Actinopyga lecanora (sea cucumber) in the
production of antimicrobial peptide. They found that after
7 h of hydrolysis, hydrolysates prepared by bromelain exhibited the highest antimicrobial activities against Pseudomonas
sp., Pseudomonas aeruginosa, and Escherichia coli compared
to other hydrolysates generated by papain, alcalase, pepsin,
trypsin, and flavorzyme. That study corroborates a previous
study performed by Salampessy et al. (2010), which showed
the antimicrobial capability of bromelain hydrolysate from
leatherjacket (Meuschenia sp.) against Staphylococcus aureus
and Bacillus cereus.
Enzymatic browning inhibition agent
The discoloration of fruits into brown color, especially apples
and pears, occurs due to the oxidation of phenols to quinones
in the presence of oxygen. Further reactions of quinones with
other substances, such as phenolic compounds and amino
acids, lead to the development of brown pigments.
Enzymatic browning may deteriorate the nutritional quality
and flavor of the fruits. To control the browning of fresh and
canned fruit, ethylene diaminetetraacetic acid (EDTA), ascorbic acid, L-cysteine, citric acid, and phosphoric acid are commonly used (Ozolu and Bayndrl 2002). However, due to
health concerns over the side effects caused by chemicalbased enzymatic browning inhibition agents, there has been
an increasing amount of research on agents derived from
plant-based sources. According to Lozano-De-Gonzalez
et al. (1993), eluate from cation exchange of the pineapple
juice is effective as a sulfite agent to inhibit browning of fresh
and dried apple rings. Later, Srinath et al. (2012) extended that
study by partitioning the fruit and crown leaf using cationexchange chromatography after ammonium sulfate precipitation. They concluded that the protease activity and browning
inhibition effects were higher in the fruit extract compared to
the crown leaf extract. Similarly, Chaisakdanugull et al.
(2007) employed pineapple juice that was fractioned by a
solid-phase C18 cartridge as an inhibitor of enzymatic browning in bananas. The study reported a 100 % inhibitory effect
of bromelain toward polyphenol oxidation (PPO). In contrast
to earlier findings, however, stem bromelain had the weakest

7288

browning inhibition effect compared with commercial L-cysteine and ascorbic acid (Tochi et al. 2009).
Animal feed
Generally, forages provide 90 % of food energy and nutrients
to ruminant animals at low cost. The variation of the nutritional value of forages is highly influenced by the climate,
forage species and cultivars, and the preservation method.
Therefore, the estimation of soluble nitrogen compounds in
rumen is pivotal to assess the forage attributes to maximize its
utilization in ruminants diets (Givens et al. 2002). The in sacco
(in situ) technique is widely used to analyze the protein
degradability of forage. Unfortunately, the technical challenges and lack of standardization limit its practicability
(Abdelgadir et al. 2013). The determination of protein degradation in the rumen by the actions of proteases can be an
alternative to replace the conventional method, which is highly expensive and time-consuming. The addition of protease to
animal feed can increase protein inversion and availability,
decrease the cost of animal feed, and increase the source of
protein. The use of bromelain in the estimation of protein
degradation of cereals, forages, hays, protein concentrates,
and silages in ruminants has been successful and produces
comparable results with the in sacco method (Polaina and
MacCabe 2007; Tomnkov and Kopen 1995).

Appl Microbiol Biotechnol (2014) 98:72837297

discolored teeth has raised the demand for tooth-whitening

products. Thus, there is heated competition between manufacturers to develop new technologies and oral care products that
overcome tooth discoloration. For tooth-whitening and extrinsic stain removal purposes, toothpaste is usually formulated
with abrasive agents that whiten teeth by removing plaque,
stains, and food debris accumulated on the tooth surface.
However, abrasive agents can accelerate tooth wear, and they
become ineffective if their particles are too large, as they
cannot be captured by toothbrush bristle (Joiner 2010).
Therefore, many types of toothpaste containing different enzymes are marketed to remove extrinsic stains (Hannig et al.
2005, 2010). The novel dentifrice efficiency can be accessed
through the lightening value. Recently, an in vitro study by
Kalyana et al. (2011) reported on the stain removal efficiency
of a dentifrice containing papain and bromelain. The
proteolytic enzymes disrupted accumulated protein on the
tooth surface, which resulted in higher lightening value after
brushing compared to the control dentifrice. Chakravarthy and
Acharya (2012) corroborated these results, finding that the
application of papain and bromelain extracts in dentifrices had
a significant lightening effect in comparison with the control
dentifrice (Colgate Regular). However, their formulation had
the limitation of a shorter shelf life than the usual dentifrice
because bromelain and papain are proteolytic in nature.
Cosmetic industry

Textile industry
Recently, the use of proteases in the silk industry has expanded enormously, especially the cocoon cooking method.
Generally, the cocoon has to be softened by cooking it in the
presence of strong alkaline agents and chemicals. However,
this conventional technique has a detrimental effect on the
quality of the silk thread produced. Thus, the development of
an effective cocoon cooking method using enzyme treatment
will reduce softening time while increasing production and
saving energy. For instance, Singh et al. (2003) reduced the
softening time from 20 h to 30 min using pineapple extract
incorporated with 9.8 mM of sodium carbonate at 60 C.
Additionally, Koh et al. (2006) reported that the pretreatment
of silk and wool with bromelain improved tactile behavior and
wettability by removing impurities and scale. These effects
increased the dye uptake of the wool and silk fibers and at the
same time maintained their tensile properties.
Tooth whitening
Tooth discoloration involves their intrinsic color and extrinsic
stains on the surface of the teeth. A number of factors that
impart extrinsic discoloration include colored foods and beverages such as tea and coffee, tobacco smoking, and tobacco
chewing (Watts and Addy 2001). The stigma attached to

The cosmetic products that have papain and bromelain as

active ingredients can effectively alleviate skin problems such
as wrinkles, acne, and dry skin. These enzymes gently digest
the protein of dead cells in the upper layer of the skin, resulting
in their replacement by younger skin cells from lower layers
(Ozlen and Chatsworth 1995). Bromelain can also reduce
bruising and swelling of skin after cosmetic injection treatments (Levy and Emer 2012).

Purification strategies of bromelain

The continuous interest in bromelain in diverse applications
has motivated many researchers to extract the enzyme from
pineapple juice and waste and characterize it. The mature
pineapple stem contains the highest concentration of bromelain (Heinicke and Gortner 1957). However, pineapple wastes
(peel, core, crown, and leaves), which are a by-product in the
pineapple processing industry, are also rich in bromelain and
are still underutilized. Therefore, Ketnawa et al. (2012),
Mutalib et al. (2012), and Nadzirah et al. (2012) investigated
the physicochemical properties and proteolytic activity of
pineapple waste. The selection of extraction buffer and
disruption method is crucial and depends on the target

Appl Microbiol Biotechnol (2014) 98:72837297

protein and its application. The optimal conditions would

maximize the extraction efficiency, minimize oxidation,
avoid denaturalization, and increase protein acquisition.
Ketnawa et al. (2012) investigated the extraction of bromelain
from pineapple peels using different extractants. They obtained the highest bromelain activity when it was extracted with
100 mM phosphate buffer with 15 mM cysteine and 2 mM
EDTA at pH 7.0. The addition of a small amount of reducing
agent, such as cysteine, and chelating agents, such as EDTA,
prevents protein oxidation and traps heavy metals that can
boost the oxidation of thiols to inhibit protease action
(Chaiwut et al. 2007; Grslund et al. 2008; Janson 2011).
The conventional extraction of bromelain from cooled pineapple juice involves grinding, filtering, and centrifugation. For
instance, Shian et al. (2005) and Gautam et al. (2010)
employed a homogenization method that disrupts the pineapple stem cells and releases bromelain using sodium acetate
solution as an extraction buffer. Later, the crude extracts are
filtered and centrifuged to remove cellular debris and insoluble. Once the desired bromelain is free from other particulate
residue, the purification continues until a desirable level of
purity is achieved, depending on its intended use.
Thorough investigations on the operational design are
needed to reduce the cost and number of purification steps
in downstream processing. Pre-purification steps such as precipitation, ultrafiltration, and extraction are most commonly
employed in the early stage to concentrate and increase the
recovery of the target protein. Once a soluble concentrate is
obtained, successive purification steps are performed to remove remaining impurities and increase the level of purity.
Occasionally, a single pre-purification is enough, but the
degree of purification will be low compared to the highresolution steps such as chromatography. Therefore, chromatography steps with ion exchange, affinity, and gel filtration
are crucial to provide higher selectivity and purity of the end
product. Some novel purification technologies have recently
been applied to the purification of bromelain. These include
reverse micellar extraction, aqueous two-phase systems, and
adsorption. These are currently being employed at a laboratory scale and have yet to be explored in large-scale processing.
Here, a brief description of ultrafiltration, precipitation, and
chromatography are provided below.
Ultrafiltration
Ultrafiltration is the best method of concentrating proteins and
has been used extensively in the lab and large-scale processes.
The ultrafiltration membrane has a molecular weight cutoff
ranging from 3 to 100 kDa, while microfiltration has a pore
diameter ranging from 0.1 to 10 m. The retentate containing
the protein of interest is retained behind the membrane and is
further recycled until the desired concentration is achieved.
The permeate is usually discarded as waste (Walsh 2002;

7289

Wheelwright 1991). Doko et al. (1991) have reported on the

application of sequential batch filtration, which included
microfiltration (8 m) and ultrafiltration (100 kDa cutoff),
followed by the additional steps of ammonium precipitation
and ultracentrifugation prior to freeze-drying to remove contaminant residues, resulting in 50 % recovery. The recovered
product contained 98 % protein. However, the proteolytic
activity of bromelain was lost during the protein adsorption
onto the ultrafiltration (UF) membranes relative to the level of
concentration reached. A similar trend of enzymatic activity
was observed when another study applied microfiltration (1.2
and 0.2 m) and ultrafiltration (10 kDa) before bioethanol
fermentation process (Gimeno et al. 2010). Apparently, the
bromelain recovery obtained by Lopes et al. (2009) (90 %)
and Hebbar et al. (2012) (92.4 %) was more than reported by
Doko et al. (1991). The additional steps of microfiltration,
RME, and ATPS before ultrafiltration caused a significant
increase in activity recovery.
Precipitation
Protein precipitation involves the addition of salts, polar solvents, nonpolar solvents, and organic polymers into cell extracts or by varying the temperature or pH. When the solubility of proteins in solution is reduced by increasing the concentration of precipitating agents, salting out (precipitation)
will occur. In a large-scale process (15 l of homogenate),
ammonium sulfate is the agent of choice for salting out the
proteins because it is effective and relatively cheap. Under
saturation (4 M ammonium sulfate), most protein will precipitate from the solution. Low temperature is favorable to avoid
denaturation during precipitation. To recover the protein of
interest, additional steps such as centrifugation and dialysis
are needed to remove contaminant proteins (Janson 2011).
Shian et al. (2005), for example, successfully precipitated 1 l
of homogenate pineapple juice at 55 % of ammonium sulfate.
Partially purified fruit bromelain was also obtained at 30
80 % saturation by Ko et al. (1996). Similarly, Devakate
et al. (2009) described the precipitation of fruit bromelain
and achieved 2.81-fold purity at 4060 % ammonium sulfate
saturation. Organic solvents such as ethanol, ketones, methanol, propanol, and other alcohols in protein precipitation have
been widely employed.
The addition of organic solvents will decrease the activity
of water, as part of the water has been replaced by organic
solvents, and cause the proteins to precipitate. Heinicke and
Gortner (1957) and Soares et al. (2011) reported the use of
acetone and ethanol as the precipitating agents, respectively.
Surprisingly, ethanol precipitation yielded 2.07-fold purification, compared to 4.44-fold obtained by ammonium precipitation (Soares et al. 2011). Likewise, Soares et al. (2012) only
managed to attain a purification factor of 2.28-fold at 3070 %
ethanol saturation. So far, organic solvent precipitation must

7290

operate at subzero temperature until the desired concentration

(10100 %) is achieved to avoid denaturation (Wheelwright
1991). Nonetheless, recently, Silva et al. (2010) improved the
quality of stem and rind bromelain recovery and activity
in ethanol precipitation with the development of a fedbatch stirred precipitation tank equipped with conventional
proportional-integral-derivative (PID) and adaptive PID
controllers. They overcame the required temperature increment as a result of heat generated upon the addition of
ethanol during precipitation. Organic polymers such as
polyethylene glycol (PEG) can precipitate protein and
obviate the need for temperature control during the process. Increasing the concentration of PEG increases the
amount of precipitated bovine serum albumin (BSA)
(Kumar et al. 2009). However, bromelain does not show
the same effect as BSA (Soares et al. 2011).
Variations in the temperature, volume of homogenate,
mixing conditions, and biochemical properties between
fruit and stem bromelain caused the discrepant saturation levels between the above studies. The decrease of
repulsive forces and the dominance of attractive forces
at the isoelectric point cause protein aggregates to reduce their solubility. Thus, by manipulating the pH,
contaminant proteins in the solution can be salted out.
Low temperature is favorable in reducing the solubility
and precipitating proteins, whereas higher temperature
denatures proteins (Walsh 2002; Wheelwright 1991).
Aqueous two-phase system (ATPS)
ATPS involves the formation of two immiscible phases after
the mixing of polymer and salts or the combination of two
incompatible polymers (PEG and dextran) in an aqueous
solution. The distribution of protein samples between these
two phases is influenced by the ionic strength, pH, temperature, molecular weight of the polymer, and type of salt. For
instance, under favorable conditions, the target protein tends
to partition into the top phase, which is less dense, less polar,
and hydrophobic (PEG phase), while contaminant proteins are
found in the lower partition, which are more dense, more
polar, and hydrophilic (dextran phase) and can be removed
through centrifugation (Walsh 2002). A considerable amount
of literature has been published on the purification of bromelain using aqueous two-phase systems. Bromelain has been
extracted from pineapple fruit by ATPS containing PEGpotassium phosphate (Babu et al. 2008; Hebbar et al. 2012)
or PEG-phosphate buffer (Ferreira et al. 2011). Bromelain has
also been extracted from pineapple wastes by ATPS. For
example, Ketnawa and Rawdkuen (2011) isolated bromelain
from pineapple peels using a PEG/magnesium sulfate system,
while Coelho et al. (2013) had purified bromelain from stems,
barks, and leaves using a PEG/ammonium sulfate system.
From all of these studies, it can be concluded that the highest

Appl Microbiol Biotechnol (2014) 98:72837297

recovery of bromelain is obtained at the highest concentration

of PEG and salts applied. This is due to the influence of
volume exclusion and the salting out effect, whereby both
the bromelain and total protein are in the top PEG-rich phases.
However, the partition of bromelain on the top phase decreases as the molecular weight of PEG increases. This is
probably due to the structural alteration of enzyme sites in
the presence of high molecular weight PEG as well as the fact
that bromelain is relatively partitioned in the bottom phase
(Babu et al. 2008; Ketnawa et al. 2010). Analogously,
thermoseparation of bromelain using an aqueous solution of
polyethylene oxide (PEO)-polypropylene oxide (PPO) block
copolymers done by Rabelo et al. (2004) had increased the
activity recovery by approximately 79.5 %, with a purification
factor of 1.25, but the values were relatively lower compared
to those of Babu et al. (2008), Ketnawa et al. (2010), and
Coelho et al. (2013).

Adsorption
The adsorption process can be defined as the binding of a
dissolved solute to a solid adsorbent. Currently, the use of
adsorption with a wide variety of adsorbents is becoming
more common in bromelain preparation. For instance, Chen
and Huang (2004) applied a magnetic nano-adsorbent using
iron oxide nanoparticles as the core and polyacrylic acid
(PAA) as the ionic exchange group. Notably, the process
successfully produced 87.4 % bromelain activity after adsorption at pH 35 and desorption at pH 7. As bromelain has the
property of having a single oligosaccharide chain attached to
the polypeptide, Gupta and Saleemuddin (2006) used affinity
binding to a Sepharose matrix pre-coupled with the lectin
concanavalin A (Con A), which resulted in 60 % bromelain
activity at pH 11. Meanwhile, Silveira et al. (2009) integrated
the expanded bed adsorption (EBA) to investigate the effect of
the expansion degree of Amberlite IRA 410 resin and the
residence time distribution, which resulted in 13-fold bromelain purification. Recently, the use of dye ligand affinity
membrane chromatography seems more promising because
it offers high selectivity, a larger surface area, and excellent
thermal and mechanical properties. The electrospun polyacrylonitrile (PAN) nanofibers developed by Zhang et al. (2010)
were chemically modified by tethering them to chitosan as a
composite bilayer membrane and Cibacron Blue F3GA (CB)
as a dye ligand. This successfully captured 161.6 mg/g of
bromelain. Sai-Nan et al. (2009) and Song et al. (2011)
employed Reactive Red 120 (Red 120) as a dye ligand
immobilized onto chitosan-coated magnetic nanoparticles
(CS-MNPs) to achieve bromelain adsorption from aqueous
solution. In these studies, 88.2 % activity of bromelain was
achieved, and the bromelain adsorption isotherm fits well with
the Freundlich model.

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7291

Reverse micelle extraction (RME)

Chromatography

The reverse micelle system is a liquid-liquid extraction technique for biomolecule separation in downstream processing.
Reverse micelle systems consist of droplets of aqueous phase
separated from an organic solvent by the presence of surfactants in the interface. The surfactants possess both hydrophobic and hydrophilic tails. The polar head (hydrophilic) is
located inside the micelle, and the nonpolar tail is in contact
with the solvent outside the micelle. The movement of protein
inside and outside the micelle can be induced by manipulating
the pH, the ionic strength of aqueous phase, and the surfactant
concentration (Wheelwright 1991). Several reports on the
application of RME for separation and purification of bromelain from pineapple fruit (Yin et al. 2011), juice (Fileti et al.
2009), and wastes (Chaurasiya and Umesh Hebbar 2013;
Hebbar et al. 2012; Umesh Hebbar et al. 2008) are available.
Yin et al. (2011) and Umesh Hebbar et al. (2008) have demonstrated that the forward extraction efficiency, purification
fold, and specific activity of bromelain are optimal at pH 7.0
8.0. The proteins transfer from the organic solvent to the
reverse micelle if the pH value of the aqueous phase is higher
than the isoelectric point of bromelain (4.6). This is because of
the electrostatic attraction that occurs when the protein has a
positive charge and the internal surface of reverse micelle has
a positive charge (cationic surfactant). Chaurasiya and Umesh
Hebbar (2013) and Umesh Hebbar et al. (2008) concur that the
concentrations of 150 mM cetrimonium bromide (CTAB) and
0.1 M sodium chloride (NaCl) for forward extraction and
0.5 M potassium bromide (KBr) in back extraction produce
high-efficiency extraction of bromelain. The protein extraction rates increase when a low ionic strength of salt and a high
concentration of surfactant are applied, which in turn favors
the forward extraction process. The high concentration of the
surfactant increases the stability and number of reverse micelles, while the high ionic strength of salt in the back extraction may reduce the enzymatic exclusion from the interior
surface of reverse micelles (Yin et al. 2011). Hebbar et al.
(2012) employed the optimized conditions for reverse micelles from Umesh Hebbar et al. (2008) and integrated the
system with an ultrafiltration process, resulting in a bromelain
activity recovery of 95.8 % and purification fold of 8.9. Fileti
et al. (2009) improved the purification factor to 4.96 by
continuous extraction of reverse micelles using a
microcolumn with pulsed caps after applying the optimum
conditions from the batch operation. Recently, affinity-based
reverse micellar extraction and separation (ARMES) has been
used to purify bromelain from pineapple core. The authors
achieved 12.32-fold purification with 185.6 % activity recovery, which was higher than those reported by Umesh Hebbar
et al. (2008), through the affinity interaction between the
bromelain and concanavalin A ligand that been incorporated
in the reverse micelle system (Kumar et al. 2011).

Chromatography is a separation method in which protein samples migrate through the column at different
rates and conditions of solid stationary and mobile
phases. The packed column in chromatography comprises two types of mechanisms: adsorption (e.g., ion
exchange, hydrophobic interaction, and affinity) and
nonadsorption (e.g., gel filtration) (Wheelwright 1991).
Among these methods, ion-exchange chromatography is
commonly used to separate bromelain from pineapple
fruit, juice and wastes. For example, Murachi and
Neurath (1960b) obtained two major components, fractions 2 and 5, which were similar in their activities
toward various substrates after ion-exchange fractionation of pineapple juice using Duolite CS101 at
pH 6.05. The purification procedure was improved by
Murachi et al. (1964) over the aforementioned method
(Murachi and Neurath 1960) through the removal of
contaminant carbohydrates and colored material. The
latter study successfully produced 0.87 g of purified
bromelain from 10 g crude commercial bromelain by
integrating gel filtration and ammonium sulfate and
acetone precipitation steps after ion-exchange purification. Similarly, Yamada et al. (1976) applied ammonium
sulfate precipitation after anion-exchange chromatography, which yielded a homogeneous fruit bromelain with
a high specific activity of 10 U/mg. Ota et al. (1964)
supplemented the observations of Murachi et al. (1964)
by further characterizing the extraneous end-group of
stem and fruit bromelain after purification by gel filtration and ion-exchange chromatography. Further fractionation by successive use of gel filtration using Sephadex
G-75 and ion exchange using carboxymethyl (CM)Sephadex and diethylaminoethyl (DEAE)-Sephacel successfully separated stem bromelain into six proteolytic
components (SBA and SBB 15) and two active components of fruit bromelain fraction A (FBA) and B
(FBB) (Ota et al. 1985). Likewise, several studies on
stem bromelain purification using cation-exchange and
affinity chromatography yielded nonglycosylated components of stem bromelain, referred to as ananain (Napper
et al. 1994; Rowan et al. 1990) and F9 (Harrach et al.
1995), as well as the glycosylated components comosain
(Napper et al. 1994), F4 and F5 (Harrach et al. 1995),
and SBA/a and SBA/b (Harrach et al. 1998).
Meanwhile, Ko et al. (1996), Devakate et al. (2009),
and Gautam et al. (2010) had employed additional steps
of ammonium sulfate precipitation and dialysis prior to
ion-exchange chromatography purification to remove
most of the impurities in the stem and fruit bromelain
samples. A summary of the purification condition of
ion-exchange chromatography is presented in Table 4.

Harrach et al. (1998)

Hale et al. (2005)

20 mM acetate buffer + 1 M NaCl
20 mM acetate buffer, pH 5

Cation

Anion (Duolite A-2)

Cation (Mono Q HR)

Cation (SP-Sepharose)

Stem bromelain

Stem bromelain

Fruit bromelain

20 mM acetate buffer + 0.1 mM EDTA, pH 5

Murachi et al. (1964)

Devakate et al. (2009)

25 mM acetate buffer + 1 M NaCl, pH 4

0.05 M potassium phosphate buffer +

1 M KCl, pH 6.1
20 mM Tris-HCl + 250 mM NaCl, pH 8
0.05 M potassium phosphate
buffer, pH 6.1
20 mM Tris-HCl, pH 8

25 mM acetate buffer, pH 4

Anion (DEAE cellulose)

Stem and fruit

bromelain
Fruit bromelain

10 g in 100 ml (0.05 M potassium phosphate

buffer, pH 6.1)
8 mg in 3 ml (20 mM Tris-HCl, pH 8)

Gautam et al. (2010)

25 mM Tris-HCl + 50 mM NaCl, pH 8

Anion (Q-Sepharose)

Cation (CM-Sephadex)
Stem bromelain

Fruit bromelain

Anion (DEAE cellulose)

Stem bromelain

3 g in 0.02 M sodium citrate buffer

saturated with phenylmercuric
acetate, pH 6.10
50 g in 0.1 M sodium phosphate buffer +
5 mM EDTA + 5 mM cysteine
10 mM Tris-HCl buffer

0.02 M potassium phosphate buffer +

100 mg sodium tetrathionate (pH 7.2)
1 g in 0.2 M potassium phosphate, pH 6.05
Anion (DEAE cellulose)

0.5 M sodium phosphate

buffer, pH 8
25 mM acetate buffer, pH 4

Bruno et al. (2006)

50 mM Tris buffer + 0.3 M NaCl, pH 8.5

Ota et al. (1985)

0.01 M sodium citrate buffer saturated
with phenylmercuric acetate, pH 6.10

0.02 M sodium citrate buffer

saturated with phenylmercuric
acetate, pH 6.10
50 mM Tris buffer, pH 8.5

Murachi and
Neurath (1960b)

Yamada et al. (1976)

8.3 mg in 2 ml (50 mM bicine buffer, pH 8.9)

Cation (CM-Sephadex)

Stem bromelain
F4 and F5
Fruit bromelain

0.02 M potassium phosphate

buffer (pH 7.2)
0.2 M potassium phosphate,
pH 6.05

Gradient, 0150 mM NaCl, F4 and F5

obtained at 50 and 60 mM of NaCl
0.4 M potassium phosphate buffer
(pH 7.2)
0.2 M potassium phosphate + 2 M KCl,
pH 6.05
50 mM bicine buffer, pH 8.9

Elution
Binding and washing
Lysis buffer
Type of ion exchange
Type of bromelain

Table 4 Purification conditions for ion-exchange chromatography

Harrach et al. (1995)

Appl Microbiol Biotechnol (2014) 98:72837297

References

7292

Future directions
Up to the present, bromelain is a plant protease of choice,
owing to its multitude of applications in industries such as
tenderization, food, beverage, and pharmaceuticals. However,
the utilization of bromelain is still in its infancy due to the
rising dispute whether pineapple crops should be designated
for growth as food or for bromelain extraction. Additionally,
as the main source of bromelain, pineapples carry issues of
space required for cultivation, seasonal growth, differences in
pharmacological activities, and accessibility of mass production. Therefore, variation in the availability of bromelain
sources, such as waste generated from pineapple processing
in the form of the crown, peel, stem, and core, is likely to be
crucial to fulfill the demand for this enzyme. Separation and
isolation of bromelain from pineapple crops are only in the
developmental stage, where challenges in technology, purity,
and the economic aspects of large-scale production still need
to be explored. A full discussion of the pros and cons of all
listed purifications is shown in Table 5.
Thanks to the advent of recombinant technology which
exploited E. coli as the workhorse for gene expression systems, the cloned bromelain gene has finally opened up the
possibility of tailor-made protein with desirable properties
(Amid et al. 2011). Despite the extensive application of protein engineering, there are still research bottlenecks that
should be alleviated in the downstream process of recombinant bromelain production. The insertion of a poly-histidine
sequence at the N- or C-terminus of the protein-coding region
seems to allow the binding of recombinant bromelain to
affinity column that has metal ions attached to it, but such a
protein might be susceptible to co-elute with the native E. coli
proteins, which are referred to as contaminants during the
purification process. Therefore, the optimization of purification conditions or re-engineering an E. coli strain by mutation
or alternative tag addition can be a realistic solution to improve the purity of recombinant bromelain and reduce the
number of purification steps. However, in order to avoid
unnecessary protein binding to the column, sometimes additional steps such as centrifugation, ultrafiltration, or precipitation are required to remove remaining host cell proteins,
polishing, and intensification of the desired protein. The multiple steps in the chromatographic method associated with the
huge amount of manufacturing cost needed ultimately will
cause a significant drawback in the large-scale downstream
process.
Among nonchromatographic methods presented in the
aforementioned literature, the aqueous two-phase systems
(ATPS) could be promising with the burgeoning interest in
bromelain research. This method was adapted successfully in
various researches that involve recombinant proteins with a
high degree of purity (Bhambure et al. 2013; Lan et al. 2013;
Omidinia et al. 2010). This purification method is easy to scale

Appl Microbiol Biotechnol (2014) 98:72837297

7293

Table 5 Comparison of different purification strategies

Type of purification

Advantages

Disadvantages

Ultrafiltration

Concentrate the protein

Efficient to remove trace contaminants

Precipitation

Long separation process

Increase protein loading will cause the ultrafiltration membrane
clogging
Need a dialysis step to remove excess salt during precipitation

Easy to scale up with simple equipment requirements

Low energy needed
Efficient to remove trace contaminants
Low cost and shorter separation time
Difficult recovery of target protein from phase-forming
Easy to scale up and produce higher yield
polymer
Efficient to remove trace contaminants
Easy to scale up and can be operated in a continuous mode Difficult recovery of target protein from surfactant-containing
No loss of native protein and activity
organic solvent
Low interfacial tension
Highly specific and scalable
Low separation efficiency and higher cost
Small sample loading capacity
Multiple chromatography steps

Aqueous two phase

Reverse micelle
System
Chromatography

up as it only comprises water, salt, and polymer which is a

biocompatible environment. With the optimized condition of
pH and composition of salt and polymer, phase separation
using centrifugation results in just one-step separation.
Nevertheless, few challenges still hampered large-scale
ATPS especially in the mechanism of phase separation (interaction between the protein, salt and polymer), reusability and
toxicity of the polymer, and mode of operation (batch,
semicontinuous, or continuous) (Azevedo et al. 2009).
Current research in the manipulation of protein affinity to a
biospecific ligand in the phase-forming polymer will favor the
selective partitioning of the target protein. Besides, in view of
the resolution to overcome drawback in the conventional
ATPS system which requires back extraction to recover salt
or polymer, the use of thermoseparating polymer could be one
promising candidate. The temperature-induced phase
partitioning method will offer cost-effectiveness and recyclability to overcome the bulk industrial waste disposal of polymer. Finally, pre-evaluation and detailed cost comparison with
other separation methods must be taken into account to determine the viability of the ATPS process in an industrial scale
(Espitia-Saloma et al. 2013).

Conclusion
Bromelain is among the protease enzymes that have been
studied extensively. With the development of recombinant
bromelain, new applications are expected in the field of pharmaceutical industry. Since the demand for large quantities of
pharmaceutical products increases tremendously, the aqueous
two-phase system offers high load capacity and easy to scale
up for separation process with no compromise in the purity
aspects. Modern methods of protein engineering combined
with high-throughput approaches such as automatic

instrumentation during data collection and analysis are

expected to enhance experimental efforts and create
systematic methods. Thus, extensive effort involving
the cooperation of many experts from various scientific
and technological fields should be devoted to identify
previously unexplored strategies to characterize recombinant bromelain for novel applications.

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