The Protein Journal
https://doi.org/10.1007/s10930-021-09974-9
Soluble Expression and Catalytic Properties of Codon‑Optimized
Recombinant Bromelain from MD2 Pineapple in Escherichia coli
Rafida Razali1 · Cahyo Budiman1
· Khairul Azfar Kamaruzaman1 · Vijay Kumar Subbiah1
Accepted: 3 March 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due
to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro;
accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro
was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into Escherichia
coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at
its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The
protein was successfully purified using single-step ­Ni2+-NTA chromatography and confirmed to be in proper folds based
on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against
N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U ­mg−1 and inhibited
by a cysteine protease inhibitor, E-64 ­(IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro
was calculated to be at 5.64 ± 0.02 × ­10–2 µM−1 ­s−1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or
metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are
important in the production of a soluble and actively stable recombinant bromelain.
Keywords Codon optimization · Bromelain · Cysteine proteases · Ananas comosus · MD2 pineapple
1 Introduction
Bromelain refers to a mixture of cysteine proteases family of
proteins obtained from pineapples (Ananas comosus). It has
a catalytic mechanism that involves the triad Cys-His-Asn/
Glu [1, 2]. This enzyme is present in many different tissues
of pineapple, but is mainly found in high concentrations in
the stem and fruit of the crop. It is known to be homologous
to papain, which is found in papaya (Carica papaya) [3].
Bromelain has gained wide interest due to its wide range of
applications in the food, pharmaceutical, cosmetic, and textile industries [4]. The use of bromelain in the food industry
has been approved by the US Food and Drug Administration
and is considered as a safe food supplement in humans [5].
* Cahyo Budiman
cahyo82@gmail.com
1
Biotechnology Research Institute, Universiti Malaysia
Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia
While there have been many studies on the application
of bromelain extract in various food and medical systems,
however, to our knowledge, the study on recombinant bromelain remains limited. Recombinant bromelain refers to
a purified single cysteine protease, instead of a mixture as
presented in crude bromelain extracts. It is obtained from
the overexpression of a gene encoding for bromelain under
a heterologous expression system. Some studies have shown
that recombinant proteins behave differently compared to
their non-recombinant (native) forms [6–8]. In this regard,
the use of recombinant technology under the heterologous
expression system for the production of bromelain is so far
challenged by the issue of expression and solubility [9–11].
Bachman [12], Srividya et al. [13], and Ikehata and Ono
[14] highlighted several strategies to overcome these issues
which mainly deal with slowing down the expression rate
of recombinant protein. Nevertheless, these strategies often
fail to address the intricacies of heterologous expression
of the protein recombinant under E. coli system due to
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R. Razali et al.
inherent codon bias. This problem arises when the codon
profile of the gene interest is not compatible with the host
either due to the existence of rare codon, G/C ratio, or other
related issues [15]. Given that bromelain is from a plant
species, codon bias is often an issue for the expression of a
plant protein under E. coli system. An alternative approach
to address this issue is to optimize the codon in the target
gene, without changing the amino acid sequences, in order
to mimic the codon preferences of the host [16–18]. Codon
optimization has been reported to be able to increase the
expression of recombinant proteins by more than 1,000-fold
[19]. As a current wave of new technologies allows us to
synthesize DNA at an affordable cost and faster rate, codon
optimization is currently doable through re-synthesizing the
whole gene sequence with the desired codon profile [15].
The codon optimization approach, nevertheless, has never
been attempted to address the production of recombinant
bromelain in E. coli.
Furthermore, as pineapple contains many genes encoding
for bromelain, the selection of the appropriate gene for further overexpression is also challenging. In spite of that, this
approach is time-saving and considerably cheaper in cost to
obtain the bromelain for various applications. Recent studies
on a whole-genome sequence of MD2 pineapple revealed
that this hybrid variety has 14 genes encoding for bromelains
of various sizes [20, 21]. Among these, one gene in particular (accession code: OAY85858.1), hereafter designated as
MD2Bro, encoded for fruit bromelain and was similar to
the previously reported fruit bromelain [21] yet with the
presence of a unique additional N- or C-terminal on its
sequence. This led to the postulation that MD2Bro may be a
good model candidate to determine the effect of recombinant
fruit bromelain on the physicochemical properties of meat as
well as the development of molecular strategies to produce
recombinant fruit bromelain in soluble and active forms.
This study describes our attempts to develop a molecular
strategy for the production of MD2Bro in soluble and active
forms. A combination of codon optimization and the use
of appropriate vector used in this study were found to be
a promising strategies for obtaining the recombinant bromelain in soluble and active forms. Of noteworthy, this is
the first soluble and active recombinant bromelain produced
from a codon-optimized synthetic gene.
2 Materials and methods
2.1 Gene optimization, Synthesis, and Expression
System Construction
The DNA sequence of fruit bromelain MD2Bro was
retrieved from the MD2 pineapple genome listed under
the GenBank accession number OAY85858.1. Codon
13
optimization was then performed with OptimumGene™
software of GenScript (NJ, USA), and subsequently, the gene
was then chemically synthesized by GenScript (NJ, USA)
and inserted into pET-32b( +) through KpnI and XhoI sites
yielding an expression system of pET-32b( +)-MD2Bro.
The construct was designed to allow the expressed MD2Bro
to possess thioredoxin (Trx)- and 6-His-tags at its N- and
C-terminal, respectively. Therefore, the fusion protein consisted of the Trx segment followed by the MD2Bro segment
and 6-His-tag, which was then designated as Trx-MD2Bro.
2.2 Overexpression
Overexpression and purification of pET-32b( +)-MD2Bro
protein were according to George et al. [9]. Briefly, pET32b(+)-MD2Bro was transformed into E. coli BL21CodonPlus (DE3) and grown overnight in Luria–Bertani
(LB) medium supplemented with 100 µg/ml ampicillin
and 25 µg·ml−1 chloramphenicol at 37 °C. The expression
of Trx-MD2Bro was then induced by 1 mM of isopropyl
β-D-1-thiogalactosidase (IPTG) once the O
­ D600nm reached
0.4—0.7, followed by further incubation at 37 °C, 180 × g
for 3 h. The cells were then harvested by centrifugation at
8,000 × g at 4 °C for 10 min. The pellet was then washed
twice and resuspended with lysis buffer (20 mM phosphate
buffer, pH 8.0) and further lysed by sonication in ice. The
soluble fraction was then separated from the cell debris by
centrifugation at 35,000 × g for 30 min at 4 °C and collected
for downstream purification. For the production of Trx protein (with no MD2Bro), only the empty pET-32b(+) plasmid was transformed into E. coli BL21-CodonPlus (DE3).
The transformant harboring empty pET-32b(+) plasmid
was cultured, and the Trx protein was expressed under the
same condition as Trx-MD2Bro. As it was expressed from
the empty pET-32b(+) plasmid, the expressed Trx protein,
therefore, contained an additional sequence (77 amino acids;
8265.91 Da) at its C-terminal.
2.3 Purification
The purification of Trx-MD2Bro was conducted using a
­Ni2+-NTA affinity chromatography according to Budiman et al. [22] with some modifications. The column was
firstly equilibrated with 20 mM Tris–HCl pH 8.0 containing
100 mM NaCl and 20 mM imidazole. The soluble fraction
which was filtered previously using 0.22 µM was loaded
into the column at the flow rate of 1 mL·min−1. The elution of bound proteins was conducted through a linear gradient of increasing concentration (0—500 mM) of imidazole in 20 mM Tris–HCl pH 8.0 containing 100 mM NaCl.
Trx protein was also purified under the same condition as
Trx-MD2Bro.
Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from…
2.4 SDS‑PAGE
Expression, solubility, and purity of the Trx-MD2Bro protein were confirmed by using 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) [23,
24]. Similarly, the purified Trx protein was also confirmed
with 15% SDS-PAGE. The gel was stained with Coomassie
Brilliant Blue (CBB) dyes [23] and visualized using a Gel
DICTM XR + imager (Biorad, CA, USA).
2.5 Western Blot
To confirm the expression of Trx-MD2Bro, the protein
bands on the gel were transferred to a nitrocellulose membrane (Amersham Biosciences, UK) using Trans-Blot
Turbo Transfer System (Bio-Rad Laboratories, Inc., USA)
with 1 × transfer buffer (25 mM Tris, 190 mM glycine,
20% methanol) for 10 min at room temperature. The membrane was then incubated for 1 h at room temperature with
Block ACE blocking reagent (Bio-Rad Laboratories, Inc.,
USA) to block non-specific binding. This was then followed
by incubation with primary antibody (Mouse Anti Penta
Histidine Tag:Hrp) (Bio-Rad Laboratories, Inc., USA) in a
ratio of 1:2000. The membrane was subsequently incubated
with secondary antibody of Goat Anti-Mouse IgG (H + L)HRP conjugate (Bio-Rad Laboratories, Inc., USA) in a ratio
of 1:5000. The membrane was later incubated with 4 × wash
buffer and 2 × PBS buffer for 5 min, each. The membrane
was developed using the Clarity™ ECL substrate (BioRad Laboratories, Inc., USA) and the images of Western
blot were visualized using ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc., USA) for 1 min at room
temperature.
2.6 Protein Concentration
The concentration of Trx-MD2Bro and Trx protein were
calculated based on the UV absorption at 280 nm with the
extinction coefficient (ε) at 0.1% (1 mg ­mL−1) of 1.65 and
0.70, respectively. This coefficient was calculated using
ε = 1,576 ­M−1 ­cm−1 for Tyr and 5,225 ­M−1 ­cm−1 for Trp at
280 nm [25].
2.7 Circular Dichroism (CD)
A J‐725 automatic spectropolarimeter (JASCO Co., Japan)
was used for the measurement of the CD spectra. The protein
was prepared in 20 mM sodium phosphate (pH 8.0) at the
concentration of 0.2 mg ­mL−1 or 0.7 mg ­mL−1 for far-UV
(200—260 nm) and near-UV (250—320 nm) CD spectra,
respectively. The measurements of the far-UV and near-UV
CD spectra were conducted in the cell with an optical path
length of 2 mm and 10 mm, respectively. Prior to the measurement, the protein was incubated at 20 °C for 30 min.
Changes of the CD value at 222 nm against varying temperatures (20 – 90 °C) were monitored to determine the thermal denaturation (unfolding curve) of the protein. The melting temperature (Tm), referring to the transition point, was
calculated from the curve fitting of the resultant CD values
versus temperature data using the least-squares method. The
mean residue ellipticity, θ, (deg·cm2·dmol−1) was calculated
by using an average amino acid relative molecular mass of
110 [26, 27].
2.8 Enzymatic Activity
The specific activity and kinetic parameters of Trx-MD2Bro
were performed according to the previous report using a
synthetic substrate of N-carbobenzoxyglycine p-nitrophenyl
ester (N-CBZ-Gly-pNP) [28]. For specific activity, the different ranges of enzyme concentrations (0.5 – 10 µg ­ml−1)
were incubated in 20 mM Tris–HCl pH 8.0 at 37 °C for
5 min with shaking prior to the addition of the substrate
(50 µM of final concentration). The product released after
a 5-min reaction was then monitored using a Perkin-Elmer
Lambda 35 UV–visible spectrometer (Massachusetts,
USA) at 340 nm. One unit of the enzyme was defined as the
amount of enzyme which produced 1 µmol of the product
per minute. The inhibition by a cysteine protease inhibitor
of trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane
(E-64) was done by measuring the specific activity of TrxMD2Bro in the presence of varying concentrations of E-64
(1 – 1000 nM). The ­IC50 value, which refers to the concentration of the sample required to reduce 50% of the catalytic
activity, was calculated using a four-parameter logistic curve
under the SigmaPlot 14.0 statistical software (CA, USA).
For the kinetic parameters, the Michaelis–Menten saturation curve and Lineweaver–Burk plots of the enzyme
were constructed for the calculation of Michaelis–Menten
constant ­(KM), maximum velocity (­ Vmax), and the turnover
number (kcat) [28]. For this purpose, the assay was done
in the presence of various concentrations of substrate (1
– 1000 µM) and 8.8 nM of Trx-MD2Bro. The assay was
done in 20 mM Tris HCl pH 8.0 at 37 °C. All experiments
were conducted in triplicates.
2.9 The Optimum Temperature and pH
The optimum temperature and pH for the catalytic activity
of Trx-MD2Bro were determined according to the previous
report [29]. For the optimum temperature, the protein was
dissolved in 20 mM Tris-HCl pH 8.0 and exposed to different temperatures ranging from 25 °C – 90 °C for 5 min
in the heating block. Meanwhile, for the optimum pH, the
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R. Razali et al.
activity was measured in 20 mM citrate buffer (pH 4.0 – 6.0)
or 20 mM Tris-HCl (pH 6.0 – 8.0) or 20 mM Gly-NaOH
(pH 8.0—pH 10.0). The highest activity obtained during
the measurement was set as 100% of the specific activity.
2.9.1 Determination of EDTA and Metal Ions Effect
The determination of ethylenediaminetetraacetic acid
(EDTA) solution and metal ions effect on catalytic activity of Trx-MD2Bro was performed according to previous
reports [30–32] with some modifications. The Trx-MD2Bro
activity assayed with the addition of different metal ions
­(Ca2+, ­Cu2+, ­Mg2+, ­Zn2+, ­Ni2+) or EDTA with a final concentration of 10 mM at the optimum temperature and pH.
3 Results
3.1 Expression and Solubility
The gene encoding fruit bromelain MD2-MBro was optimized by increasing the GC content of MD2Bro gene from
47.40 to 55.98%. This value was in the range of a favorable
GC content for E. coli host cells [33]. In addition, to prevent
premature translational termination, the removal of AT-rich
regions in the new sequence was also performed. Lastly,
re-adjustment of the codon adaptation index (CAI, 0.37) on
the MD2Bro gene was performed yielding a final CAI value
of 0.96 which indicated that 96% of the codon profile was
compatible with the codon preferences of the host. A higher
CAI index was reported to indicate higher suitability for the
expression of the gene of interest in the heterologous host
[34]. The optimized MD2Bro protein was expressed in E.
coli as a fusion protein (Trx-MD2Bro) in a fully soluble
form with an apparent size of 56 kDa (Fig. 1a). Western blot
analysis using a 6-His-tag primary antibody also produced
a remarkable signal on the band that corresponds to the size
of Trx-MD2Bro which confirmed that the expressed protein was indeed Trx-MD2Bro (Fig. 1a). Furthermore, the
protein was also successfully purified using a single-step
­Ni2+-NTA chromatography (Fig. 1b). The identification of
the purified band was also confirmed by Western blot analysis, which indicated that the band was indeed Trx-MD2Bro
(Fig. 1b). The purification yield of MD2Bro in this study
was estimated at 20 mg from 1 L culture, with the detail of
the purification profiles shown in Table 1. The purification
of Trx protein from empty pET-32b( +) was also successful as indicated by a single band under 15% SDS-PAGE
(Fig. 1c) with the yield of 9 mg from 1 L culture. To note,
the apparent size of the purified Trx protein (20 kDa) was
higher than the theoretical size of Trx (11.62 kDa). This discrepancy is due to additional sequences originated from the
plasmid (77 amino acids, with the size of about 8.26 kDa).
13
The specific activity of MD2Bro toward N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) substrate was
6.13 ± 0.01 U ­mg−1, which was 20-fold higher than the crude
extract’s activity (Table 1).
Of note, the thioredoxin tag was not cleaved in this
experiment due to limited resources on our side and the risk
of degradation upon the cleavage. Nevertheless, purified
Trx protein was confirmed to have no activity towards the
N-CBZ-Gly-pNP substrate. Figure 2 showed that no yellow
color was formed when the substrate was incubated in the
presence of purified Trx protein, which indicated no cleavage products in the reaction. Also, there was no absorbance
reading using UV–Vis spectrophotometer at 340 nm for the
solution. Meanwhile, an obvious yellow color was visible
in the presence of Trx-MD2Bro, which suggested that the
substrate was cleaved to produce a free form of chromogenic pNP moiety. These results confirmed that the activity
obtained is genuinely generated by Trx-MD2Bro. Note that,
the catalytic activity of MD2Bro was remarkably inhibited
by E-64, a cysteine protease-specific inhibitor, in a concentration-dependent fashion (Fig. 3), with an apparent ­IC50
value of 74.38 ± 1.65 nM. This indicated that MD2Bro was
indeed a cysteine protease member.
3.2 CD Spectra Properties
Far-UV CD spectra (Fig. 4a) revealed that Trx-MD2Bro
exhibited an α + β protein-like spectrum with a minimum
value at 214 – 219 nm, suggesting that the purified TrxMD2Bro produced through our system was correctly folded.
The estimated helical content of MD2Bro was about 35%
as calculated from its spectrum based on the method of Wu
et al. [35]. In addition, Fig. 4b shows the near UV CD spectrum of MD2Bro that reflects the three‐dimensional environments of aromatic residues such as Trp (270–290 nm), Tyr
(280–300 nm), and Phe (255–270 nm). Figure 4b showed
three strong and distinct signal peaks at 255, 270, and
280 nm which correspond to Phe, Trp, and Tyr residues of
MD2Bro, respectively. This indicates that the expressed and
purified MD2Bro obtained was properly folded with tight
tertiary packing of aromatic side chains. It was interesting
to note that the amino acid sequence of MD2Bro (in a fusion
form with thioredoxin) showed that the protein contained 11
Trp, 23 Tyr, and 21 Phe. Figure 4a-b also showed that the farUV and near-UV CD spectra of Trx protein were remarkably
different from that of Trx-MD2Bro. Far-UV CD spectrum of
Trx protein was lower than that of MD2Bro, which indicated
lesser helical content than MD2Bro (Fig. 4a). Besides, the
signal of the near-UV CD spectrum of Trx protein was also
lower than MD2Bro, which indicated that fewer aromatic
residues are present as compared to MD2Bro. Furthermore,
the unfolding curve of Trx-MD2Bro which reflected temperature-induced structural changes in Trx-MD2Bro at 222 nm
Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from…
Fig. 1 a Expression check of Trx-MD2Bro by 15% SDS-PAGE
and Western blot. Lane 1: Before IPTG induction; Lane 2: After
IPTG induction; Lane 3: Soluble fraction obtained after the sonication; Lane 4: Insoluble fraction obtained after the sonication. The
band corresponds to Trx-MD2Bro is indicated by the arrow; b 15%
SDS-PAGE and Western blot analysis of purified Trx-MD2Bro after
Table 1 Purification profile
of Trx-MD2Bro indicating its
activity and yield
Step
Volume (mL)
Soluble frac60
tion (Crude)
32
Ni2+-NTA
affinity chromatography
­Ni2+-NTA chromatography. P lane indicates the purified protein. The
band corresponds to Trx-MD2Bro is indicated by the arrow. c Purified Trx protein under 15% SDS-PAGE. P lane indicates the purified protein. The band corresponds to Trx protein is indicated by the
arrow
Total protein (mg)
Total activity (U)
Specific
activity
(U ­mg−1)
Yield (%)
Purification
(fold)
499
149.51
0.30
100
1.0
20
122.60
6.13
82
20.4
13
R. Razali et al.
indicates a single thermal transition and fits the two-state
model well (Fig. 5). Accordingly, the thermal melting point
(Tm) of MD2Bro was calculated to be 54.93 ± 0.2 °C. Figure 5 also showed the unfolding curve of Trx protein, which
is remarkably different from that of Trx-MD2Bro, with the
Tm value of 62.71 ± 2.85 °C.
3.3 Kinetic Parameters of Trx‑MD2Bro
Fig. 2 a Catalytic reaction mixture in the absence of enzyme (only
buffer); b Catalytic reaction mixture in the presence of purified Trx
protein (100 µg.mL−1); c Catalytic reaction mixture in the presence of
purified Trx-MD2Bro (1.0 µg.mL−1). N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) was used as a substrate in the reaction mixture
The kinetic parameters of Trx-MD2Bro were derived
using increasing concentrations of N-CBZ-Gly-pNP.
Michaelis–Menten curve was constructed (Fig. 6a),
and the ­KM and ­Vmax for N-CBZ-Gly-pNP were determined using Lineweaver–Burk plots (Fig. 6b). Accordingly, the purified Trx-MD2Bro exhibited catalytic activity with a ­KM of 34.24 ± 1.02 µM, kcat of 1.93 ± 0.05 ­s−1,
­V max of 0.017 ± 0.0005 µM ­s −1 , and k cat /K M of
5.64 ± 0.02 × ­10–2 µM ­s−1.
3.4 Temperature‑ and pH‑dependent Activities of
Trx‑MD2Bro
Fig. 3 Concentration-dependent inhibition of Trx-MD2Bro by
cysteine protease inhibitor E-64. The activity in the absence of E-64
inhibitor was adjusted as 100%
The catalytic activity of Trx-MD2Bro towards N-CBZGly-pNP measured at various temperatures indicates that
the highest activity was observed at 50 °C (Fig. 7a). This
seems to suggest that 50 °C is the optimal temperature
for the catalytic activity of Trx-MD2Bro. Meanwhile,
at temperatures higher than 85 °C, the catalytic activity
remains, albeit only less than 20% of the activity compared to the optimal temperature (Fig. 7a). On the other
hand, at 25 °C, the residual activity was about 40% of the
optimal temperature. The result seems to suggest that this
Fig. 4 a Far-UV and b Near-UV CD spectrum of the purified Trx-MD2Bro (black circle) and Trx protein (gray circle)
13
Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from…
3.5 Effect of Metal Ions on Catalytic Activity
of Trx‑MD2Bro
Fig. 5 Unfolding curve of the purified Trx-MD2Bro (black circle)
and Trx protein (gray circle) observed under the changes of CD value
at 222 nm from 20–90 °C
enzyme remains active, albeit with very low activity, at
temperatures lower than 25 °C.
Meanwhile, when the activity of Trx-MD2Bro was
measured at different pH, it was found that the recombinant protein had a broad working pH (pH 4.0–pH 10.0)
with optimum activity observed at pH 6.0 (Fig. 7b).
The declining catalytic activity at alkaline pH was more
extreme as compared to that at the acidic level (Fig. 7b).
Residual activity at pH 4.0 was more than 50% which
suggested that this enzyme remains active even at low pH.
The addition of EDTA had remarkably decreased the activity of Trx-MD2Bro which suggested that the recombinant
protein may require metal ions for its activity (Fig. 8). Nevertheless, the identity of the metal ions that are required for
the enzyme activity remains yet to be investigated. Figure 8
further showed that the addition of M
­ g2+, ­Ni2+, and C
­ a2+
ions remarkably increased the activity of the Trx-MD2Bro
up to 176%, 111%, and 118%, respectively. This suggested
that all the three types of metal ions are possible cofactors
for MD2Bro. While this may be so, there may be a possibility that the increased level of bromelain activity seen here
could also be due to some other metal ions inherent in the
buffer. This, nevertheless, needs to be further investigated.
4 Discussions
In this study, the gene encoding MD2Bro was synthetically
produced and the protein was expressed using an E. coli system. The use of synthetic gene in this study was performed
to avoid the tedious and lengthy process of constructing
the cDNA encoding MD2Bro. Besides, the synthetic gene
allows the codon optimization process to be done to ensure
that the gene is compatible with the expression machinery
of the host cells (E. coli). The differences in codon usage
between the source organism and the host cells were found
to be the major factors associated with expression problems
(expressibility and solubility) in heterologous expression
system [36]. Indeed, various attempts in the production of
bromelain through heterologous expression had ended up
Fig. 6 a Michelis-Menten curve; b Lineweaver–Burk plot of the purified Trx-MD2Bro
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R. Razali et al.
Fig. 7 a Temperature- and b pH-dependent activities of the purified Trx-MD2Bro. The highest activity was adjusted as 100%
Fig. 8 Effect of EDTA and metal ions on the activity of purified TrxMD2Bro. The activity without any metal ions or EDTA was used as
control and adjusted as a 100% catalytic activity
with a low production level and insolubility [9]. Codon optimization was widely reported to overcome the issues related
to expressibility and solubility of the target proteins under
heterologous expression system [37–48], but has never been
used for the production of recombinant bromelain.
The optimized gene of MD2Bro is considerably compatible for E. coli as a host cell. This is based on GC content and
CAI index of the optimized gene which were in the range
for E. coli [33, 34]. Besides, the absence of AT-rich regions
in the optimized gene was also preferable for the expression in E. coli. The optimization was only performed at the
DNA level without any changes at the translated amino acid
sequences. Therefore, it is believed that the expressed protein should not be affected by the optimization. The result
in Fig. 1a clearly showed that MD2Bro was successfully
heterologously expressed in E. coli, in a fusion form of TrxMD2Bro, in a soluble form which is believed to be due to
the gene optimization approach. Indeed, some reports on
the insoluble expression of bromelain under E. coli system
were conducted without the optimization step ended up with
13
failure on expression or having solubility issue [9]. Besides,
thioredoxin tag fused at the N-terminal of MD2Bro was
also believed to contribute to the solubility of the expressed
protein. The success of thioredoxin in enhancing solubility in other recombinant proteins was also supported by a
study conducted by Yasukawa et al. [49]. Interestingly, the
recombinant bromelain without the codon optimization step
expressed in a thioredoxin fusion form remains in the insoluble form [9]. This indicated that a combination of the codon
optimization and thioredoxin fusion steps formed an important strategy in the production of recombinant bromelain in
a soluble form.
Noteworthy, the apparent size of Trx-MD2Bro as
shown in SDS-PAGE was about 56 kDa (Fig. 1b), which
was remarkably higher than that of the theoretical size of
MD2Bro (39 kDa) calculated from its amino acid sequence.
This discrepancy was due to the existence of two tags of
thioredoxin and 6-His-tags. In this study, the tags were not
removed due to the high risk of protein degradation during the cleavage of the tags and limited resources. Besides,
there was no report on the proteolytic activity of Trx or
6-His-tags; thus, it is believed that the presence of these
tags should have no contribution to the activity of TrxMD2Bro. Indeed, Fig. 2 clearly demonstrates that there
was no Trx protein activity against the N-CBZ-Gly-pNP
substrate used in this study. Furthermore, the size of TrxMD2Bro (56 kDa) was also comparable to the accumulative
size of MD2Bro (38 kDa) and Trx fragment with additional
sequence (20 kDa). This indicated that the MD2Bro gene
was fully translated into an intact polypeptide. BLAST analysis of MD2Bro indicated that the protein was organized into
two segments of inhibitor I29 superfamily and peptidase C1
superfamily. Ramle et al. [50] reported that these segments
(known as I29 and C1 lobes) were found in various fruit
bromelain structures, in which the C1 lobe was structurally
Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from…
more conserved than the I29 lobe. Nevertheless, both lobes
exist in the protein, which indicated no peptide removal during the translation of the fruit bromelain gene. In fact, the
cleft formed by these lobes was found to be important for
the catalytic sites configuration [50, 51].
It was interesting to note that the purification yield of TrxMD2Bro in this study was about 20 mg from 1 L culture,
which is extremely higher than Iffah et al. [52]. Unfortunately, previous studies that involved recombinant bromelain
by George et al. [9] and Amid et al. [10] did not report the
purification yield for comparison. The yield of purification
was found to be roughly 82% which was relatively high yield
for the bromelain purified using a chromatography technique
[53]. In particular, this value was slightly lower than the
yield of non-recombinant purified bromelain reported by
Yin et al. [54], but remarkably higher than the other recombinant bromelain [9, 10, 52]. In addition, the purification
fold obtained in this study was found to be higher than that
obtained from other chromatography techniques [52, 53].
The comparative analysis among several bromelains implied
that the efficiency of bromelain productions varied depending on the sources of the bromelain and purification techniques [53].
The purified Trx-MD2Bro was also shown to be in a
proper folded form based on CD spectra measurement
(Fig. 4). It is noted that the shape of far- and near-UV CD
spectra of Trx-MD2Bro may also be contributed by the Trx
tag. Earlier, Schneider et al. [55] reported the contribution
of the thioredoxin tag to the far-UV CD spectrum of the
protein. The presence of a Trx tag might contribute to the
far-UV CD spectrum (Fig. 4a) as reported earlier [55]. The
helical content predicted from the amino acid sequence of
MD2Bro was only about 33%. An additional sequence of
Trx segment increased the helical content to 36%, which is
comparable to the helical content calculated from the farUV CD spectrum. This confirmed that the spectrum of TrxMD2Bro not only originated from the MD2Bro, but also
from the Trx tag.
Notably, the far-UV CD spectrum of Trx-MD2Bro was
shown not to be identical to the other bromelains signal
[27, 56, 57]. This suggested that the spectrum might be
unique to each different variant types of bromelain. Nevertheless, all far-UV CD spectra of other bromelains also
demonstrated a typical of α + β proteins signal [27, 56, 57].
Similar typical far-UV spectra signals were also reported
in other cysteine protease members, including papain [58,
59], chymopapain [60], ficin [61] and baupain, a papain-like
enzyme from Bauhinia forficata leaves [62]. These results
have suggested that Trx-MD2Bro folded into the canonical
folding motif of other papain family members. Meanwhile,
the near-UV CD spectrum of Trx-MD2Bro (Fig. 4b) was a
positive signal indicating that the residues were in a folded
three-dimensional structure. To note, both far- and near-UV
of MD2Bro and Trx protein were found to be remarkably
different (Fig. 4a and b). This might be indicated that the
MD2Bro was indeed in a folded form in its fusion form (TrxMD2Bro). It is unlikely to assume that, in the Trx-MD2Bro
fusion form, only thioredoxin was in a folded form, while the
MD2Bro segment was unfolded. If this was the case, the farand near-UV CD spectra of Trx-MD2Bro should be identical
to the spectra of thioredoxin only. Nevertheless, the far- and
near-UV CD spectrum of Trx-MD2Bro was undoubtedly a
combination spectrum of the tag (thioredoxin) and the main
protein (MD2Bro). Besides, Trx-MD2Bro should also be
catalytically inactive due to the unfolded state of MD2Bro
in this fusion.
Interestingly, the in silico prediction using CYS_REC
server (http://www.softb ​ e rry.com/berry​ . phtml ​ ? topic​
=cys_rec&group​=progr​ams&subgr​oup=propt​) revealed
three possible S–S bonds in MD2Bro (Cys144-Cys184,
Cys178-Cys217, and Cys274-Cys325). While it was known
that the production of proteins with S–S bonds has been
considered difficult in E. coli [63], this study demonstrated
that MD2Bro had no issue with the formation of these S–S
bonds in the cytoplasmic region and hence was able to be
fully soluble, folded, and catalytically active. This result is
intriguing as the S–S bond containing proteins was usually
translocated into the periplasmic region for the proper S–S
bond formation [63]. Besides, the formation of S–S bonds
in the cytoplasm is an exceedingly rare event [64]. However,
several studies have reported that some S–S bonds that are
not required for the stability of the native state have been
detected in some cytoplasmic proteins, including ribonucleotide reductase, the transcription factors OxyR and RsrA,
the Hsp33 chaperone, and P22 tail spike endorhamnosidase
[65, 66]. In addition, Basette et al. [67] convincingly demonstrated that the cytoplasm could be rendered sufficiently
oxidizing to allow the efficient formation of native disulfide
bonds without compromising cell viability. Nevertheless,
whether the three S–S bonds exist in MD2Bro remains to
be experimentally confirmed.
It was interesting to note that the Tm value of TrxMD2Bro was considerably higher than favorable growth
temperatures in pineapple farms (18 – 32 ˚C) [68]. It
remains unknown if the Tm of Trx-MD2Bro is associated
with its biological roles in the pineapple fruit. The Tm value
obtained in this study was comparable to that reported by
Habib et al. [69]. Nevertheless, this value was about 20 ˚C
lower than that reported by Haq et al. [57]. This discrepancy may be due to the use of salt by Haq et al. [57] which
could have stabilized the protein by preventing unspecific
hydrophobic interaction. In addition, the Tm value of TrxMD2Bro was also lower than the glass transition temperature
(Tg) [70]. This is acceptable as Tg of the material is known
to be constantly higher than the melting temperature of the
material [71]. To note, in a fusion form of Trx-MD2Bro,
13
R. Razali et al.
the Trx tag possibly stabilized MD2Bro. Nevertheless, the
stabilization was not significant as the Tm of Trx-MD2Bro
was lower than that of Trx protein. This, however, has to be
experimentally confirmed.
The temperature-dependent activity of Trx-MD2Bro
strongly suggests that MD2Bro behaves as a mesophilic
activity, where it optimally works in the range of moderate temperature (Fig. 7a). To note, the Tm value of TrxMD2Bro calculated from the thermal unfolding was found
to be higher than 50 °C. At the melting temperature, protein
is in the transition of folding–unfolding state. Accordingly,
Trx-MD2Bro is believed to be still in the folded state at the
temperature 50 °C (lower than its melting temperature), and
thus, it is acceptable to have 50 °C as the optimum temperature for the Trx-MD2Bro. The broad working pH (pH 5.5
– pH 7.5) also suggested that acidic pH is more preferable
for Trx-MD2Bro to be active. Noteworthy, as Trx protein
was proven to have no activity (Fig. 2), it is, therefore, convincing enough to assume that the temperature-dependent
activity indeed reflected the activity of MD2Bro only, without the interference of thioredoxin.
The reduction of the catalytic activity of Trx-MD2Bro
by EDTA (Fig. 8) is in good agreement with the previous
reports on the study of other cysteine proteases [32, 73, 74].
Meanwhile, another study on bromelain reported that EDTA
did not significantly affect its activity [75]. Similar results
were also reported on the activity of bromelain-like cysteine
protease from Billbergia pyramidalis [76], Triticum aestivum
[77], Calostropis procera [30], and Curcuma longa [78].
This indicated that the inhibitory effect of EDTA is apparently not the general characteristic for cysteine protease as
found in other studies.
Further, Fig. 8 also implies that ­Mg2+, ­Ni2+, and ­Ca2+
ions may be the cofactors for Trx-MD2Bro which is in good
agreement with previous studies [31, 79–82]. The study by
Kaur et al. [72] explained that calcium ions stabilizing the
secondary structure of the bromelain, thus, promote the
enzyme activity. Furthermore, Haq et al. [82] proposed
that the effect of C
­ a2+ ions in the structure and function
of bromelain might also arise from other factors including
the changes on the shielding charge of protein and water
structural changes. In addition, C
­ a2+ ions may also affect
the binding of the protein to other ions (specific or nonspecific) that are present in its surrounding microenvironment [82]. Unfortunately, no detailed study on the effect
of ­Mg2+ and ­Ni2+ on bromelain activity is available. Broderick [83] reported that metal ions are required by at least
30% of thousands of enzymatic in function from Lewis acid
catalysis to redox catalysis to electron transfer. In addition,
metal ions provide strong electrophilic center which add to
the enzyme’s functionality. Meanwhile, Z
­ n2+ and C
­ u2+ were
found to decrease the bromelain activity by 41% and 76%,
respectively. Similar results were also reported for the effect
13
of these two ions on bromelain [31, 79–81, 84]. In addition,
Kaur et al. [72] reported the inhibitory effects of C
­ u2+ on
papain-like cysteine. The inhibition by these ions may be
due to the formation of coordination bond between the ions
and catalytic sulfhydryl group [79, 84, 85].
5 Conclusion
A combination of codon optimization and expression vector
selection was found to be an effective strategy to produce
recombinant fruit bromelain MD2Bro, in a fusion with Trx
tag, in soluble and active forms. The functional properties of
fusion form of Trx-MD2Bro were confirmed by its remarkable catalytic activity toward a synthetic substrate and its
dependency toward pH, temperature, and metal ions. The
findings from this work point towards the possibility of a
large-scale production of recombinant bromelain in soluble
and active forms.
Acknowledgment This study is financially supported by the research
grant of SGI0065-2018. The authors thank Dr. K. Takano and F.A.
Fahrudin for technical help and valuable comments on the manuscript.
Funding The Innovation Grant Scheme of SGI0065-2018.
Declarations
Conflicts of interest Authors declare no conflict of interest.
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