Research Article

Received: 5 October 2013 Revised: 23 December 2013 Accepted article published: 6 January 2014 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.4304

Optimal conditions for deproteinizing natural

rubber using immobilized alkaline protease
Siriwan Junoi,a Yusuf Chistib and Nanthiya Hansupalaka∗

Abstract
BACKGROUND: Natural rubber latex contains allergenic proteins. Therefore, processes for deproteinizing the latex are needed.
An immobilized enzyme process for deproteinization is reported. An optimal protocol was first developed for immobilizing
a protease on cellulose−chitosan composite beads. The beads were then used in developing an optimal deproteinization
treatment. The main effects and the interactions of the factors for the two processes were identified using a two-level full
factorial experimental design.

RESULTS: Under optimal conditions (15% cellulose in beads, immobilizing reaction pH of 9, immobilization period of 24 h), the
beads attained the highest specific activity of 1685.3 U g−1 beads. Using these beads, the optimal conditions for deproteinization
of the latex were: an enzyme loading of 0.1 parts per hundred of rubber (phr), a sodium dodecyl sulfate concentration of 20 phr,
30◦ C, and a treatment period of 12 h. The nitrogen content of the rubber was reduced to 0.012% from an initial value of 0.3%.
The enzyme beads were operationally stable and could be reused for at least five cycles.

CONCLUSIONS: The optimized treatment effectively deproteinized the natural rubber. The final product was free of peptides.
This deproteinization treatment was more effective than the conventional urea-based treatment performed for comparison.

c 2014 Society of Chemical Industry

Keywords: alkaline protease; cellulose; chitosan; deproteinization; enzyme immobilization; natural rubber latex

INTRODUCTION recovered and reused.14 In principle, good contact between the

Natural rubber (NR) latex is preserved using ammonia. In addition
to ammonia, the preserved latex contains small rubber particles
dispersed in water and the nonrubber components such as
proteins, carbohydrates and lipids.1 The proteins occur in the
aqueous phase as well as bound to the surface of the rubber
particles.2 These proteins are potent allergens2 and, therefore, the
products made from NR can be allergenic.3 Deproteinization of
NR reduces allergenic reactions.2,4,5 Also, the proteins attached to
the NR particles interfere with processes used in modifying the
properties of rubber.6,7 Protein-free natural rubber is known as
deproteinized natural rubber, or DPNR.
Rubber deproteinization methods include treatment with
enzymes, urea, surfactants, and acids and alkalis.4 – 6,8 Proteolytic
enzymes remove proteins by cleaving them into small
peptides.9 – 11 Urea and surfactants denature proteins to facilitate
their removal by washing. Acids and alkalis are both denaturants
and may also hydrolyze proteins. Urea treatment and enzymatic
treatment are equally effective and can reduce the total nitrogen
content of rubber to a lower level than the other treatment
methods. However, after a urea treatment, all urea must be
removed, or high levels of residual nitrogen may be detected
in the treated rubber as urea itself contains nitrogen.12 Also,
the urea treatment does not actually break down proteins, but
denatures them13 so that they are easier to wash away. Any
residual denatured protein retains the potential to be allergenic.
Current enzymatic treatments use soluble enzymes that are lost
after a single use. An alternative is to use enzymes immobilized
on polymer beads11 because a bead-bound enzyme can be easily
proteins on the NR particles and the surface immobilized enzyme
on beads is feasible as the rubber particles are extremely small
(100 nm to 2 µm)1 compared with the beads.11
An enzyme may be attached to a polymer bead by durable
covalent bonds. This form of immobilization can provide high
surface loading of the enzyme,15,16 but is generally irreversible.16,17
Immobilization generally improves enzyme stability relative to the
dissolved enzyme.16,18 Some enzyme activity may be lost during
immobilization and some of the bound enzyme may be inactive
because of steric hindrance of the active sites. Notwithstanding
these problems, covalently immobilized enzymes are used in
large-scale industrial processes.16,17
This work is concerned with deproteinization of natural rubber
using an alkaline protease immobilized on chitosan–cellulose
composite beads. Chitosan has the necessary functional groups to
covalently bond with the enzyme under mild reaction conditions.
Chitosan beads tend to have a low mechanical strength, but
∗
Correspondence to: Nanthiya Hansupalak, Center of Excellence for Petroleum,
Petrochemicals and Advanced Materials, Department of Chemical Engineering,
Faculty of Engineering, Kasetsart University, 50 Ngam Wong Wan Road, Jatujak,
Bangkok 10900, Thailand. E-mail: fengnyh@ku.ac.th
a Center of Excellence for Petroleum, Petrochemicals and Advanced Materials,
Department of Chemical Engineering, Faculty of Engineering, Kasetsart
University, 50 Ngam Wong Wan Road, Jatujak, Bangkok, 10900, Thailand
b SchoolofEngineering,PN456,MasseyUniversity,PrivateBag11222,Palmerston
North, New Zealand

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this can be improved by crosslinking with epichlorohydrin and
other bifunctional compounds.11,16,19 If epichlorohydrin under
mild alkaline conditions is used for the crosslinking, links form
mainly between the hydroxyl groups and most of the amine
functional groups in chitosan remain available for covalent binding
to the enzyme through the amine–aldehyde linkages formed
using glutaraldehyde.20 Chitosan beads may be further reinforced
by blending the polymer with a small amount of microcrystalline
cellulose (≤15% w/w).21 Chitosan–cellulose composite beads
have been shown to be mechanically and chemically more
stable than chitosan beads.21,22 Both chitosan and cellulose are
compatible at the molecular level as their chemical structures
are similar,22,23 and they both are nontoxic, inexpensive, and
biodegradable.14
Alkaline protease immobilized covalently on epichlorohydrin
crosslinked chitosan beads has been previously used to
deproteinize natural rubber.11 The present study examines an
alternative composite bead matrix of cellulose–chitosan for
enzyme immobilization for deproteinizing natural rubber. As
cellulose does not have the functional groups that can bind
to glutaraldehyde,24,25 the agent used in attaching the enzyme
to the beads, its presence reduces the frequency of the potential
enzyme binding sites on the surface. This reduces the surface
loading of the enzyme on the beads to prevent overcrowding
and can actually improve deproteinization efficacy,26 as discussed
later.
To minimize the experimental effort required for developing
an optimal immobilized enzyme catalyst bead and an optimal
protocol for the deproteinization of NR with these beads, a two-
level full factorial experiment design was used. This design was
later proved to be suitable as the resulting linear models with
interaction terms could sufficiently describe the two processes. The
reusability of the immobilized enzyme beads for deproteinization
was examined. The deproteinization performance of the optimized
enzymatic process was compared with the performance of the
urea treatment, the method used conventionally to deproteinize
natural rubber.
METHODS AND MATERIALS
Materials
Alkaline protease (from Bacillus sp. with a specific activity
of ≥16 U g−1 ; EC number 232-752-2) and microcrystalline
cellulose (Avicel PH101) were purchased from Sigma–Aldrich
(www.sigmaaldrich.com). Fine grade chitosan flakes (90.5%
deacetylated; molecular mass of ∼500 kDa) were purchased from
Elan Corp., Thailand. NR latex with a high preservative ammonia
content and a solid content of 60% (w/w) was purchased from
Thailand Natural Rubber Research Institute (Bangkok, Thailand).
All other reagents were of analytical grade and used as received.
All solutions were prepared by using deionized water.
Preparation of immobilizing beads
Chitosan and microcrystalline cellulose with a total weight of 0.3
g were dissolved in a 1% (w/w) aqueous acetic acid solution with
stirring at room temperature for 3 h to obtain a 0.3% mixed
polymer solution. The solution was degassed under vacuum and
added dropwise to a 2 mol L−1 NaOH solution using a syringe with
a 22G needle.11 This produced chitosan–cellulose hydrogel beads
(diameter ∼2–3 mm). The beads were allowed to harden in the
NaOH solution for ∼75 min. The hardened beads were washed
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S Junoi, Y Chisti, N Hansupalak
with deionized water until the pH of the effluent was neutral. Next,
the beads were added to a flask containing a mixture of a NaOH
solution (0.1% w/w NaOH, 12 mL), epichlorohydrin (5 mL) and
ethanol (3 mL). The flask was gently stirred for 2 h while being held
in a water bath at 50◦ C. The beads were then recovered and washed
sequentially with deionized water, 0.1 mol L−1 hydrochloric acid
and 0.1 mol L−1 NaOH. A final deionized water wash was used
to remove all traces of alkali. The washed beads were stored in
deionized water until use as enzyme supports. The storage period
did not exceed one day.
Covalent immobilization of the enzyme on the beads
Prior to enzyme immobilization, the beads (∼3 g fresh weight)
were treated for 1 h with 10 mL of 2% w/w glutaraldehyde solution
at 30◦ C while being shaken. The activated beads were washed
three times with borax-NaOH buffer of a certain pH and then
incubated (30◦ C, 3 h) with 20 mL of a protease solution (1 mL
stock of protease mixed with 19 mL of borax-NaOH buffer of the
same pH as the wash buffer) while being shaken. The beads still
suspended in the protein solution were stored at 4◦ C for a specified
period, the immobilization period. Before use, the beads with the
immobilized protease were washed with a 2 mol L−1 NaCl solution
and deionized water.
Assay of protease activity
The protease activity was measured using casein as the substrate.27
The immobilized enzyme was incubated with 3 mL of a 0.6% casein
solution at 55◦ C for 10 min. Then, the reaction was stopped by
adding 3.2 mL of a 10% trichloroacetic acid solution. The resulting
mixture was held at room temperature to allow precipitation
to take place. The amount of tyrosine in the supernatant was
measured by a spectrophotometer (Thomas Scientific, Genesys
10S; Swedesboro, NJ, USA) at a wavelength of 665 nm. One unit
(U) of the enzyme activity was defined as the amount of enzyme
that produced 1 µg of tyrosine per min under the experimental
conditions. The bead activity (U g−1 beads) was defined as the
activity of the immobilized enzyme per gram dry weight of the
beads.
Deproteinization of NR
NR latex containing 100 g of dry rubber was mixed (10 min,
continuous stirring) with a specified amount of sodium dodecyl
sulfate (SDS) dissolved in 167 g of deionized water. This mixture was
incubated with a specified amount of the immobilized protease
at a specified temperature for 12 h. The enzyme beads were then
removed by sieving through a steel mesh screen with 1 mm
openings. The remaining mixture was centrifuged (9000g, 1 h) to
recover the cream fraction (the deproteinized rubber particles).
The cream was washed by resuspending in a 1% SDS solution of the
same volume as the original latex sample. The washed cream was
recovered by centrifugation as above. The washed deproteinized
rubber particles were dried to a constant weight in an oven at
60◦ C. The product was the immobilized enzyme deproteinized
natural rubber, or IE-DPNR.
The removal of protein was assessed by measuring the total
Kjeldahl nitrogen (ASTM D3533-90; Standard Test Method for
Rubber–Nitrogen Content) in the deproteinized rubber and the
control sample, i.e. the washed rubber particles recovered from the
untreated latex. In addition, FTIR spectra of treated and untreated
rubber particles were recorded (Perkin-Elmer Spectrum-1
FTIR Spectrometer, www.perkinelmer.com) to check for the
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presence of protein nitrogen. The enzymatic deproteinization
results were compared with the urea-based deproteinization. The
latter was performed in house, as described by Kawahara et al.5
and Pukkate et al.6 The urea deproteinized natural rubber samples
were designated as U-DPNR.
Reusability of the immobilized protease
The reusability of the immobilized enzyme beads for
deproteinization was assessed by repeatedly using the beads
that had been prepared by the immobilization protocol that had
been identified as optimal, as discussed later. After each use, the
enzyme beads were recovered and washed repeatedly with borax-
NaOH buffer (pH 10). The rubber deproteinization reaction in each
use cycle was carried out under the set of conditions that had
been established to be most effective. The number of replicates
was three.
The washed and dried rubber particles recovered after the
treatment were measured for their total nitrogen content. The
efficacy of nitrogen removal was taken to equate to the efficacy of
protein removal. The efficacy of nitrogen removal was calculated
as follows:
Ninitial − Nfinal
Efficacy (%) = × 100
Ninitial
where Ninitial and Nfinal are the total nitrogen content (%, w/w) of
the dried rubber before and after deproteinization, respectively.
Experimental design and statistical analyses
Based on the literature8,21,28,29 and preliminary screening
experiments (data not shown), the six process factors shown in
Table 1 were identified as being important in influencing enzyme
immobilization and latex deproteinization.
Variation of these six potential factors (Table 1) simultaneously
using a two-level full factorial design required 2k experiments
(k = number of factors), or 64 experiments excluding the replicates.
To reduce the number of experiments, the enzyme immobilization
and latex deproteinization were carried out independently, but
sequentially. Enzyme immobilization was developed first and
later the deproteinization protocol was developed. This reduced
the total number of experiments to 16 (Table 1), excluding the
replicates, or 32 with replicates.
The development of the deproteinizing scheme used the
enzyme beads that had been prepared in accordance with an
immobilization protocol that had previously been established to
achieve a high enzyme activity in the beads. Each experiment was
conducted in duplicate; the averaged data are shown in Table 1.
These average values were then used in the regression analysis to
develop a multiple-regression equation for the three independent
variables (x 1 , x 2 , x 3 ),30 as follows:
Y = β0 + β1 x1 + β2 x2 + β3 x3 + β12 x1 x2 + β13 x1 x3
+ β23 x2 x3 + β123 x1 x2 x3 (2)
In Equation (2), Y is the response variable (i.e. bead activity or
total nitrogen content of the rubber) and β 0 is a constant. β 1–3
in Equation (2) are regression coefficients of the corresponding
main effects. β 12 , β 13 and β 23 in Equation (2) are the coefficients
of two-way interaction effects. β 123 is the coefficient for the three-
way interactive effect. The values of x 1 , x 2 , and x 3 may be either
the uncoded values or the coded values (the values of regression
coefficients and the constant must be changed accordingly). A
(1)
trial version of Minitab 16 (www.minitab.com) was used for all
statistical analyses. The use of two-level full factorial design to
determine optimal conditions and to evaluate the significant effect
of experimental factors and their interactions has been reported
in other applications.31,32
RESULTS AND DISCUSSION
Protease immobilization
For these experiments, the response was the enzyme activity of the
beads. The factors used in studying the response were the amount
of microcrystalline cellulose in the immobilization matrix (A), the
pH of borax–NaOH buffer used during immobilization (B), and the
length of the immobilization period (C) (Table 1). These factors had
been selected based on the results of the screening experiments
(data not shown) which showed that the peak enzyme activity in

Table 1. The two-level full factorial designsa and results

Run 1 2 3 4 5 6 7 8

Protease immobilization
Cellulose in matrix (A, %) 5 (−1) 15 (1) 5 (−1) 15 (1) 5 (−1) 15 (1) 5 (−1) 15 (1)
pH of borax–NaOH buffer (B) 9 (−1) 9 (−1) 11 (1) 11 (1) 9 (−1) 9 (−1) 11 (1) 11 (1)
Immobilization time (C, h) 12 (−1) 12 (−1) 12 (−1) 12 (−1) 24 (1) 24 (1) 24 (1) 24 (1)
Bead activityb (U g−1 beads) 1518.9 1545.5 1518.5 1500.7 1492.4 1685.3 1539.9 1511.5
Rubber deproteinization
Enzyme loadingc (D, phr) 0.01 (−1) 0.10 (1) 0.01 (−1) 0.10 (1) 0.01 (−1) 0.10 (1) 0.01 (−1) 0.10 (1)
SDS amount (E, phr) 10 (−1) 10 (−1) 20 (1) 20 (1) 10 (−1) 10 (−1) 20 (1) 20 (1)
Incubation temperature (F, ◦ C) 30 (−1) 30 (−1) 30 (−1) 30 (−1) 60 (1) 60 (1) 60 (1) 60 (1)
Total nitrogen contentd (%w/w) 0.023 0.017 0.018 0.012 0.023 0.019 0.017 0.022
a Coded values in parentheses.
b
The standard deviation was always less than ±12 U g−1 beads.
c
The loading of the dissolved enzyme is the weight of the protease solution (specific activity ≥16 U g−1 solution) added per 100 g of the rubber
solids in the latex, or parts per hundred (phr) of rubber. The loading of the immobilized enzyme is the equivalent weight of dissolved enzyme solution
obtained by equating the total enzyme activity (U) of immobilized enzyme beads used in the reaction mixture to that of the dissolved enzyme per
100 g of the rubber solids.
d
The standard deviation ranged between 0.000 and 0.001%.

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Figure 1. Scanning electron microscope images of the surfaces of the beads containing: (a) no cellulose; and (b) 15% cellulose.
the beads occurred at a cellulose concentration of 10%. In an earlier
study, Azeredo et al.21 suggested that the cellulose concentration
should not exceed 15% as at higher concentrations the cellulose
agglomerates in the chitosan matrix.
A further consideration is that the crosslinking agent
glutaraldehyde binds to the amino groups of chitosan in the
immobilization matrix and various other functional groups on
the protein33 being immobilized. Increasing the concentration
of cellulose in the matrix results in a dilution of the chitosan
amino groups on the surface of the beads. There appears to be an
optimal density of the chitosan amino groups on the bead surface
to immobilize the protease. Too few amino groups on the surface
of the beads reduce the number of potential immobilization sites.
Whereas too many amino groups on the immobilization matrix
mean that a protease molecule can bind to multiple points on the
matrix. Binding of the protease at too many points may reduce
access of the substrate to the active site of the enzyme, or leave
the enzyme molecule too rigid to be active.34
Based on the earlier screening results, a cellulose concentration
range of 5–15% (equivalent to a chitosan concentration range of
95–85%) was used in the experiments. In the concentration range
5–15%, cellulose was fully compatible with chitosan as previously
reported by others.22,35 In this concentration range, scanning
electron microscope (SEM) images (Fig. 1; JSM–5410 scanning
electron microscope) of the surface of the beads indicated an
absence of phase separation. Thus, the SEM image with 15%
cellulose in the beads was identical to the image for the beads
without cellulose (Fig. 1). Furthermore, the beads with any level
of cellulose (>0%) withstood equally well the working conditions
used in this study. Thus, no broken beads were observed in any
experiment.
The reaction pH was selected as one of the factors because
pH influences the enzyme stability and also the ionization of the
functional groups that are involved in the immobilization reaction.
An alkaline pH range was used (Table 1) as the enzyme involved
was an alkaline protease.
Immobilization time was one of the factors as extending the
time has been reported to increase the quantity of the enzyme
bound to the immobilizing matrix.36 – 38 At some point in time the
maximum attainable binding is achieved.36,37 Further extending
the reaction time can actually reduce the enzyme activity.38 In
screening experiments, lengthening the reaction time beyond a
certain value was found to reduce the enzyme activity (data not
shown).
The settings of the factors A–C used in the various experimental
runs are shown in Table 1. Table 1 also shows the measured activity
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S Junoi, Y Chisti, N Hansupalak
of the beads produced in the different runs. Based on Table 1, the
estimated coefficients for the response model (Equation (2)) are
shown in Table 2. As all the P-values in Table 2 are <0.05, all the
individual factors (A–C) and their interactive effects (AB, AC, BC,
ABC) significantly influenced the response at the 95% confidence
level. The analysis of variance of the response model is shown
in Table 3. The response is significantly affected (P < 0.05) by the
main effects of the individual factors and by their interactions
(Table 3). With the various constants in Equation (2) substituted
with the regression coefficients of Table 2, the response model
becomes:
Bead activity (U g−1 beads) = 1539.08 + 21.66A − 21.45B
+ 18.18C − 33.21AB + 19.46AC
− 10.14BC − 22.1ABC (3)
The values of the factors in the above equation are the coded
values.
Equation (3) had a high correlation coefficient (R2 = 0.994)
implying that 99.4% of the variability in the response was a
consequence of the deliberate variation of the factors in the
course of the experiments. The ANOVA results in Table 3 confirm
that Equation (3) adequately fits the data as all P-values are less
than 0.05.
In addition, the residuals relating to Equation (3) were about
evenly distributed along a straight line (Fig. 2(a)), demonstrating
a normal distribution of the residuals. The predicted response
values were in good agreement with the observed values,
as shown in Fig. 2(b). In view of all this evidence, the
response model in Equation (3) is appropriate for describing the
observed data.
A positive regression coefficient in Equation (3) indicates a
positive influence of the corresponding factor on the response,
whereas a negative regression coefficient indicates a negative
influence of the relevant factor. In all cases, the magnitude of
a regression coefficient indicates the strength of the influence
of the associated factor on the response. Therefore, both the
amount of cellulose in the bead matrix (A) and the length of
the immobilization period (C) positively affected the response
(Equation (3)). The effect of the amount of cellulose on the
response was a little stronger than the effect of the immobiliza-
tion time. The pH of the buffer (B) during immobilization had a
negative effect on the response (Equation (3)); that is, the enzyme
activity immobilized on the beads decreased as the pH increased
from the low to the high level. A pH of greater than 9 likely
increased enzyme denaturation.
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Table 2. Estimated coefficients for bead activity (Equation (3)) and total nitrogen content (Equation (4))

Coefficienta
Term Coded value Uncoded value SE b t-value P-value

Protease immobilization (bead activity)

Constant 1539.08 2130.32 1.58 972.26 0.000
A 21.66 −73.56 1.58 13.68 0.000
B −21.45 −57.23 1.58 −13.55 0.000
C 18.18 −60.24 1.58 11.48 0.000
AB −33.21 6.62 1.58 −20.98 0.000
AC 19.46 8.02 1.58 12.29 0.000
BC −10.14 5.68 1.58 −6.41 0.000
ABC −22.10 −0.74 1.58 −13.96 0.000
Rubber deproteinization (total nitrogen content)
Constant 0.018813 2.67×10−2 0.000283 66.48 0.000
D −0.001188 9.44×10−3 0.000283 −4.20 0.003
E −0.001637 −2.97×10−4 0.000283 −5.79 0.000
F 0.001250 5.37×10−5 0.000283 4.42 0.002
DE 0.001063 −9.28×10−3 0.000283 3.75 0.006
DF 0.001550 −2.37×10−3 0.000283 5.48 0.001
EF 0.000800 −6.44×10−6 0.000283 2.83 0.022
DEF 0.001050 3.11×10−4 0.000283 3.71 0.006
a
Estimated by regression of the data in coded and uncoded units.
b
Standard error.

Table 3. Analysis of variance for bead activity and total nitrogen content

Source DF a SSb MSc F P-value

Protease immobilization (bead activity)

Main effects 3 20 151.8 6717.26 167.54 0.000
2-way interactions 3 25 349.6 8449.88 210.75 0.000
3-way interactions 1 7817.7 7817.65 194.98 0.000
Residual error 8 320.7 40.09
Total 15 53 639.8
Rubber deproteinization (total nitrogen content)
Main effects 3 904.7×10−7 3.02×10−5 23.54 0.000
2-way interactions 3 667.4×10−7 2.23×10−5 17.36 0.001
3-way interactions 1 176.4×10−7 1.76×10−5 13.77 0.006
Residual error 8 102.5×10−7 1.28×10−5
Total 15 1851.0×10−7
a Degrees of freedom.
b
Sum of squares.
c
Mean square.

The objective of the experiments was to identify the
immobilizing conditions for attaining the maximum activity in
the beads. Run 6 in Table 1 gave beads with the highest activity
of 1685.3 U g−1 beads. These had been produced under the
following conditions: a cellulose content in the beads of 15%, the
reaction buffer pH of 9, and an immobilizing time of 24 h. These
conditions were taken to be the best experimental conditions
for producing the beads. The enzyme immobilized under these
conditions was then used to determine the best conditions for the
deproteinization of NR as explained in the next section.
Rubber deproteinization
Development of the NR deproteinization process focused on the
following factors (Table 1): the loading (D) of the enzyme in the
reaction mixture, the amount of the surfactant SDS in the reaction
mixture (E) and the incubation temperature (F).
Increasing the loading of enzyme in the reaction mixture is
known to positively influence the protein removal in NR being
deproteinized with a dissolved enzyme.39 Too low a loading of
dissolved enzyme is insufficient for protein removal, but too high
a loading is not economical.39 For the dissolved enzyme, a loading
range of between 0.0001 and 20% (w/w) of the solid rubber
content in the mixture has been reported.39 This is equivalent
to 0.0001–20 parts of enzyme solution per 100 g of the rubber
particles, or 0.0001–20 phr (parts per hundred rubber).
A surfactant is usually added to stabilize the NR latex during
deproteinization. Any kind of surfactant (i.e. anionic, cationic, or
nonionic surfactants) can be used,9,40 but a cationic surfactant can

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99 99
(a) (a)

90 90

Percent probability
Percent probability

70 70

50 50

30 30

10 10

1 1
-10 -8 -6 -4 -2 0 2 4 6 8 10 -0.003 -0.002 -0.001 0.000 0.001 0.002 0.003
Residual Residual

1750
0.024

Predicted nitrogen content (%, w/w)

(b) (b)
Predicted activity (U/g beads)

1700 0.022

1650 0.020

0.018
1600
0.016
1550
0.014

1500 0.012

0.010
1450 0.010 0.012 0.014 0.016 0.018 0.020 0.022 0.024
1450 1500 1550 1600 1650 1700 1750
Observed nitrogen content (%, w/w)
Observed activity (U/g beads)
Figure 3. (a) Normal probability plot of the residuals. (b) The predicted
Figure 2. (a) Normal probability plot of the residuals. (b) The predicted
total nitrogen content (Equation (4)) versus the observed values.
bead activity (Equation (3)) versus the observed activity.

(P < 0.05; Table 2) at the 95% confidence level. Using the

destabilize the latex if an unsuitable amount of the surfactant is
used.9 SDS is an anionic surfactant and, therefore, is expected
to have a stabilizing effect on the latex irrespective of the
concentration of the surfactant.
Compared with nonionic surfactants, anionic surfactants
are more effective in solubilizing the oligopeptides in the
rubber latex.9,40 Therefore, the use of anionic surfactants is
preferred in a deproteinization process. A suitable concentration
range for anionic surfactants of 0.001 to 20 phr has been
suggested in latex deproteinization mixtures involving dissolved
proteases.41
Use of an optimal incubation temperature is essential for
rapid enzymatic deproteinization. Too high a temperature would
denature the enzyme and too low a temperature will slow the
deproteinization reaction. Immobilization of the enzyme is likely
to enhance its temperature stability34 and, therefore, the optimal
deproteinization temperature using the immobilized enzyme may
be different compared with the optimal temperature for the
dissolved form of the same enzyme. Activity and longevity of
immobilized enzyme preparations are known to be temperature
dependent.28,42
The settings used for the factors D–F in the eight experimental
runs and the measured response of the total residual nitrogen
in the rubber samples, are shown in Table 1. The estimated
coefficients for the model equation (Equation (2)) and the relevant
ANOVA are shown in Table 2 and Table 3, respectively.
The data in Table 2 confirm that the main factors (D, E,
and F) and their interactions (two-way interactions = DE, DF, EF;
three-way interaction = DEF) significantly influence the response
regression coefficients shown in Table 2, Equation (2) could be
rewritten as:
Total nitrogen content (%, w/w) = 0.018813 − 0.001188D
− 0.001637E + 0.00125F + 0.001063DE + 0.00155DF
+ 0.0008EF + 0.00105DEF (4)
The values of the factors in the above equation are the coded
values.
Equation (4) had a high correlation coefficient value (R2 = 0.95)
and the ANOVA results (Table 3) all had P-values <0.05. Therefore,
Equation (4) satisfactorily predicted the response. In addition,
the residuals relating to Equation (4) were normally distributed
(Fig. 3(a)). Furthermore, the predicted and the observed responses
were linearly correlated by a line with a unit slope (Fig. 3(b)). All
this suggested that the model (Equation (4)) closely followed the
observed data.
Of all the main factors and the interaction factors in Equation
(4), only the enzyme loading (D) and the SDS amount (E), had a
negative impact on the response, i.e. raising the enzyme loading
or the SDS quantity in the reaction mixture reduced the response.
The regression coefficients for all the factors (Equation (4)) were of
a similar magnitude and, therefore, the factors had a comparable
influence on the response. These results agreed well with the
literature39,41 as an increase in the enzyme loading and/or the
SDS amount improved the removal of protein from the rubber
particles, to reduce the total nitrogen content of the rubber

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c 2014 Society of Chemical Industry J Chem Technol Biotechnol (2014)
Rubber deproteinization using immobilized protease www.soci.org

sample. Furthermore, the results suggested that the immobilized

alkaline protease was denatured at a temperature of greater than
30◦ C, resulting in a diminished enzyme activity and, consequently,
an elevated level of the total residual nitrogen.28,42
The objective of the experiments was to identify the
deproteinizing conditions that minimized the total nitrogen
content in the rubber. The conditions used in Run 4 in Table 1
achieved the minimum measured total nitrogen content of
0.012%. These were taken as the optimal conditions for
deproteinization. The conditions were: an enzyme loading of
0.1 phr, an SDS concentration of 20 phr and an incubation
temperature of 30◦ C.
We previously reported on the use of the same alkaline protease
immobilized on epichlorohydrin crosslinked chitosan beads for
deproteinizing natural rubber.11 For beads of the same size and
density as in the current work, the optimally produced crosslinked
chitosan beads11 had an enzyme loading of 2789 µg g−1 beads,
or nearly 2.4-fold greater than a loading of 1159 µg g−1 beads
observed in the present study. Therefore, as expected, a dilution of
the enzyme binding sites on the surface of the cellulose-containing
beads substantially reduced the enzyme loading relative to
beads made exclusively of crosslinked chitosan. Nevertheless,
the specific activity of the beads of the present work was 1685.3
U g−1 beads, or nearly 60% greater than the specific activity of
1043 U g−1 beads previously reported for the crosslinked chitosan
beads.11 Thus, a reduced surface loading of the enzyme on the
beads actually improved the activity of the beads. This may be
explained as follows: excessive loading of the enzyme is known
to cause overcrowding which can reduce activity via mutual
hindering of the active sites.29 Enzymes are expensive. Therefore,
an immobilization matrix that reduces surface loading without
sacrificing the specific activity and stability is useful.
Validation of regression models
The regression models for the bead activity (Equation (3)) and
the total nitrogen content of the washed NR (Equation (4)) were
validated for selected combinations of factors (Table 4) within
the design space. Each experiment was performed in triplicate.
In all cases, the experimentally measured responses were in close
agreement with the predictions of the models (Equation (3) and
Equation (4)) as shown in Table 4. Therefore, the linear models
which included the interaction effects demonstrated a satisfactory
predictive capability, confirming the suitability of the optimal con-
ditions for the immobilization and the deproteinization processes.
Comparison of deproteinization methods
Table 5 compares the total nitrogen contents of the rubber
samples that had been deproteinized using urea (U-DPNR), the
immobilized enzyme beads (IE-DPNR), and the nonimmobilized
dissolved enzyme (DE-DPNR). Nitrogen content data is also
shown for the rubber sample that had not been deproteinized
(NR). The data were obtained from experiments performed in
triplicate.
The nitrogen content of all three DPNR samples were <15%
of the nitrogen content of the untreated sample (NR, Table 5).
Compared with the other deproteinization treatments, the
immobilized enzyme treatment (IE-DPNR, Table 5) reduced the
nitrogen level the most, to just 4% of the level in untreated NR.
Compared with the literature data8 for deproteinization
with a dissolved protease (DE-DPNR), the immobilized enzyme
preparation used in this work (IE-DPNR; Table 5) was superior.
Table 4. Validation of regression models for bead activity (Equation
(3)) and total nitrogen content (Equation (4))
Response
Conditions Predicteda
Measured
Bead activity (U g−1 beads)
A = 10%, B = 11, C = 18 h 1518 1518 ± 10
A = 8%, B = 9, C = 24 h 1549 1550 ± 7
A = 10%, B = 10, C = 18 h 1528 1539 ± 11
Total nitrogen content (%, w/w)
D = 0.07 phr, E = 20.0 phr, F = 30◦ C 0.016 0.014 ± 0.001
D = 0.04 phr, E = 16.7 phr, F = 30◦ C 0.018 0.018 ± 0.000
D = 0.07 phr, E = 16.7 phr, F = 50◦ C 0.019 0.019 ± 0.000
a Calculated using Equation (3) and Equation (4) and the coefficients
for uncoded inputs.
Table 5. Total nitrogen content of various rubber samples
Sample Total nitrogen content (%, w/w)
NR 0.300 ± 0.000
U-DPNR 0.040 ± 0.000
IE-DPNR 0.012 ± 0.000
DE-DPNR 0.020*
*From Ichikawa et al.8
The optimal enzyme loading determined in the present work
was lower than the optimal loading determined for the soluble
enzyme.8 This was possibly because of the different alkaline
protease used by Ichikawa et al.8 in determining their optimal
loading, Ichikawa et al.8 varied one factor at a time. Therefore,
any interactive effects of factors on deproteinization were
ignored.
In Table 5, the relatively high total nitrogen content of the
U-DPNR may be partly attributed to traces of residual urea in
the sample, as a complete removal of urea can be difficult.12 Also,
some of the surface protein denatured by urea may have remained
attached to the rubber particles and this may have contributed to
a relatively high final nitrogen level.
The removal of protein from the treated NR was confirmed using
FTIR spectroscopy. The FTIR spectra of the U-DPNA, the IE-DPNR
and the untreated NR are compared in Fig. 4. The spectrum for
the untreated NR (Fig. 4) has a broad absorption band at ∼3295
cm−1 that is associated with large peptides, or proteins.5 This
band does not exist in the enzyme treated sample IE-DPNR (Fig. 4),
indicating a lack of large peptides. In the spectra of the U-DPNR
and NR (Fig. 4) absorption bands are expected at 3320 cm−1 due to
possible presence of monopeptides and dipeptides,43 – 45 but are
likely masked by the broad band at ∼3295 cm−1 that is associated
with NH stretching in proteins.
Although the urea based deproteinization requires only 1 h
compared with 12 h needed for deproteinization with immobilized
enzyme, the enzymatic method appears to remove nearly all
protein. A shortening of the treatment time with the immobilized
enzyme to less than 12 h did not achieve satisfactory deproteiniza-
tion (data not shown). Compared with the 24 h (35◦ C) required for
treatment with the dissolved enzyme,8 the immobilized enzyme
method (12 h treatment at 30◦ C) was much superior.

J Chem Technol Biotechnol (2014)

c 2014 Society of Chemical Industry wileyonlinelibrary.com/jctb
 www.soci.org S Junoi, Y Chisti, N Hansupalak

(a) NR on chitosan–cellulose composite beads were found to be the

following: 15% (w/w) cellulose in the beads, an immobilizing
reaction pH of 9 and an immobilization period of 24 h. Under these
conditions, the enzyme activity immobilized on the beads was
(b) U-DPNR 1685.3 U g−1 beads. Using the enzyme beads produced under
the above mentioned conditions, the optimal conditions for
the deproteinization of the NR were the following: an enzyme
(c) IE-DPNR loading of 0.1 phr, an SDS concentration of 20 phr, an incubation
temperature of 30◦ C and a treatment time of 12 h.
Using the optimal deproteinization protocol, the total nitrogen
content of the NR was reduced from an initial value of 0.3%
to 0.012%, a 96% reduction. No peptides could be detected
in the rubber samples that had been deproteinized using the
immobilized enzyme. Deproteinization with immobilized enzyme
using the optimal protocol was more effective in nitrogen
removal than the urea based deproteinization. The enzyme beads
prepared and used under the identified best conditions were
physically stable for up to at least five cycles of repeated use
in deproteinization. Thus, a blend of microcrystalline cellulose
(15% w/w) and chitosan (85%) crosslinked with epichlorohydrin
provided physically strong composite beads for covalently
3600 3400 3200 3000 2800 2600 2400
attaching proteases using glutaraldehyde as the linking agent.
Wavenumber (cm-1)
In view of its ability to essentially fully deproteinize the NR, the
Figure 4. FTIR spectra of natural rubber (NR), urea deproteinized natural immobilized enzyme treatment is recommended in preference to
rubber (U-DPNR) and the immobilized enzyme deproteinized natural the urea based treatment. The latter does not assure a complete
rubber (IE-DPNR). elimination of potential allergens from the rubber.

100
ACKNOWLEDGEMENTS
Financial support from the following sources is gratefully
Nitrogen removal efficacy (%)

80 acknowledged: Center of Advanced Studies in Industrial

Technology, Faculty of Engineering, Kasetsart University; Center of
Excellence for Petroleum, Petrochemicals and Advanced Materials,
60 S&T Postgraduate Education and Research Development Office
(PPAM); and Kasetsart University Research and Development
Institute (KURDI). Dr. Ben Embley assisted with proofreading.
40

20
1 2 3 4
Use cycle number
Figure 5. Reusability of the optimally prepared immobilized protease
beads in deproteinization under the optimal conditions. The standard
deviation values are smaller than the dimensions of the symbols.
Reusability of immobilized enzyme beads
Enzyme beads that had been produced under the previously
identified optimal conditions were used in repeated cycles for
deproteinizing the NR latex. Each deproteinization cycle was
carried out under the earlier identified best treatment conditions.
The results are shown in Fig. 5. For up to five cycles of repeated
use, the beads barely lost deproteinization activity, demonstrating
excellent stability and reusability.
CONCLUSIONS
The statistical analysis showed that all the experimental factors
and their interactions significantly influenced the enzyme
immobilization and the NR deproteinization processes. The best
conditions for the covalent immobilization of the alkaline protease
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