Article pubs.acs.org/IECR
Deproteinization of Natural Rubber Using Protease Immobilized on Epichlorohydrin Cross-linked Chitosan Beads Sirawan Prasertkittikul,† Yusuf Chisti,‡ and 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 ‡ School of Engineering, PN 456, Massey University, Private Bag 11 222, Palmerston North, New Zealand ABSTRACT: Deproteinization of natural rubber using an alkaline protease from Bacillus sp. immobilized on cross-linked chitosan beads is reported. Conditions were identified for attaining a high activity of the immobilized enzyme on the beads. The optimally produced enzyme beads were then used to establish the conditions for effective deproteinization of natural rubber. A two-level full factorial design was used to quantify the main effects and the interaction of the factors influencing the formation of the catalytic beads and the rubber deproteinization process. Results showed that by using the optimal deproteinization process, the total nitrogen level in the rubber was reduced from 0.35% to 0.013% w/w, or a >96% nitrogen removal. In contrast, the urea method of deproteinization achieved a lower nitrogen removal of 0.05) on the response. Table 4 further confirms the strength of the main effects and the twoway interactions on the bead activity (p < 0.05). The results, therefore, support the model eq 3 as being significant and adequate for predicting the response. The maximum enzyme activity in the beads was achieved at the conditions of run 4 (Table 2): a glutaraldehyde solution concentration of 6% (w/v), a reaction buffer pH of 11, and an immobilization period of 2 h at 30 °C. The beads produced under these conditions had an activity of 1043.4 U/g and a protein loading of 25% (i.e., 25% of the protein in the original enzyme solution was attached to the beads) as measured by the Bradford protein assay using bovine serum albumin as the standard.36 For this run, the model predicted activity (eq 3) was 1050.2 U/g beads, or within ∼1% of the measured value. The enzyme beads produced under these optimal conditions were
Degrees of freedom. bSum of squares. cMean square.
activated sites on the support matrix are occupied by the enzyme, a further extension of the immobilization period provides no added benefit. A determination of the optimal immobilization period was necessary. The effects of the concentration of glutaraldehyde, the pH of the borax−NaOH buffer used during immobilization, and the duration of the immobilization reaction on the activity immobilized in the beads were examined in eight experimental runs. The immobilization temperature was held constant at 30 °C. The levels of the various factors in the eight runs and the measured response are shown in Table 2. Table 3 provides the estimated coefficients for the model (eq 2) and the relevant pvalues. A p-value indicates the significance of a factor. Only factors with p-values of less than 0.05 are deemed as significant at the 95% confidence level. In Table 2, all of the main factors (A, B, and C) and two of the two-way interactions (AC and BC) had a significant impact (p < 0.05) on the enzyme activity of 11726
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study for use with the immobilized enzyme was consistent with the earlier recommendations.31 The choice of the reaction temperature is important as too low a temperature would slow the rate of deproteinization, whereas too high a temperature would rapidly denature the enzyme. Activity and stability of immobilized enzymes are known to be strongly affected by temperature.41−43 Therefore, the temperature of deproteinization was one of the experimental factors. The levels of the various factors used in the deproteinization experiments and the associated response of the total residual nitrogen in rubber are shown in Table 2. Table 3 provides the estimated coefficients for the model (eq 2) and the relevant pvalues. In Table 3, only two of the main factors (i.e., D and E) and three of the interactions (i.e., DE, EF, and DEF) have pvalues of less than 0.05. Therefore, these main factors and interactive effects have a significant influence on the response at the 95% confidence level. Hence, the factor F and the interaction DF (Table 3) can be disregarded from further consideration. This is confirmed by the analysis of variance shown in Table 4. Using the coefficients of the significant factors and interactions (Table 3), the response model (eq 2) can be rewritten as follows: total nitrogen content (%, w/w) = 2.000 × 10−2 + 5.500 × 10−4D + 2.426 × 10−3E − 2.424 × 10−3DE + 6.000 × 10−4EF + 1.009 × 10−3DEF
(4)
The values of D, E, and F in the above equation are coded values. Equation 4 had a high correlation coefficient (R2) of 0.97, confirming that more than 97% of the variability in the response could be explained by the deliberate changes in the values of the factors during the experiments. The residuals relating to eq 4 were fairly normally distributed as evidenced by their scatter around the straight line in Figure 2a. A direct linear relationship with a unit slope existed between the response predicted with eq 4 and the experimentally measured data (Figure 2b), further confirming the high reliability of the regression model (eq 4). The minimum total nitrogen content in the treated rubber occurred at the conditions of run 1 (Table 2). These conditions were as follows: an enzyme loading of 0.01 phr, an SDS amount of 10 phr, a temperature of 30 °C, and treatment time of 12 h. Under these conditions, the total nitrogen content of the rubber was reduced to 0.013% from an initial level of 0.35%, or a >96% nitrogen removal. Although SDS treatment without enzymes has been used previously to deproteinize natural rubber,40 it proved ineffective and nearly 36% of the total nitrogen present in the untreated sample remained after the treatment. As shown in Figure 3, the immobilized enzymes beads produced using various concentrations of glutaraldehyde (a reaction buffer pH of 11, an immobilization period of 2 h, 30 °C) reduced the residual nitrogen in the rubber to different levels when used under the above identified optimal conditions of deproteinization. The beads with a 6% concentration of glutaraldehdye were clearly the most effective as they had the highest activity of the enzyme per unit mass of the beads (Figure 3). 3.3. Validation of Regression Models. The regression models for bead activity (eq 3) and total nitrogen content (eq 4) were validated within the design space, for the sets of
Figure 1. (a) Normal probability plot of the residuals and (b) the predicted bead activity (eq 3) versus the observed activity.
then used in the various experiments for the deproteinization of the natural rubber. 3.2. Rubber Deproteinization. Immobilized enzyme beads that had been prepared under the above identified optimal conditions were used in the deproteinization of natural rubber. The effects of the enzyme loading in the reaction mixture, the amount of the surfactant SDS, and the reaction temperature were examined. The reaction time was always 12 h. The above three factors were selected for the study in view of their known effects on deproteinization carried out using dissolved proteases. For example, increasing the concentration of the dissolved enzyme in the deproteinization treatment of natural rubber has been found to enhance protein removal.37,38 Too low a loading of the dissolved enzyme is insufficient for removing proteins, and too high a loading is not wanted because of the cost of the enzyme.37 For dissolved enzyme, an effective loading range of between 0.0001% and 20% of the solid rubber content, or 0.0001−20 phr, has been reported for rubber deproteinization. Surfactants are often used to stabilize the rubber latex during deproteinization. Anionic, cationic, and nonionic surfactants can be used as stabilizers,16,39,40 but the anionic surfactant SDS has been previously found to be better than some of the other surfactants for use in conjunction with enzymatic deproteinization.16 Surfactants also act as rinsing agents and help in washing away of the proteins detached from the surfaces of the rubber particles. For anionic surfactants such as SDS used in combination with dissolved proteases, Tanaka et al.31 have recommended a suitable concentration range of between 0.001 and 20 phr. The SDS concentration range evaluated in this 11727
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Table 5. Validation of Regression Models for Bead Activity and Total Nitrogen Content response predicteda
conditions Bead Activity (U/g beads) A = 4%, B = 10, C = 2 h A = 8%, B = 11, C = 2 h A = 8%, B = 10, C = 5 h Total Nitrogen Content (%, w/w) D = 0.04 g, E = 10.0 g, F = 30 °C D = 0.07 g, E = 10.0 g, F = 30 °C D = 0.07 g, E = 16.7 g, F = 40 °C
996.4 1093.3 942.2 0.017 0.020 0.021
exp 1056.6 1109.7 958.3 0.018 0.021 0.024
a
Calculated using eqs 3 and 4 for bead activity and total nitrogen content, respectively.
and the variously deproteinized natural rubber samples is shown in Table 6. Samples deproteinized with urea (U-DPNR), Table 6. Total Nitrogen Content of the Rubber Samples
a
sample
total nitrogen content (%, w/w)
NR U-DPNR IE-DPNR DE-DPNR
0.350 0.050 0.013 0.020a
From Ichikawa et al.18
the immobilized enzyme (IE-DPNR), and the dissolved enzyme (DE-DPNR) all had ≤14.3% of the nitrogen content of the untreated natural rubber (Table 6). As compared to the other deproteinization methods, the treatment with immobilized enzyme removed the highest amount of nitrogen at >96%. Proteases actually cleave peptide bonds44 and, therefore, progressively reduce the size of the protein fragments attached to the surface of the rubber particles until no peptide bonds remain and essentially all protein has been removed. The extent of deproteinization achieved with the immobilized enzyme in this work was higher than has been reported in the literature for deproteinization with the dissolved enzyme.18 This was likely because of the different protease used by Ichikawa et al.18 In addition, the deproteinization conditions used by Ichikawa et al.18 had been established using the onefactor-at-a-time method, therefore, ignoring any interactive effects of the factors on the deproteinization process. The relatively high total nitrogen content in the urea treated sample (U-DPNR) may be partly a result of the residual urea in the sample, as urea can be difficult to remove.45 Urea treatment denatures proteins,46 but does not cleave the peptide bonds. Therefore, large denatured peptides that are potentially immunogenic may be left on the rubber particles treated with urea. The presence of proteins on the surface of the natural rubber and the deproteinized rubber samples was further assessed by using FTIR spectroscopy of films of samples. The relevant spectra are shown in Figure 4. In the spectrum for the untreated natural rubber (NR; Figure 4), the absorption hump at 3290 cm−1 is due to long peptides, or proteins.9 This absorption band is barely seen in the spectrum of the sample that had been treated with the immobilized enzyme (IE-DPNR; Figure 4), confirming an absence of proteins in the treated samples. In the urea treated sample, a relatively small absorption band appears at 3320 cm−1 (U-DPNR; Figure 4).
Figure 2. (a) Normal probability plot of the residuals and (b) the predicted total nitrogen content in rubber (eq 4) versus the observed values.
Figure 3. Total residual nitrogen content as a function of glutaraldehyde concentration used in the immobilization process. Experiments were performed in duplicate. In all cases, the pH of the reaction buffer was 11 and the length of the treatment period (i.e., the immobilization period) was 2 h. The treatment temperature was 30 °C. A low level of residual nitrogen is indicative of highly active beads used.
conditions given in Table 5. As shown in Table 5, the measured responses were in excellent agreement with the responses predicted using the model equations. This proved the reliable predictive capabilities of the models. 3.4. Comparison of Deproteinization Methods. The nitrogen content of the untreated washed natural rubber (NR) 11728
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Figure 4. FTIR spectra of (a) natural rubber; (b) urea deproteinized natural rubber; and (c) the immobilized enzyme deproteinized natural rubber.
This indicates the presence of mono- and dipeptides in the sample.12,47,48 Therefore, the urea treatment did not fully remove proteins from the rubber. In the spectrum of the urea treated sample, the broad band at 3320 cm−1 may also be masking any absorbance at 3290 cm−1 associated with long peptides and proteins. Treatment of the rubber with the immobilized enzyme required a relatively long incubation time (12 h) as compared to 1 h required by the urea treatment. Nevertheless, the immobilized enzyme treatment is preferable to the urea treatment, as the former assures an effective removal of proteins, which is important in certain applications. The treatment with the dissolved enzyme required 24 h,18 but this may be shortened by a different choice of the enzyme and the use of a higher enzyme concentration. Notwithstanding this, unlike immobilized enzyme, the dissolved enzyme cannot be reused, and its potentially immunogenic residue can contaminate the rubber. 3.5. Reusability of Enzyme Beads. Under the above identified best conditions for deproteinization (section 3.1), using the enzyme beads that had been produced under the optimal conditions (section 3.2), the beads were used in repeated cycles of deproteinization of the natural rubber latex. The efficacy of the beads for deproteinization was barely affected by repeated use for at least five consecutive use cycles (Figure 5). The slight loss in activity of ∼10% of the initial activity after five use cycles may be ascribed to protein leakage and possible denaturation. This notwithstanding, a repeated use of the beads prepared as in this work is feasible. No mechanical damage to the beads was seen after five use cycles.
Figure 5. Reusability of the optimally prepared immobilized enzyme beads in deproteinization under the optimal conditions.
immobilized enzyme beads used in this treatment had been prepared under conditions that maximized the enzyme activity in the beads. These conditions were as follows: a glutaraldehdye concentration of 6% w/v, a pH of 11 (borax−NaOH buffer), and an immobilization period of 2 h at 30 °C. The catalyst beads prepared under these conditions had an activity of 1050 U/g and could be used repeatedly for at least five deproteinization cycles. The immobilized enzyme deproteinization treatment was more effective than the urea treatment.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
4. CONCLUSIONS The use of the alkaline protease covalently immobilized on reinforced chitosan beads was extremely effective in deproteinization of the natural rubber latex. The nitrogen content of the natural rubber could be reduced by more than 96% with the enzymatic deproteinization treatment carried out for 12 h under the optimal conditions of 30 °C, an enzyme loading of 0.01 phr, and a SDS surfactant loading of 10 phr. The
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from the following sources is gratefully acknowledged: Center of Advanced Studies in Industrial Technology, Faculty of Engineering, Kasetsart University; Center of Excellence for Petroleum, Petrochemicals and 11729
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Advanced Materials, S&T Postgraduate Education and Research Development Office (PPAM); Kasetsart University Research and Development Institute (KURDI); and Rubber Research Institute of Thailand. Dr. Ben Embley assisted with proofreading.
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