Abstract: Background: Interaction between -NH2 group of chitosan to -CHO groups of glutaraldehyde is critical in lipase immobilization using chitosan as a matrix and glutaraldehyde as a linker. Since the Deacetylation Degree (DD%) of chitosan represent the number of -NH2 group, it is important to understand the effect of the DD% value of chitosan on the effectiveness of immobilization. Chitosan beads and powder have different accessible -NH2 group, therefore, the influence of the form of chitosan is also interesting to be investigated. Materials and Methods: Three chitosans with different DD prepared with various repetition of deacetylation. This chitosan was set in beads and powder form then cross-linked to lipase (porcine’s pancreas) using glutaraldehyde as across-linking agent. The amount of the immobilized lipase as well as the hydrolytic activity of lipase was observed to determine the effectiveness of the immobilization. The thermal stability and reusability of this immobilized lipase were also investigated. Results: The optimum DD% value of powder chitosan using in lipase immobilization is at 90% DD with the optimum condition by using 1.5% of glutaraldehyde at pH 6.0 with efficiency up to 89%. The optimum DD% of beads chitosan is at 81% DD with the optimum condition by using 0.5% of glutaraldehyde at pH 6 with efficiency up to 53%. The immobilization at optimum condition of powder chitosan capable in retaining 32% of the activity, while the beads chitosan could keep 60% of the activity based on a comparison of the specific activity data. The result is corresponding to the kinetic data, where in beads the KM value is 100 mM higher than that of the free enzyme with 86 mM in KM value. Immobilization of lipase improves the stability of the enzyme with no significant reduction of activity up to five times of reuse and thermal stability up to 50°C. Conclusion: The DD% value of chitosan plays an essential role in lipase immobilization using crosslink method with glutaraldehyde as alinker with medium DD% give the best result. Immobilization of lipase improves the stability of the enzyme leading to reusability and thermal of the enzyme.
INTRODUCTION
Lipase (triacylglycerol hydrolases, EC 3.1.1.3) is a biocatalyst offering three interesting activities including hydrolytic, esterification and transesterification. Hydrolytic activity of lipase is usefulfor food industries and surfactant production, while esterase and transesterase activity are widely studied for the production of new emerging biodiesel production1-4. The primary challenge in lipase industries application is to maintain the enzyme stability in term of its activity after reaction for reuseis given enzyme normally used in solution form and possibly that the enzyme might have been polluted by the products5. A way to overcome the problems is through immobilization of the enzyme using cross-link technique.
In cross-linking immobilization method, selection of the matrix as well as the linker suitable to the enzyme is critical factors. Chitosan, a polymer mixture of N-acetyl glucosamine and glucosamine has become an attractive material to be functioned as a matrix in enzyme immobilization6-8. Chitosan is usually produced by deacetylation process of chitin, a polymer of N-acetylglucosamine, which presents in the shells of crab sand shrimp. Deacetylation process results in a various number of removed acetyl group leading to a various number of level of deacetylation of produced chitosan, which characterized as different Deacetylation Degree (DD level). Deacetylation Degree (DD) correspond to the number of -NH2 groups present in the chitosan. In lipase cross-link immobilization, -NH2 groups of chitosan will bind to the -NH2 reactive of the enzyme with the help of the linker which. One of the groups that can bind covalently to the -NH2 is -CHO group from analdehyde or -COOH from an acid linker. The linker should also be a bi-functional compound, since it has to be capable to covalently bind to the matrix and the enzyme at the same time. Some linkers which are widely used are citric acid and glutaraldehyde. Glutaraldehyde posses two -CHO group, widely used because it is cheap and easy and quick to prepare9. Glutaraldehyde is capable of crossing-link chitosan and enzyme through Schiff base reaction (imine substitution, -CH = NR) with -NH2 group on the enzyme and chitosan. Glutaraldehyde concentration as crosslinker compound during immobilization process must be carefully considered since the character of glutaraldehyde could prevent protein binding due to the possibility of making double imine structure with the matrix emerging from using a higher concentration of glutaraldehyde. High concentration of glutaraldehyde could also hamper the activity of the immobilized enzyme since -CHO group could also make alkoxide structure with an enzyme containing -OH group in the active site.
Cross-linking immobilization using chitosan-glutaraldehyde system succeed if the -CHO group of glutaraldehyde could bridge a -NH2 group of chitosan and -NH2 group of the enzyme. This fact offer advantage for hydrolytic lipase, since the active site of the enzyme consists of three amino acids of aspartic acid, serine and histidine, which will not be interfered with cross-linking. However, the number of -NH2 group of chitosan could hinder the effectiveness of crosslink of the enzyme. The low number of -NH2, which represents by less DD value of chitosan could lead to low immobilization effectiveness, while if the number of -NH2 group is too high, represent by the larger number of DD value, could cause self-crosslink (chitosan-glutaraldehyde-chitosan link) leading to low immobilization effectiveness. In immobilization, chitosan size and form determine the effectiveness of immobilization10,11. Chitosan usually applied both in powder and bead form. Therefore, the study of the effect of DD of chitosan as matrix should be conducted to both of chitosan form to find the most effective condition.
MATERIALS AND METHODS
Materials: Lipase used in this study was porcine’s pancreas lipase (Merck), while the chitin was isolated from crab shell obtained from local restaurants in Yogyakarta. Testing of the enzyme activity was done using commercial palm cooking oil (Bimoli), while the determination of enzyme as protein was carried out using Bovine Serum Albumin (BSA) as a reference (Sigma-Aldrich). Other main chemicals used in this study were glutaraldehyde, n-hexane, ethanol, sodium hydroxide and acetic acid (Merck) all are in pro analysis grade.
Methods
Preparation of chitosan with various DD level: Chitin was prepared from crab shell through deproteination and demineralization process according to Islam et al.12. Chitosan was prepared by deacetylation process of chitin using NaOH according to Dutta et al.13. To obtain chitosan with various DD%, the resulted chitosan was repeatedly deacetylated with the same method. Chitin and chitosan powders with various deacetylation process were then characterized using FTIR to determine their DD%14. Characterization was also performed to determine the moisture content, ash content and total N content to both chitin and chitosan using AOAC methods15.
Chitosan beads were prepared using inverse phase method16. Chitosan solution was prepared in concentration of 3% (b/v) by dissolving various DD% chitosan in acetic acid 1% (v/v). Chitosan solution was added through a syringe needle (26 G) to beaker glass 250 mL filled with 100 mL of coagulant (solution of 1 M NaOH in 26% ethanol). The mixture was allowed to stand for 3 h to form a spherical gel before separated by filtration. The beads were washed using aquadest until the filtrate became neutral.
Immobilization enzyme in chitosan powder: Matrix was prepared by putting one gram of chitosan powder in a 50 mL flacon bottle then added it with various amount of glutaraldehyde (parameter was optimized) as cross-linked agent and 5 mL 0.05 M phosphate buffers at various pH (parameter to be optimized). The mixture was then allowed to stand at room temperature for 10 min and then filtered using filter paper. Lipase solution was prepared by dissolving 100 mg lipase in 10 mL phosphate buffer 0.05 M at various pH in 50 mL flacon bottle. The chitosan which had previously been treated with glutaraldehyde was added to the lipase solution. The mixture was allowed to stand at room temperature for 1 h, followed by filtration using filter paper and wash using aquadest5. The filtrate and washing filtrate were combined and subjected for protein content determination using Biuret protein assay. The amount of the immobilized enzyme was calculated by subtracting the initial enzyme with the total remaining enzyme present in the combined filtrate and washing solution.
Immobilization enzyme in chitosan bead: This step was performed based on previously published method17 with slight modification. Chitosan beads were activated first using cross-linked agent, glutaraldehyde. One gram of wet chitosan beads was added into 3 mL buffer solution at various pH (parameter to be optimized). Glutaraldehyde was used in various concentration (parameter to be optimized). The activation process was performed by standing the mixture at room temperature for 10 min. The beads were then washed with aquadest. Lipase solution was prepared by dissolving 100 mg lipase in 10 mL phosphate buffer 0.05 M with various pH in 50 mL flacon bottle. The chitosan beads, which had been previously treated with glutaraldehyde were added to the lipase solution. The mixture was allowed to stand at room temperature for 1 h, followed by filtering using filter paper and washing using aquadest. The filtrate, as well as the washing filtrate were combined and subjected for protein content determination using Biuret method. The amount of the immobilized enzyme was calculated by subtracting the initial enzyme with the total remaining enzyme present in the combined filtrate and washing solution.
Study of optimum condition for immobilization: Optimum condition on both forms of chitosan as a matrix in lipase immobilization was investigated. The optimum pH condition for enzyme immobilization was determined by dissolving enzyme (100 mg lipase in 10 mL in 100 falcon tube) followed by immobilization. Both processes were performed at pH of 5.0, 6.0, 7.0, 8.0 and 9 of 0.05 M phosphate buffer. Optimum pH was determined by comparing the concentration of dissolved lipase among various pH, as well as percentage of immobilized enzyme among the condition, which was determined by measuring enzyme concentration of using Biuret methods.
The optimum mole ratio of chitosan-glutaraldehyde to the amount of immobilized enzyme was obtained by performing immobilization of the lipase to chitosan in both powder and beads, which were previously treated with various glutaraldehyde. The ratio powder chitosan or bead chitosan to the glutaraldehyde in matrix preparation was set by using a different concentration of glutaraldehyde (0, 0.5, 0.75, 1.00, 1.50, 2.00 and 2.5%) in matrix preparation. The mixture was then processed according to the immobilization protocol. The optimum ratio was concluded based on the amount of the immobilized enzyme determined based on enzyme concentration calculated by Biuret methods.
Enzyme activity testing: Lipase hydrolytic activity of both in free and immobilized enzyme was determined using a volumetric method based18 with light modification. One gram palm oil was put into 10 mL volumetric flasks, added by 1 μL aquadest and diluted by n-hexane. The solution was then transferred to flacon bottle 50 mL and added with 100 mg of free lipase or immobilized lipase in 1 g chitosan powder or chitosan bead. The mixture was then stirred for 5 h at 37°C. The enzyme was separated from the product by filtering the mixture using filter paper. The filtrate was added with 10 mL of ethanol along with 2-3 drops of phenolphthalein indicator before titrated with standardized NaOH 0.05 M. The reaction system without the present of lipase or immobilized lipase was used as blank. The unit activity of lipase was defined as the amount of fatty acid (in micromole), which resulted in every minute of reaction. The specific activity of the lipase compared total unit of the enzyme with the amount of the enzyme in gram.
Thermal stability test of immobilized enzyme: The thermal stability test was performed by heating the enzyme before activity testing. All enzymes, free and immobilized ones were heated to a different temperature of 30, 40, 45 and 50°C for 20 min. After heating, the hydrolytic activity of the enzymes was determined as described in activity testing and then the specific activity of each enzyme were calculated. The thermal stability of the enzymes was concluded by comparing the specific activity of the enzyme as a function of heating temperature and specific activity of the unheated enzyme.
Reusability test of immobilized enzyme: Enzymes, free and immobilized lipases were used in palm oil hydrolysis as described in activity testing. After the reaction, enzymes were separated from the reaction system by filtration. The enzymes were washed using aquadest then re-used as a catalyst for new reaction system. This process was repeated for several times. The activity of the enzyme for each reaction was calculated as specific activity. The specific activity data was compared between the first, second, third, fourth to fifth uses to conclude the liability of the immobilized enzyme compared to the free enzyme.
Determination kinetics parameter (KM and Vmax) of immobilized enzyme: The KM and Vmax determination was carried out by performing activity tests of the enzyme in various concentration of substrate under the optimum condition. The palm oil substrate concentration ([S]) was varied from 0.025, 0.05, 0.075, 0.10-0.125 M for chitosan powder immobilized enzyme and from 0.03, 0.08, 0.16, 0.32-0.64 M for chitosan bead immobilized enzyme. The rate of reaction (V) was calculated by measuring fatty acid as a product in mol unit per hour of reaction. The data of 1/V was plotted against 1/[S] resulted in Lineweaver-Burk curve for each enzyme. The slope represented KM/Vmax, while the intercept of the curve represented 1/Vmax.
RESULTS AND DISCUSSION
Deacetalytaliton degree of chitosan: Chitosan with various DD% was obtained by variation of some NaOH reflux repetitions during chitin deacetylation. The result of deacetylation of the chitin could be seen at Table 1, while its associated IR spectra are shown in Fig. 1. Due to the limitation of the capacity, the preparation of the chitosan for immobilization, the matrix in the form of powder and chitosan beads were performed separately result in different DD%. Table 1 also shows that the deacetylation process using NaOH reflux seemed to give poor precision in term of yield as well as quality or DD% of the chitosan product. It could be due to different chitin as raw materials, which were also resulted from crab shell. The calculation of the DD% was based on the infra-red spectra as represented by Fig. 1.
| Fig. 1: | Comparison of FTIR spectra among a: Crab shell, b: Chitin from crab shell, c: Chitosan resulted from chitin deacetylation with once NaOH reflux, d: Chitosan resulted from chitin deacetylation with twice NaOH reflux and e: Chitosan resulted from chitin acetylation with three times NaOH reflux |
| Table 1: | Deacetylation degree of chitosan with various NaOH reflux repetition used in powder and beads matrix |
Figure 1 also shows different characteristics of IR spectra among crab shell, chitin and chitosan. Chemically, crab shell is a mixture of the complex organic compound. Therefore, the obtained peaks of IR did not show the sharp peak. Meanwhile, the deacetylation of chitin to form chitosan could be observed at ν 1700 cm-1, which represented vibration of C=O bond carbonyl group. For chitin, the peak was sharp with high absorbance since the acetyl group was still present in a significant amount. The absorbance of this ν was decreased as DD% of chitosan increase due to a reduction of acetyl group12.
Optimum pH condition of lipase immobilization: As predicted, the amount of the immobilized lipase in chitosan was influenced by the DD% of the chitosan matrix. The change of pH was capable of transforming the -NH2, protonated to deprotonated as well as control interaction between chitosan-glutaraldehyde and lipase. In general, chitosan in bead form gives the higher efficiency of the immobilization as Table 2.
| Table 2: | Influence of pH and DD to the percentage of immobilized lipase |
Immobilization of lipase at pH 6 to the chitosan with 81% DD gave the highest efficiency of immobilization regarding the amount of lipase protein could be absorbed to the chitosan matrix. Neutral pH and low ionic strength contributed in keeping -NH2 group in unprotonated leading to the formation of anamide bond with enzyme -COOH, instead of ionic interaction between -+NH3 with -COO19.
Optimization of glutaraldehyde as linker to chitosan resulted in an optimum condition of effective immobilization (Table 3). In general, the pattern of immobilization follows condition at the pH study, especially in bead chitosan matrix, where the chitosan with DD% 81% (medium) gives optimum result. The optimum glutaraldehyde concentration for bead immobilization is 1.5% to the chitosan. A rough calculation of this percentage equal to 1:10 mole ratio of -CHO of glutaraldehyde to -NH2 of chitosan. Glutaraldehyde at a concentration of 1.5% was also able to give the best immobilization of lipase in the coupling of chitosan and gelatin20.
Both in study of influence of pH and in study of the glutaraldehyde, the bead form of chitosan matrix gave particular pattern with certain optimum condition, while the powder form of the matrix did not. The interaction between lipase to the chitosan beads through glutaraldehyde performed better than that through the powder form did. It appeared that the bead had more space for lipase and glutaraldehyde to access the -NH2 group of chitosan. In powder form, the irregular structure seemed to avoid maximum interaction of -NH2 group of chitosan with lipase through glutaraldehyde.
The goal of the immobilization was not only to get as much as possible enzyme attached to the matrix, but also to get highly stable activity of the immobilized enzyme. Table 4 shows that immobilized enzyme has lower specific activity compared to free enzyme.
| Table 3: | Influence of glutaraldehyde to the percentage of immobilized lipase |
| Table 4: | Activity of the immobilized lipase |
It means that attachment of lipase to the chitosan through lipase affected the structure of the protein. Since none of the triad catalytic -NH2 of lipase involved, it means that there was no reduction of some active sites in each lipase molecule. It seemed that the interaction of lipase with chitosan involved the amino acid with -NH2 side chain responsible for the structure determining the interaction of substrate (triacylglycerol) to the active site of lipase. Since the data of specific activity on different conditions of immobilized enzyme were consistent with the data of the amount of immobilized lipase, lipase immobilized on medium DD% chitosan in beads form could give the highest activity. It means that the mode of binding of lipase to chitosan are same among all condition. The DD% did not correlate linearly to the effectiveness of the immobilization process. At low DD%, it seemed that the number of -NH2 group was not sufficient to attach with glutaraldehyde as alinker to lipase. When DD% of the chitosan is too high, the effectiveness of immobilization is also reduced. High DD% means a large number of -NH2 group leading to competition with -NH2 group of lipase to attach to the glutaraldehyde. As a comparison, the previous study reported that using entrapment technique, the amount of lipase was maximum at the highest DD% tested (92% DD), but the maximum activity of the immobilized enzyme was reported at medium DD%21. The optimum condition at medium DD% was also reported in as tudy of immobilization in hydrophobically modified chitosan22.
The highest specific activity of the immobilized enzyme is approximately 60% compared to free enzyme. The immobilization process could be claimed as a success, based on the comparison to the previous result using different technique21.
Reusability and thermal stability of immobilized lipase
Reusability: One of the primary goal of immobilization is to improve enzyme stability, leading to reused of the enzyme. Figure 2 shows reusability study of free lipase compared to immobilized lipase of chitosan in bead form. In general, immobilized lipase has lower loss of activity when compared to the free one. In all DD% of chitosan matrix, the activity stays more than 75% of its original activity, compared to loss up to 80% of free enzyme activity only in three times reused of the enzyme. Similar result previously reported for lipase immobilization into silica gel could retain the activity up to 75% after five reaction cycle and 62% after ten reaction cycle23. Higher DD% gives better stability to the enzyme, which could be related to the structure of the bead. Higher DD% gives more stable structure capable of protecting enzyme.
Thermal stability: The stability of the immobilized enzyme is also expressed by its thermal stability. Thermal stability is important because the limitation of the thermal stability of free enzyme leads to the need of the particular condition for enzyme storage. The thermal stable enzyme also has an advantage from possibility to perform their action at high temperature in which theoretically will create even higher reaction rate.
Figure 3 shows that the activity of the immobilized enzymes did not change significantly after treatment at a temperature up 50oC. In contrast, the activity of free enzyme decreased after treatment at 45°C. Heating at 40°C prior reaction apparently increased the activity of both free enzyme and immobilized enzyme. It seemed that the heat treatment improved the structure of the lipase leading to better interaction with the substrates. This result also gave abetter result if compared to immobilization of lipase in chitosan-gelatin system, where immobilized enzyme lost up to 20% of its activity after treatment at 50°C20. However, the better result was reported using glyoxyl-agaroseimmobilization system, which produced stability up to 60°C24.
| Fig. 2: | Reusability of immobilized lipase |
| Fig. 3: | Thermal stability of the immobilized enzyme |
Kinetic parameter of immobilized lipase: Two main parameters in studying kinetic of enzyme reaction are KM and Vmax. Michaelis-Menten and Lineweaver plot based on data of reaction rate at avarious substrate concentration in the approach could be used to determine the value of both parameters (Fig. 4). Table 5 shows the KM and Vmax values. The highest Vmax was found in free enzyme, while the lowest KM was discovered in the free enzyme as well. Free lipase has more free active sites and natural structure of the lipase. Therefore, the highest Vmax from this enzyme was as expected. The KM value represents the affinity of the enzyme to the substrate. The lower KM value corresponds to the higher affinity. The natural structure and higher number of an active site of free lipase also correspond to its high affinity to the substrate leading to lower KM value. The increase of KM value, which is only approximately 25% (chitosan bead DD 81%), relatively low means that the affinity of the immobilized enzyme to the substrate was not significantly different from the free enzyme. Other study using silica gel as matrix showed the increase of the KM of immobilized lipase23 up to 400%. Among immobilized condition, the kinetic parameter data supports the conclusion that immobilized lipase in chitosan with medium DD% gives the best result with the lowest KM value.
| Fig. 4(a-b): | Kinetic parameter determination, (a) Plot Michaelis-Menten and (b) Lineweaver Burk plot |
| Table 5: | Comparison of kinetic parameter of the enzyme |
The Vmax data was not significantly different between lipase immobilized at medium DD% and high DD%, but both gave higher Vmax than lipase immobilized at chitosan with low DD%.
CONCLUSION
The DD% value of chitosan plays an essential role in lipase immobilization using crosslink method with glutaraldehyde as a linker with the optimum condition at medium DD% value (81%) and 1.5% of glutaraldehyde concentration at pH 6.0. The bead form of chitosan gives the better result of immobilization due to more space or substrate to access active site of the immobilized lipase. Immobilization of lipase improves the stability of the enzyme with no significant reduction of activity up to five times of reuse and thermal stability up to 50°C, which is much better than free enzyme.
ACKNOWLEDGMENT
This study is financially supported by the Directorate General of Higher Education, Minister Education and Culture of Indonesia through LPPM-UGM competitive grant contract No. of LPPM-UGM/382/LIT/2014. The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.