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Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents
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Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents Ng Lin Cieh, Safwan Sulaiman, Mohd Noriznan Mokhtar ∗ , Mohd Nazli Naim Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
a r t i c l e
i n f o
Article history: Received 15 November 2016 Received in revised form 23 February 2017 Accepted 27 February 2017 Available online xxx Keywords: Cyclodextrin glucanotransferase (CGTase) Bleached kenaf microfiber Covalent immobilization Spacer arm Ligand
a b s t r a c t Enzyme immobilization via covalent binding provides a strong interaction between enzyme and support material. In this study, the effect of different coupling agents (spacer arms and ligands) in cyclodextrin glucanotransferase (CGTase) immobilization on bleached kenaf microfiber as a support matrix was investigated. The immobilized CGTase properties such as storage stability, thermal stability and reusability were evaluated. Immobilized CGTases on microfiber resulted in 0.162–0.24 U/mg-fiber when 55.6 U/mL of CGTase activity was initially added during the immobilization. The highest storage stability (60 ◦ C) was shown by CGTase that was immobilized with ethylenediamine and o-phthalaldehyde, whereby 60% of its activity remained after 15 days. Its high stability was also confirmed by the lowest deactivation constant, kd that was obtained at 25 ◦ C (0.0161 day−1 ) and 60 ◦ C (0.0361 day−1 ). The CGTase immobilized using ethylenediamine and glutaraldehyde has shown the best retention of enzyme activity up to 72.72% after 12 cycles of batch reaction. The results indicate that kenaf microfiber has potential to be applied as a support for enzyme immobilization and its enzymatic properties were affected by the coupling agents. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Enzymes are biocatalysts that boost up the rate of chemical reactions by reacting with substrates at their active site. Cyclodextrin glucanotransferase, CGTase (E.C.2.4.1.19) is an important enzyme in converting long complex chain of starch into various kinds of cyclodextrins (CDs) via cyclization reaction, which is also called as intramolecular transglycosylation reaction [1,2]. In cyclization reaction, a linear oligosaccharide will attach to the CGTase active site pocket. This oligosaccharide consisted of glucose units, linked by ␣-(1,4) glycosidic bond. The enzyme will react on non-reducing end glucose molecule on the oligosaccharide, to form a cyclic oligosaccharide, which is also called CDs [2,3]. Besides cyclization, CGTase can also catalyze another 3 reactions which are disproportionation, hydrolysis and coupling [4,5]. CGTases are produced by several types of bacteria but mainly by the bacteria of the genus Bacillus with the parameters that can affect its production the most are pH and temperature [6].
∗ Corresponding author. E-mail address: [email protected] (M.N. Mokhtar).
CGTase produces 3 main types of CD, which are ␣-, -, and ␥-CD with a trace amount of larger CDs [7]. ␣-CD is a cyclicmaltooligosaccharide that consists of 6 D-glucose units that are linked by ␣-(1,4) glycosidic bonds, whereas - and ␥- CD consist of 7 and 8 D-glucose units, respectively [2,8,9]. The CDs produced have a shape of a hollow truncated cone whose outer layer shows good hydrophilicity, whereas the internal cavity being hydrophobic. This allows CDs to form inclusion complexes with many kinds of guest molecule, which possess hydrophobic nature [10]. Due to this special characteristic, CDs have been widely used and applied in many fields such as in pharmaceuticals, instrumental analysis and cosmetics [11–13]. In comparison with free CGTase, the immobilized CGTase allows continuous and repeated operation [14]. In addition, enzyme immobilization also simplifies the handling of the enzyme as it is mostly used as solid rather than in solution form. Besides that, the immobilization also enables easier and better separation of the enzyme from the products and thus preventing contamination of the product by the enzyme [15]. Other than these, the physical and chemical properties of the enzymes such as 3D conformational structure, enzyme stabilities, kinetic properties, specificity, selectivity and resistance to inhibition are also improved usually upon immobilization [16].
http://dx.doi.org/10.1016/j.procbio.2017.02.025 1359-5113/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: N.L. Cieh, et al., Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.02.025
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There are several immobilization methods available [17] and one of the popular methods is by covalent binding, which it provides rigid bonds between enzyme and support, as well as it minimizes enzyme leaching after repeated use, compared to other immobilization methods [18]. The immobilized enzymes are very likely to retain its structure and have better performance than free enzymes under drastic conditions. For example, higher optimal temperature and some distorting reagent may significantly decrease free enzyme activity while inducing a lesser effect on the activity of immobilized enzymes [19]. Therefore, the use of stabilizing agent to prevent bond breakage after immobilization is often not required [20]. Different immobilization strategies are used to serve certain purposes and functions such as enzyme rigidification, prevention of enzyme aggregation and prevention of diffusional problems. Generally in covalent binding, most of the strategy involves is activation of the functional group that presents on the support matrix [21,22]. The activated functional group will then react with the functional group presents on the enzyme (e.g. amine group) [22]. Selection of proper coupling agents during immobilization is of utmost importance as the binding efficiency and the properties of the immobilized enzyme are also affected by distance between enzyme and support [23]. When compared to direct immobilization of enzyme on any support (e.g. adsorption), the use of coupling agents that act as spacer arms and ligands often provide better performance of the immobilized enzyme such as specific activity and recovered activity [24,25]. This is because spacer arm increases the distance between support and enzyme molecule and subsequently minimize the effect of steric hindrance and diffusional resistance [23]. Meanwhile, the use of ligands can help increase the efficiency of enzyme molecules to attach on end-terminal of ligand-spacer arm-support interaction. Several previous studies proved that suitable selection of chemical coupling would effect to the properties of immobilized enzyme especially in enzyme activity, stability and reusability [26–28]. In facts, covalent immobilization of enzyme could form an intense multipoint covalent binding on support, which provide a rigidity toward immobilized enzyme structure and enhance the stability, compared to the single point binding [23]. Selecting suitable support materials is also importance for enzyme immobilization purpose. Parameters such as mechanical resistance, pore diameter and specific surface area are need to be considered as a suitable support material [29]. All these parameters can affect the properties of the immobilized enzymes. Previous studies were reported for support matrix in CGTase immobilization such as Eupergit C [30], PVC [31], silica microspheres [32], Accurel MP 1000 [33], divinyl sulphone-agarose [34], core-shell polymeric support [35], poly-styrene-divinylbenzene matrices [36] and glyoxal-agarose beads [37]. Lignocellulosic fiber is expected to have high potential to be used as support for enzyme immobilization. For example, it has been studied by Souza et al. [38], where cashew apple bagasse fiber was used as support material in the immobilization of lipase B from Candida Antarctica, resulting an excellent performance in storage stability and reusability of the enzyme. In this study, bleached kenaf bast microfiber extracted from kenaf bast was used in covalent immobilization. The selection of bleached kenaf bast microfiber is due to its high potential for future applications in industrial biotechnology [22,39]. CGTase from Bacillus macerans that mainly produces ␣-CD [40–42] was immobilized on the bleached kenaf microfiber by using various coupling agents such as hexamethylenediamine (HMDA) and ethylenediamine (EDA) as spacer arms, whereas glutaraldehyde (GA) and o-phthalaldehyde (OPA) as ligands. The properties of the immobilized CGTase with different coupling agents such as
reusability and storage stability were then investigated in this study. 2. Materials and methods 2.1. Materials Kenaf bast fiber was supplied by the Lembaga Kenaf dan Tembakau Negara, Malaysia. CGTase from Bacillus macerans with activity of ∼600 U/mL (∼4 mg protein/mL) was purchased from Amano Enzyme Inc. (Japan). HMDA, EDA and OPA were bought from Sigma Aldrich (Malaysia). Water soluble potato starch powder was bought from Fluka (Switzerland). GA 25% was bought from Ajax Finechem (Australia) while sodium chlorite and ␣-CD were bought from Acros Organics (USA). Other chemicals used in this research were reagent grade. 2.2. Bleaching of kenaf bast fiber Kenaf bast fiber was firstly cut into approximately 5 cm in length. Then, the fiber was bleached and mercerized according to the method described by Tee et al. [43]. 20 g fiber was immersed in 640 mL of heated water (70 ◦ C) and then 4 mL of CH3 COOH and 8 g of NaClO2 were added. In each subsequent hour, the same amount of CH3 COOH and NaClO2 were added to the solution, stirred and left to bleach the fiber in the beaker. This was repeated for 5 h. The delignified holocellulose was then washed and filtered with distilled water until the filtrate become colorless. The obtained holocellulose was then immersed in 500 mL of 5% w/v NaOH solution at room temperature for 2 h. After that, the brown solution formed was filtered and 500 mL of diluted CH3 COOH (1.47% w/v) was added to neutralize the fiber. The mixture was left to settle for 5 mins. This was followed by rinsing the fiber with distilled water and then filtration. This washing process was repeated until the pH become neutralized (pH 7.0). The bleached kenaf fiber was finally dried in the conventional oven at 105 ◦ C for 24 h. 2.3. Support activation Two types of spacer arm were used in this study; HMDA and EDA. The coupling of bleached kenaf microfiber with HMDA was done according to the method described by Chang and Shaw [44]. Similar method was repeated when EDA was used. Then, the fiber that had already been coupled with the spacer arm was activated by ligands, which were GA and OPA. To activate the fiber with GA, 1 g of the fiber-spacer arm was immersed into 20 mL of GA (0.05 M) in phosphate buffer (0.1 M, pH 8.0). The mixture was stirred at 4 ◦ C for 12 h. After that, the activated support was washed with distilled water and stored at 4 ◦ C before further use. For OPA activation, 1 g of the fiber-spacer arm was immersed into 20 mL of OPA (0.05 M) in ethanol and the mixture was stirred for 6 h at room temperature. 2.4. Surface morphology characterization The morphological changes of kenaf microfiber before and after covalent coupling with spacer and ligand were observed by Hitachi S3400N SEM at 5 kV accelerated voltage. Carbon coating was applied to each sample prior to the observation under SEM. 2.5. CGTase immobilization CGTase with different activities, 13.9, 27.8, 41.7 and 55.6 U/mL (with protein concentration of 0.18 ± 0.02, 0.36 ± 0.034, 0.54 ± 0.06, 0.72 ± 0.08 mg/mL, respectively) were prepared separately by dilution of enzyme stock solution with phosphate
Please cite this article in press as: N.L. Cieh, et al., Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.02.025
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buffer (0.05 M, pH 6.0, final volume 100 mL). The specific activity of all samples was calculated as ∼77.07 U/mg protein. Then, to each diluted 100 mL CGTase solution, 2 g of the activated fiber was added. These mixtures were then stirred at 4 ◦ C for 12 h. The immobilized CGTase activity and protein content were measured. 2.6. Protein loading determination The amount of protein was determined by analysis at 595 nm using UV–vis spectrophotometer according to the Bradford method [45]. BSA was used as standard protein. During preparation of enzyme immobilization, the fiber was washed with water and each filtrate was analyzed for the amount of free protein. The washing process was repeated until no protein was detectable in the washing filtrate. The amount of protein loaded was then calculated as: Protein Loading (%) =
Po − Pf Po
× 100%
(1)
where Po represents the amount of protein (mg) added and Pf represents the total amount of protein (mg) detected in all filtrates. 2.7. CGTase assay The activity of free and immobilized CGTase was analyzed according to the method described by Lejeune et al. [46] with slight modification. Free CGTase or immobilized CGTase was added to 4 mL of 5% starch solution (in phosphate buffer, 0.05 M, pH 6.0). The enzymatic reaction was performed at 60 ◦ C for 10 min. To 1 mL of the reaction mixture, 49 L of 6.0 M HCl was added to stop the CGTase activity. Then, 1.43 mL of methyl orange solution (0.06 mM) was added. Each sample was then cooled at 16 ◦ C for 10 min. The reading of the sample was taken by UV–vis spectrophotometer at the wavelength of 505 nm with the extinction coefficient of ␣CD is −0.255 (mg/mL)−1 cm−1 . 1 unit of enzyme activity (U) was defined as the amount of enzyme that produced 1 mole of ␣-CD per minute. 2.8. Effect of temperature on CGTase activity 20 mg of fiber with immobilized CGTase and equivalent activity of free CGTase were incubated in 4 mL of 5% starch solution in phosphate buffer (0.05 M, pH 6.0) for 10 min. The reaction was carried out for different temperatures ranging from 30 ◦ C to 90 ◦ C and the CGTase activity was assayed as mentioned previously. 2.9. Storage stability and deactivation study of CGTase Free and immobilized CGTase were stored under 3 different temperatures, 4 ◦ C, 25 ◦ C and 60 ◦ C. At day initial, day 1, day 2, day 6, day 8, day 11, day 14 and day 15, the activity of CGTase was assayed as described previously. To study deactivation kinetic of CGTase, it is considered that the rate of deactivation is a first order reaction [47] as shown: dCE = −kd · CE dt
(2)
where CE and kd are CGTase activity (U/mL or U/mg-fiber) and deactivation constant (day−1 ), respectively. After integration, it gave: ln
CE = −kd · t CE0
(3)
kd can be estimated based on the slope from graph (ln CE /CE0 versus t). Then, kd can be represented as Arrhenius equation: Ed
kd = kd0 · e− R·T
(4)
3
Therefore, after linearization, it gave: ln kd = −
Ed 1 · + ln kd0 R T
(5)
where Ed and kd0 are deactivation energy (kJ/kmole) and initial deactivation constant (day−1 ), respectively. They can be estimated from the graph of ln kd against 1/T. 2.10. Production of ˛-CD 50 mg of fiber with immobilized CGTase and almost equivalent activity of free CGTase were added separately to 100 mL of 5% starch solution in phosphate buffer (0.05 M, pH 6.0). The reaction was then conducted for 24 h at 60 ◦ C. Aliquots of the sample were taken at 10 min, 30 min, 2 h, 9 h, 14 h, 19 h, 22 h and 24 h and each of them was analyzed for ␣-CD concentration as previously described. 2.11. Operational stability of immobilized CGTase 20 mg of the support that consists of immobilized CGTase was added to 6 mL of 5% starch solution in phosphate buffer (0.05 M, pH 6.0). The mixture was incubated at 60 ◦ C for 30 min. After the reaction, the immobilized CGTase was collected by centrifugation at 10,000 rpm for 10 min while the supernatant was analyzed for ␣CD production. The precipitate, recovered fiber with immobilized CGTase was washed with the same buffer, and centrifuged. The supernatant was removed and then immobilized CGTase was reincubated by adding 6 mL of newly prepared starch solution to start a new cycle. This was repeated for a total of 12 cycles. 3. Results and discussion 3.1. Surface morphology Fig. 1a shows the SEM image of bleached kenaf bast microfiber with the diameter of 10–15 m. The surface of the fiber is slightly smooth and uniform. However, some agglomerations were observed upon coupling with HMDA as shown in Fig. 1b. This could be due to the presence of the spacer arms on the fiber, which is the HMDA. When the fiber was further activated with OPA, more agglomerations were observed as in Fig. 1c. The extra agglomerations observed on the surface of the fiber could be the OPA, which is the ligand. The presence of the spacer arm, ligand and CGTase on the surface of the fiber has been confirmed by Sulaiman et al. [48]. In their study, 1,12-dodecanediamine and glutaraldehyde were used as spacer arms and ligands, respectively. From FTIR analysis, it indicates that the interaction of spacer arm-ligand toward support and CGTase molecules has been covalently immobilized. HMDA and EDA act as spacer arms, and GA and OPA act as ligands, which all of them have the same functional groups and chemical classes (diamines and dialdehydes) as reported by that previous study [48]. A possible mechanism can be adopted to understand the reaction of covalent immobilization on the surface of microfiber. In this study, ( NH2 ) group located at both ends of the spacer arms as they were all homobifunctional linkers. The first end-terminal of NH2 group would then react with ( OH) in the carboxyl group of the support, which was exposed from oxidation of cellulose during bleaching process [49]. Thus, the amide bond ( CONH ) was formed between the support and the spacer arm as shown in Fig. 2a [50,51]. After that, other end-terminal of ( NH2 ) group on the spacer arm will react with the functional group of the ligand, ( CHO) to form ( CH N ) covalent bond as in Fig. 2b. Hence, other end-terminal of ( CHO) group will attach to CGTase molecule by reacting with the ( NH2 ) group on CGTase to form ( CH N ) covalent bond as in Fig. 2c [25,52]. This finally completed the immobilization procedure.
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Fig. 1. SEM image of a) Bleached kenaf bast microfiber, b) Fiber coupled with HMDA, c) Fiber coupled with HMDA and OPA.
3.2. Protein loading and activity recovery of immobilized CGTase The amount of CGTase activity added during immobilization has a significant effect on protein loading and activity recovery of immobilized CGTase. Fig. 3a shows that the protein loading percentage for all immobilized CGTases decreased with the increase of CGTase activity added during the immobilization. At lower CGTase activity that was added, higher ratio of coupling agent to CGTase leads to high percentage of protein loading due to higher chance for available CGTase molecule to bind with coupling agent. As the CGTase activity added was increased, it decreases the percentage of protein loading due to limitation of coupling agent that was available for the binding. In Fig. 3b, the immobilized CGTase activity increased steadily when the free CGTase added during immobilization was increased from 13.9 U/mL to 41.7 U/mL. However lesser increment can be observed in all the immobilized CGTases when 55.6 U/mL of CGTase activity was added, resulting 0.162–0.24 U/mg-fiber of their activity. This is probably because overcrowding of CGTase molecules has started to occur on the surface of bleached kenaf bast fiber. Overcrowding or agglomeration may develop steric hindrance some of the enzyme active sites during the assay, which reduces the reaction between the immobilized CGTase and the substrate [31]. The specific activity of immobilized CGTase shows the increasing trend when higher activity of free CGTase was added. As shown in Fig. 3c, (EDA-GA)-CGTase depicts the highest specific activity in this study (29.4 U/mg-protein). From Fig. 3, it can be observed that spacer arm, HMDA resulted in higher protein loading percentage than EDA. HMDA is a longer aliphatic chain of carbon atom, which is able to create a longer of interaction distance compared to EDA. The longer interaction dis-
tance would easily increase the chance to the interaction of ligand, as well as enhance the implication of CGTase binding [53]. Besides, OPA was also found to be a better ligand than GA in increasing protein loading percentage. A possible reason for this is the structural difference between GA and OPA. The ring structure of OPA may enable greater interaction between functional groups of OPA and enzyme. As a result, more OPA molecules are available for enzyme binding compared to GA which has a linear structure. Thus, the interaction between microfiber-(HMDA-OPA) and CGTase resulted in the highest protein loading. Meanwhile, microfiber-(EDA-GA)CGTase resulted in higher residual enzyme activity and its specific activity. It seems that, the EDA-GA resulted in less alteration of CGTase molecule during the immobilization compared to other spacer arm-ligand combinations in this study. 3.3. Effect of temperature on CGTase activity Many studies have found out a common finding which is immobilization will tend to alter the properties of immobilized enzymes [54]. Rigidification of enzyme structure resulted from immobilization may bring changes in some of the enzymatic properties [19]. The results have become more obvious when the effect of temperature on free and immobilized CGTases activity were compared as in Fig. 4. The activity of both free and immobilized CGTases increased when the temperature was increased up to their optimum point. However, a shift of optimum temperature can be observed in all the immobilized CGTases (optimum at 70 ◦ C) when compared to free CGTase (optimum at 60 ◦ C). In addition, the immobilized CGTases were able to maintain higher relative activity than native CGTase at high temperatures which was above 70 ◦ C. These results indicate thermal stability of enzyme that has been obtained by immobiliza-
Please cite this article in press as: N.L. Cieh, et al., Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.02.025
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5
O C OH + H2N CH2C4H8CH2 NH2
O H C N CH2C4H8CH2 NH2 + H2O
Support
Support
a)
(HMDA)
Support
Support
b) H H O H + C N CH2C4H8CH2 NH2 O C C3H6 C O (GA)
H H O H C N CH2C4H8CH2 N C C3H6 C O
+ H2O
Support
Support
c) H H O H C N CH2C4H8CH2 N C C3H6 C O + H2N
H H O H C CH C H CH C H N C 2 4 8 2 N 3 6 C N
CGTase
CGTase
+ H2O
Fig. 2. Mechanism of a) coupling of spacer arm on bleached kenaf bast microfiber, b) coupling of ligand on fiber with spacer arm, c) enzyme immobilization on the activated fiber. Table 1 Deactivation constant and deactivation energy of free and immobilized CGTase on kenaf microfiber. Spacer arm-ligand
Deactivation constant, kd (day−1 ) ◦
Free CGTase HMDA-GA HMDA-OPA EDA-GA EDA-OPA
◦
Initial deactivation constant, kd0 (day−1 )
Deactivation energy, Ed (kJ/kmole)
3.86 × 109 148.62 25.09 15.32 32.12
64370 19377 16726 16081 18810
◦
4 C
25 C
60 C
0.0026 0.0331 0.0171 0.0138 0.0091
0.0227 0.0592 0.0309 0.0244 0.0161
0.2929 0.1360 0.0584 0.0450 0.0361
tion. Greater stability of covalent immobilization could prevent the inactivation of CGTase molecules at high degree of temperature. Through covalent binding, multipoint covalent attachment was also very likely to occur during the immobilization. The presence of multiple covalent bonds will fix the relative positions of all the functional groups of enzyme, which are involved in immobilization even when the immobilized CGTase was exposed in distorting agents such as extreme heat, solvent and pH [55]. Besides, ionization of amino group and other reactive groups of enzymes during immobilization may also occur since the enzyme was immobilized in buffer solution. This ionization of reactive groups will improve its penetrability and thus it improves the stability of the immobilized CGTase [56]. Therefore, the immobilized CGTase has lower rate of denaturation compared to free CGTase especially at high temperature. 3.4. Storage stability and deactivation study of CGTase Fig. 5 shows the result of storage stability for free CGTase and all immobilized CGTases at 3 different storage temperatures, 4 ◦ C, 25 ◦ C and 60 ◦ C. From the figure, free CGTase and all the immobi-
lized CGTases show the best retention of enzyme activity at the lowest storage temperature, 4 ◦ C. However, as the storage temperature increases to 25 ◦ C and 60 ◦ C, the activity retention for both free CGTase and all the immobilized CGTases decreased. The storage stability decreased was due to the deactivation of enzyme caused by prolonged of storage time and exposure to high temperature. From the figure, it can also be observed that the performance of free CGTase is better than immobilized CGTase when they were stored at 4 ◦ C. The activity of free CGTase shows almost constant at 4 ◦ C and decreased about 80% at 25 ◦ C (at the end of day 15). However, at higher storage temperature (60 ◦ C), all the immobilized CGTases show better performance than free CGTase. Free CGTase denatured completely since day 11 while all the immobilized CGTases still retained their respective catalytic activity. This result shows that the decrease of activity in free CGTase is higher than any of the immobilized CGTase at higher temperature. The deactivation constant, kd at 4 ◦ C, 25 ◦ C and 60 ◦ C for free and all the immobilized CGTases were obtained from the slope of graphs (Eq. (3)) and tabulated in Table 1. Using kd , Fig. 6 was plotted based on Eq. (5); Ed and kd0 of free and immobilized CGTase can be estimated from the slope and the y-intercept of the lines,
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6
b) 70
0.30
60
0.25
Immobilized CGTase activity (U/mg fibre)
Protein loading (%)
a)
50 40 30 20 10
0.20 0.15 0.10 0.05 0.00
0 0
10
20
30
40
50
0
60
10
20
30
40
50
60
CGTase added (U/mL)
CGTase added (U/mL)
c) Specific activity of immoblized CGTase (U/mg protein)
35 30 25 20 15 10 5 0 0
10
20
30
40
50
60
CGTase added (U/mL) Fig. 3. Effect of amount of CGTase added during immobilization on a) protein loading, b) activity recovery, and c) specific activity of CGTase. Symbols: (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)-CGTase; (), (EDA-OPA)-CGTase. The immobilization procedure was conducted at pH 6.0 and 4 ◦ C.
120
Relative activity (%)
100 80 60 40 20 0
20
30
40
50
60
70
80
90
100
Temperature (oC) Fig. 4. Relative activity of free and immobilized CGTases at different temperatures. Symbols: (), Native CGTase; (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)-CGTase; (), (EDA-OPA)-CGTase. The assay was conducted at pH 6.0.
respectively. The values of Ed and kd0 for free and immobilized CGTases were also summarized in Table 1. It shows that free CGTase has better stability than the immobilized CGTase when stored at 4 ◦ C and 25 ◦ C. It can be seen that free CGTase has the lowest kd (0.0026 day−1 ) at 4 ◦ C. However, at 60 ◦ C, the kd of free CGTase is the highest (0.2929 day−1 ) compared to all the immobilized CGTases. Among the immobilized enzymes, CGTase immobilized with EDA-OPA shows the lowest kd at 25 ◦ C (0.0161 day−1 ) and 60 ◦ C (0.0361 day−1 ). The lower deactivation constant, results in the higher stability of the enzyme. It seems that the immobilization has altered its conformation structure, resulting in a gradual decrease its residual activity during the storage (even it was stored at 4 ◦ C). Thus, the CGTase immobilization is recommended to be prepared freshly before use. However, all the immobilized CGTases have better thermal stability than free CGTase when they were stored at 60 ◦ C. EDA-OPA was the best combination of spacer and ligand in maintaining the storage stability of the immobilized CGTase while HMDA-GA provides the
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b) 100
100
90
90
80
80
Relative activity (%)
Relative activity (%)
a)
7
70 60 50 40 30
70 60 50 40 30
20
20
10
10 0
0 0
2
4
6
8
10
12
14
0
16
2
6
4
8
10
12
14
16
Time (days)
Time (days)
c) 100
Relative activity (%)
90 80 70 60 50 40 30 20 10 0 0
2
4
6
8
10
12
14
16
Time (days) Fig. 5. Storage stability at different temperatures a) 4 ◦ C, b) 25 ◦ C, and c) 60 ◦ C. Symbols: (), Native CGTase; (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)-CGTase; (), (EDA-OPA)-CGTase. The assay was conducted at pH 6.0 and 60 ◦ C.
least storage stability to the immobilized CGTase. This is due to the influence of different coupling agents used. HMDA is a longer spacer arm and hence lower rigidity can be transmitted to the immobilized enzymes through multipoint covalent attachment compared to EDA [29]. For the ligands, GA has a linear structure while OPA is globular. Hence the distance between support and enzyme will be longer when GA is used as the ligand. Due to this, lesser protection could be provided by the support matrix to the enzyme when compared to using OPA as ligand. This study is very important for getting insight about operational stability especially when the immobilized enzyme was used for several times or/and certain period of time. Initial deactivation constant (kd0 ) for free CGTase is extremely higher (3.86 × 109 day−1 ) compared to the immobilized CGTases (15.32–148.62 day−1 ). This indicates that the activity of free CGTase is high sensitive by temperature change.
3.5. Production of ˛-CD Fig. 7 shows the production of ␣-CD for 24 h by free and immobilized CGTase. The ␣-CD production by all CGTases increased significantly during the first 30 min in which free CGTase, immobilized CGTase with HMDA-GA, HMDA-OPA, EDA-GA and EDA-OPA achieved approximately 77%, 72%, 51%, 74% and 72% of their maximum production, respectively. After that, the production of ␣-CD by all the CGTase slowed down and eventually reached their plateau. The time required to reach maximum production was about 14 h for free CGTase and approximately 19 h for immobilized CGTase. The reasons for the production to stop in the end include inhibition by its product and substrate limitation [57]. A substantial amount of ␣-CD may act as the inhibitor and occupy the active site, thus it will cause the conformation change and lead to the inactivation of CGTase molecule.
Please cite this article in press as: N.L. Cieh, et al., Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.02.025
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Fig. 6. Determination of initial deactivation constant and deactivation energy of free and immobilized CGTases. Symbols: (),Free CGTase; (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)-CGTase; (), (EDA-OPA)-CGTase. The assay was conducted at pH 6.0 and 60 ◦ C.
Fig. 8. Reusability of CGTase immobilized with different coupling agents. Symbols: (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)- CGTase; (), (EDA-OPA)-CGTase. The reaction was conducted at pH 6.0 and 60 ◦ C using 5% starch.
temperature and 70 ◦ C–90 ◦ C for reaction temperature) when compared to free CGTase. This is due to the structure rigidification of the immobilized CGTase developed by the presence of covalent bonds and multipoint covalent attachments after immobilization. Besides that, when the same spacer arm is used, OPA acts as a better ligand when compared to GA in improving the thermal stability of the immobilized CGTase. In terms of enzyme activity and reusability, EDA-GA is the best coupling agent combinations which resulted the highest activity retention, specific activity and reusability (0.23 U/mg-fiber, 29.4 U/mg-protein and 72.72%, respectively).
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3.6. Reusability of immobilized CGTase In this study, the reusability of the immobilized CGTase was investigated by carrying out 12 cycles of batch reaction and the results are shown in Fig. 8. It was observed that after 12 cycles, CGTase immobilized on microfiber using EDA-GA retained the highest residual activity (72.72%) and followed by CGTase immobilized with HMDA-OPA (66.10%), EDA-OPA (58.35%) and HMDA-GA (52.20%). From the result, it can be observed that the residual activity of all the immobilized CGTase decreased with the number of cycle up to 10 cycles. The main reason that can be related to the reduction of CGTase residual activity includes the presence of irreversible product inhibitor [57]. Within the reaction period (total cycle times was less than 24 h), based on the thermal deactivation study, the loss of residual activity due to alteration on its conformation structure by immobilization is not significant. 4. Conclusions Bleached kenaf bast microfiber has potential to be used as a support for enzyme immobilization. Immobilized CGTase generally has improved stability at high temperature (60 ◦ C for storage
The authors would like to express thanks to the Ministry of Higher Education Malaysia and Universiti Putra Malaysia for the sponsorship of this work under Fundamental Research Grant Scheme 03-01-14-1426FR/5524510 and Putra Grant GPIPS/2014/9438708, respectively. References [1] A.S.S. Ibrahim, A.A. Al-Salamah, A.M. El-Toni, M.A. El-Tayeb, Y.B. Elbadawi, Cyclodextrin glucanotransferase immobilization onto functionalized magnetic double mesoporous core–shell silica nanospheres, Electron. J. Biotechnol. 17 (2014) 55–64. [2] C. Moriwaki, L.R. Ferreira, J.R.T. Rodella, G. Matioli, A novel cyclodextrin glycosyltransferase from Bacillus sphaericus strain 41: production, characterization and catalytic properties, Biochem. Eng. J. 48 (2009) 124–131. [3] M.T. Martı´ın, M.A. Cruces, M. Alcalde, F.J. Plou, M. Bernabé, A. Ballesteros, Synthesis of maltooligosyl fructofuranosides catalyzed by immobilized cyclodextrin glucosyltransferase using starch as donor, Tetrahedron 60 (2004) 529–534. [4] D. Svensson, P. Adlercreutz, Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides: control of product distribution by flow rate adjustment, J. Mol. Catal. B Enzym. 69 (2011) 147–153. [5] N. Atanasova, T. Kitayska, D. Yankov, M. Safarikova, A. Tonkova, Cyclodextrin glucanotransferase production by cell biocatalysts of Alkaliphilic bacilli, Biochem. Eng. J. 46 (2009) 278–285. [6] F.S.T. Pinto, S.H. Flôres, M.A.Z. Ayub, P.F. Hertz, Production of cyclodextrin glycosyltransferase by alkaliphilic Bacillus circulans in submerged and solid-state cultivation, Bioprocess Biosyst. Eng. 30 (2007) 377–382. [7] Y. Terada, M. Yanase, H. Takata, T. Takaha, S. Okada, Cyclodextrins are not the major cyclic alpha-1, 4-glucans produced by the initial action of cyclodextrin glucanotransferase on amylose, J. Biol. Chem. 272 (1997) 15729–15733. [8] J. da N. Schöffer, M.P. Schöffer, R.C. Klein, Continuous production of -cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan, Carbohydr. Polym. 98 (2013) 1311–1316. [9] K.M. Goh, N.M. Mahadi, O. Hassan, R.N.Z.R.A. Rahman, R.M. Illias, A predominant -CGTase G1 engineered to elucidate the relationship between protein structure and product specificity, J. Mol. Catal. B Enzym. 57 (2009) 270–277.
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Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents Ng Lin Cieh, Safwan Sulaiman, Mohd Noriznan Mokhtar ∗ , Mohd Nazli Naim Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
a r t i c l e
i n f o
Article history: Received 15 November 2016 Received in revised form 23 February 2017 Accepted 27 February 2017 Available online xxx Keywords: Cyclodextrin glucanotransferase (CGTase) Bleached kenaf microfiber Covalent immobilization Spacer arm Ligand
a b s t r a c t Enzyme immobilization via covalent binding provides a strong interaction between enzyme and support material. In this study, the effect of different coupling agents (spacer arms and ligands) in cyclodextrin glucanotransferase (CGTase) immobilization on bleached kenaf microfiber as a support matrix was investigated. The immobilized CGTase properties such as storage stability, thermal stability and reusability were evaluated. Immobilized CGTases on microfiber resulted in 0.162–0.24 U/mg-fiber when 55.6 U/mL of CGTase activity was initially added during the immobilization. The highest storage stability (60 ◦ C) was shown by CGTase that was immobilized with ethylenediamine and o-phthalaldehyde, whereby 60% of its activity remained after 15 days. Its high stability was also confirmed by the lowest deactivation constant, kd that was obtained at 25 ◦ C (0.0161 day−1 ) and 60 ◦ C (0.0361 day−1 ). The CGTase immobilized using ethylenediamine and glutaraldehyde has shown the best retention of enzyme activity up to 72.72% after 12 cycles of batch reaction. The results indicate that kenaf microfiber has potential to be applied as a support for enzyme immobilization and its enzymatic properties were affected by the coupling agents. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Enzymes are biocatalysts that boost up the rate of chemical reactions by reacting with substrates at their active site. Cyclodextrin glucanotransferase, CGTase (E.C.2.4.1.19) is an important enzyme in converting long complex chain of starch into various kinds of cyclodextrins (CDs) via cyclization reaction, which is also called as intramolecular transglycosylation reaction [1,2]. In cyclization reaction, a linear oligosaccharide will attach to the CGTase active site pocket. This oligosaccharide consisted of glucose units, linked by ␣-(1,4) glycosidic bond. The enzyme will react on non-reducing end glucose molecule on the oligosaccharide, to form a cyclic oligosaccharide, which is also called CDs [2,3]. Besides cyclization, CGTase can also catalyze another 3 reactions which are disproportionation, hydrolysis and coupling [4,5]. CGTases are produced by several types of bacteria but mainly by the bacteria of the genus Bacillus with the parameters that can affect its production the most are pH and temperature [6].
∗ Corresponding author. E-mail address: [email protected] (M.N. Mokhtar).
CGTase produces 3 main types of CD, which are ␣-, -, and ␥-CD with a trace amount of larger CDs [7]. ␣-CD is a cyclicmaltooligosaccharide that consists of 6 D-glucose units that are linked by ␣-(1,4) glycosidic bonds, whereas - and ␥- CD consist of 7 and 8 D-glucose units, respectively [2,8,9]. The CDs produced have a shape of a hollow truncated cone whose outer layer shows good hydrophilicity, whereas the internal cavity being hydrophobic. This allows CDs to form inclusion complexes with many kinds of guest molecule, which possess hydrophobic nature [10]. Due to this special characteristic, CDs have been widely used and applied in many fields such as in pharmaceuticals, instrumental analysis and cosmetics [11–13]. In comparison with free CGTase, the immobilized CGTase allows continuous and repeated operation [14]. In addition, enzyme immobilization also simplifies the handling of the enzyme as it is mostly used as solid rather than in solution form. Besides that, the immobilization also enables easier and better separation of the enzyme from the products and thus preventing contamination of the product by the enzyme [15]. Other than these, the physical and chemical properties of the enzymes such as 3D conformational structure, enzyme stabilities, kinetic properties, specificity, selectivity and resistance to inhibition are also improved usually upon immobilization [16].
http://dx.doi.org/10.1016/j.procbio.2017.02.025 1359-5113/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: N.L. Cieh, et al., Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.02.025
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There are several immobilization methods available [17] and one of the popular methods is by covalent binding, which it provides rigid bonds between enzyme and support, as well as it minimizes enzyme leaching after repeated use, compared to other immobilization methods [18]. The immobilized enzymes are very likely to retain its structure and have better performance than free enzymes under drastic conditions. For example, higher optimal temperature and some distorting reagent may significantly decrease free enzyme activity while inducing a lesser effect on the activity of immobilized enzymes [19]. Therefore, the use of stabilizing agent to prevent bond breakage after immobilization is often not required [20]. Different immobilization strategies are used to serve certain purposes and functions such as enzyme rigidification, prevention of enzyme aggregation and prevention of diffusional problems. Generally in covalent binding, most of the strategy involves is activation of the functional group that presents on the support matrix [21,22]. The activated functional group will then react with the functional group presents on the enzyme (e.g. amine group) [22]. Selection of proper coupling agents during immobilization is of utmost importance as the binding efficiency and the properties of the immobilized enzyme are also affected by distance between enzyme and support [23]. When compared to direct immobilization of enzyme on any support (e.g. adsorption), the use of coupling agents that act as spacer arms and ligands often provide better performance of the immobilized enzyme such as specific activity and recovered activity [24,25]. This is because spacer arm increases the distance between support and enzyme molecule and subsequently minimize the effect of steric hindrance and diffusional resistance [23]. Meanwhile, the use of ligands can help increase the efficiency of enzyme molecules to attach on end-terminal of ligand-spacer arm-support interaction. Several previous studies proved that suitable selection of chemical coupling would effect to the properties of immobilized enzyme especially in enzyme activity, stability and reusability [26–28]. In facts, covalent immobilization of enzyme could form an intense multipoint covalent binding on support, which provide a rigidity toward immobilized enzyme structure and enhance the stability, compared to the single point binding [23]. Selecting suitable support materials is also importance for enzyme immobilization purpose. Parameters such as mechanical resistance, pore diameter and specific surface area are need to be considered as a suitable support material [29]. All these parameters can affect the properties of the immobilized enzymes. Previous studies were reported for support matrix in CGTase immobilization such as Eupergit C [30], PVC [31], silica microspheres [32], Accurel MP 1000 [33], divinyl sulphone-agarose [34], core-shell polymeric support [35], poly-styrene-divinylbenzene matrices [36] and glyoxal-agarose beads [37]. Lignocellulosic fiber is expected to have high potential to be used as support for enzyme immobilization. For example, it has been studied by Souza et al. [38], where cashew apple bagasse fiber was used as support material in the immobilization of lipase B from Candida Antarctica, resulting an excellent performance in storage stability and reusability of the enzyme. In this study, bleached kenaf bast microfiber extracted from kenaf bast was used in covalent immobilization. The selection of bleached kenaf bast microfiber is due to its high potential for future applications in industrial biotechnology [22,39]. CGTase from Bacillus macerans that mainly produces ␣-CD [40–42] was immobilized on the bleached kenaf microfiber by using various coupling agents such as hexamethylenediamine (HMDA) and ethylenediamine (EDA) as spacer arms, whereas glutaraldehyde (GA) and o-phthalaldehyde (OPA) as ligands. The properties of the immobilized CGTase with different coupling agents such as
reusability and storage stability were then investigated in this study. 2. Materials and methods 2.1. Materials Kenaf bast fiber was supplied by the Lembaga Kenaf dan Tembakau Negara, Malaysia. CGTase from Bacillus macerans with activity of ∼600 U/mL (∼4 mg protein/mL) was purchased from Amano Enzyme Inc. (Japan). HMDA, EDA and OPA were bought from Sigma Aldrich (Malaysia). Water soluble potato starch powder was bought from Fluka (Switzerland). GA 25% was bought from Ajax Finechem (Australia) while sodium chlorite and ␣-CD were bought from Acros Organics (USA). Other chemicals used in this research were reagent grade. 2.2. Bleaching of kenaf bast fiber Kenaf bast fiber was firstly cut into approximately 5 cm in length. Then, the fiber was bleached and mercerized according to the method described by Tee et al. [43]. 20 g fiber was immersed in 640 mL of heated water (70 ◦ C) and then 4 mL of CH3 COOH and 8 g of NaClO2 were added. In each subsequent hour, the same amount of CH3 COOH and NaClO2 were added to the solution, stirred and left to bleach the fiber in the beaker. This was repeated for 5 h. The delignified holocellulose was then washed and filtered with distilled water until the filtrate become colorless. The obtained holocellulose was then immersed in 500 mL of 5% w/v NaOH solution at room temperature for 2 h. After that, the brown solution formed was filtered and 500 mL of diluted CH3 COOH (1.47% w/v) was added to neutralize the fiber. The mixture was left to settle for 5 mins. This was followed by rinsing the fiber with distilled water and then filtration. This washing process was repeated until the pH become neutralized (pH 7.0). The bleached kenaf fiber was finally dried in the conventional oven at 105 ◦ C for 24 h. 2.3. Support activation Two types of spacer arm were used in this study; HMDA and EDA. The coupling of bleached kenaf microfiber with HMDA was done according to the method described by Chang and Shaw [44]. Similar method was repeated when EDA was used. Then, the fiber that had already been coupled with the spacer arm was activated by ligands, which were GA and OPA. To activate the fiber with GA, 1 g of the fiber-spacer arm was immersed into 20 mL of GA (0.05 M) in phosphate buffer (0.1 M, pH 8.0). The mixture was stirred at 4 ◦ C for 12 h. After that, the activated support was washed with distilled water and stored at 4 ◦ C before further use. For OPA activation, 1 g of the fiber-spacer arm was immersed into 20 mL of OPA (0.05 M) in ethanol and the mixture was stirred for 6 h at room temperature. 2.4. Surface morphology characterization The morphological changes of kenaf microfiber before and after covalent coupling with spacer and ligand were observed by Hitachi S3400N SEM at 5 kV accelerated voltage. Carbon coating was applied to each sample prior to the observation under SEM. 2.5. CGTase immobilization CGTase with different activities, 13.9, 27.8, 41.7 and 55.6 U/mL (with protein concentration of 0.18 ± 0.02, 0.36 ± 0.034, 0.54 ± 0.06, 0.72 ± 0.08 mg/mL, respectively) were prepared separately by dilution of enzyme stock solution with phosphate
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buffer (0.05 M, pH 6.0, final volume 100 mL). The specific activity of all samples was calculated as ∼77.07 U/mg protein. Then, to each diluted 100 mL CGTase solution, 2 g of the activated fiber was added. These mixtures were then stirred at 4 ◦ C for 12 h. The immobilized CGTase activity and protein content were measured. 2.6. Protein loading determination The amount of protein was determined by analysis at 595 nm using UV–vis spectrophotometer according to the Bradford method [45]. BSA was used as standard protein. During preparation of enzyme immobilization, the fiber was washed with water and each filtrate was analyzed for the amount of free protein. The washing process was repeated until no protein was detectable in the washing filtrate. The amount of protein loaded was then calculated as: Protein Loading (%) =
Po − Pf Po
× 100%
(1)
where Po represents the amount of protein (mg) added and Pf represents the total amount of protein (mg) detected in all filtrates. 2.7. CGTase assay The activity of free and immobilized CGTase was analyzed according to the method described by Lejeune et al. [46] with slight modification. Free CGTase or immobilized CGTase was added to 4 mL of 5% starch solution (in phosphate buffer, 0.05 M, pH 6.0). The enzymatic reaction was performed at 60 ◦ C for 10 min. To 1 mL of the reaction mixture, 49 L of 6.0 M HCl was added to stop the CGTase activity. Then, 1.43 mL of methyl orange solution (0.06 mM) was added. Each sample was then cooled at 16 ◦ C for 10 min. The reading of the sample was taken by UV–vis spectrophotometer at the wavelength of 505 nm with the extinction coefficient of ␣CD is −0.255 (mg/mL)−1 cm−1 . 1 unit of enzyme activity (U) was defined as the amount of enzyme that produced 1 mole of ␣-CD per minute. 2.8. Effect of temperature on CGTase activity 20 mg of fiber with immobilized CGTase and equivalent activity of free CGTase were incubated in 4 mL of 5% starch solution in phosphate buffer (0.05 M, pH 6.0) for 10 min. The reaction was carried out for different temperatures ranging from 30 ◦ C to 90 ◦ C and the CGTase activity was assayed as mentioned previously. 2.9. Storage stability and deactivation study of CGTase Free and immobilized CGTase were stored under 3 different temperatures, 4 ◦ C, 25 ◦ C and 60 ◦ C. At day initial, day 1, day 2, day 6, day 8, day 11, day 14 and day 15, the activity of CGTase was assayed as described previously. To study deactivation kinetic of CGTase, it is considered that the rate of deactivation is a first order reaction [47] as shown: dCE = −kd · CE dt
(2)
where CE and kd are CGTase activity (U/mL or U/mg-fiber) and deactivation constant (day−1 ), respectively. After integration, it gave: ln
CE = −kd · t CE0
(3)
kd can be estimated based on the slope from graph (ln CE /CE0 versus t). Then, kd can be represented as Arrhenius equation: Ed
kd = kd0 · e− R·T
(4)
3
Therefore, after linearization, it gave: ln kd = −
Ed 1 · + ln kd0 R T
(5)
where Ed and kd0 are deactivation energy (kJ/kmole) and initial deactivation constant (day−1 ), respectively. They can be estimated from the graph of ln kd against 1/T. 2.10. Production of ˛-CD 50 mg of fiber with immobilized CGTase and almost equivalent activity of free CGTase were added separately to 100 mL of 5% starch solution in phosphate buffer (0.05 M, pH 6.0). The reaction was then conducted for 24 h at 60 ◦ C. Aliquots of the sample were taken at 10 min, 30 min, 2 h, 9 h, 14 h, 19 h, 22 h and 24 h and each of them was analyzed for ␣-CD concentration as previously described. 2.11. Operational stability of immobilized CGTase 20 mg of the support that consists of immobilized CGTase was added to 6 mL of 5% starch solution in phosphate buffer (0.05 M, pH 6.0). The mixture was incubated at 60 ◦ C for 30 min. After the reaction, the immobilized CGTase was collected by centrifugation at 10,000 rpm for 10 min while the supernatant was analyzed for ␣CD production. The precipitate, recovered fiber with immobilized CGTase was washed with the same buffer, and centrifuged. The supernatant was removed and then immobilized CGTase was reincubated by adding 6 mL of newly prepared starch solution to start a new cycle. This was repeated for a total of 12 cycles. 3. Results and discussion 3.1. Surface morphology Fig. 1a shows the SEM image of bleached kenaf bast microfiber with the diameter of 10–15 m. The surface of the fiber is slightly smooth and uniform. However, some agglomerations were observed upon coupling with HMDA as shown in Fig. 1b. This could be due to the presence of the spacer arms on the fiber, which is the HMDA. When the fiber was further activated with OPA, more agglomerations were observed as in Fig. 1c. The extra agglomerations observed on the surface of the fiber could be the OPA, which is the ligand. The presence of the spacer arm, ligand and CGTase on the surface of the fiber has been confirmed by Sulaiman et al. [48]. In their study, 1,12-dodecanediamine and glutaraldehyde were used as spacer arms and ligands, respectively. From FTIR analysis, it indicates that the interaction of spacer arm-ligand toward support and CGTase molecules has been covalently immobilized. HMDA and EDA act as spacer arms, and GA and OPA act as ligands, which all of them have the same functional groups and chemical classes (diamines and dialdehydes) as reported by that previous study [48]. A possible mechanism can be adopted to understand the reaction of covalent immobilization on the surface of microfiber. In this study, ( NH2 ) group located at both ends of the spacer arms as they were all homobifunctional linkers. The first end-terminal of NH2 group would then react with ( OH) in the carboxyl group of the support, which was exposed from oxidation of cellulose during bleaching process [49]. Thus, the amide bond ( CONH ) was formed between the support and the spacer arm as shown in Fig. 2a [50,51]. After that, other end-terminal of ( NH2 ) group on the spacer arm will react with the functional group of the ligand, ( CHO) to form ( CH N ) covalent bond as in Fig. 2b. Hence, other end-terminal of ( CHO) group will attach to CGTase molecule by reacting with the ( NH2 ) group on CGTase to form ( CH N ) covalent bond as in Fig. 2c [25,52]. This finally completed the immobilization procedure.
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Fig. 1. SEM image of a) Bleached kenaf bast microfiber, b) Fiber coupled with HMDA, c) Fiber coupled with HMDA and OPA.
3.2. Protein loading and activity recovery of immobilized CGTase The amount of CGTase activity added during immobilization has a significant effect on protein loading and activity recovery of immobilized CGTase. Fig. 3a shows that the protein loading percentage for all immobilized CGTases decreased with the increase of CGTase activity added during the immobilization. At lower CGTase activity that was added, higher ratio of coupling agent to CGTase leads to high percentage of protein loading due to higher chance for available CGTase molecule to bind with coupling agent. As the CGTase activity added was increased, it decreases the percentage of protein loading due to limitation of coupling agent that was available for the binding. In Fig. 3b, the immobilized CGTase activity increased steadily when the free CGTase added during immobilization was increased from 13.9 U/mL to 41.7 U/mL. However lesser increment can be observed in all the immobilized CGTases when 55.6 U/mL of CGTase activity was added, resulting 0.162–0.24 U/mg-fiber of their activity. This is probably because overcrowding of CGTase molecules has started to occur on the surface of bleached kenaf bast fiber. Overcrowding or agglomeration may develop steric hindrance some of the enzyme active sites during the assay, which reduces the reaction between the immobilized CGTase and the substrate [31]. The specific activity of immobilized CGTase shows the increasing trend when higher activity of free CGTase was added. As shown in Fig. 3c, (EDA-GA)-CGTase depicts the highest specific activity in this study (29.4 U/mg-protein). From Fig. 3, it can be observed that spacer arm, HMDA resulted in higher protein loading percentage than EDA. HMDA is a longer aliphatic chain of carbon atom, which is able to create a longer of interaction distance compared to EDA. The longer interaction dis-
tance would easily increase the chance to the interaction of ligand, as well as enhance the implication of CGTase binding [53]. Besides, OPA was also found to be a better ligand than GA in increasing protein loading percentage. A possible reason for this is the structural difference between GA and OPA. The ring structure of OPA may enable greater interaction between functional groups of OPA and enzyme. As a result, more OPA molecules are available for enzyme binding compared to GA which has a linear structure. Thus, the interaction between microfiber-(HMDA-OPA) and CGTase resulted in the highest protein loading. Meanwhile, microfiber-(EDA-GA)CGTase resulted in higher residual enzyme activity and its specific activity. It seems that, the EDA-GA resulted in less alteration of CGTase molecule during the immobilization compared to other spacer arm-ligand combinations in this study. 3.3. Effect of temperature on CGTase activity Many studies have found out a common finding which is immobilization will tend to alter the properties of immobilized enzymes [54]. Rigidification of enzyme structure resulted from immobilization may bring changes in some of the enzymatic properties [19]. The results have become more obvious when the effect of temperature on free and immobilized CGTases activity were compared as in Fig. 4. The activity of both free and immobilized CGTases increased when the temperature was increased up to their optimum point. However, a shift of optimum temperature can be observed in all the immobilized CGTases (optimum at 70 ◦ C) when compared to free CGTase (optimum at 60 ◦ C). In addition, the immobilized CGTases were able to maintain higher relative activity than native CGTase at high temperatures which was above 70 ◦ C. These results indicate thermal stability of enzyme that has been obtained by immobiliza-
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5
O C OH + H2N CH2C4H8CH2 NH2
O H C N CH2C4H8CH2 NH2 + H2O
Support
Support
a)
(HMDA)
Support
Support
b) H H O H + C N CH2C4H8CH2 NH2 O C C3H6 C O (GA)
H H O H C N CH2C4H8CH2 N C C3H6 C O
+ H2O
Support
Support
c) H H O H C N CH2C4H8CH2 N C C3H6 C O + H2N
H H O H C CH C H CH C H N C 2 4 8 2 N 3 6 C N
CGTase
CGTase
+ H2O
Fig. 2. Mechanism of a) coupling of spacer arm on bleached kenaf bast microfiber, b) coupling of ligand on fiber with spacer arm, c) enzyme immobilization on the activated fiber. Table 1 Deactivation constant and deactivation energy of free and immobilized CGTase on kenaf microfiber. Spacer arm-ligand
Deactivation constant, kd (day−1 ) ◦
Free CGTase HMDA-GA HMDA-OPA EDA-GA EDA-OPA
◦
Initial deactivation constant, kd0 (day−1 )
Deactivation energy, Ed (kJ/kmole)
3.86 × 109 148.62 25.09 15.32 32.12
64370 19377 16726 16081 18810
◦
4 C
25 C
60 C
0.0026 0.0331 0.0171 0.0138 0.0091
0.0227 0.0592 0.0309 0.0244 0.0161
0.2929 0.1360 0.0584 0.0450 0.0361
tion. Greater stability of covalent immobilization could prevent the inactivation of CGTase molecules at high degree of temperature. Through covalent binding, multipoint covalent attachment was also very likely to occur during the immobilization. The presence of multiple covalent bonds will fix the relative positions of all the functional groups of enzyme, which are involved in immobilization even when the immobilized CGTase was exposed in distorting agents such as extreme heat, solvent and pH [55]. Besides, ionization of amino group and other reactive groups of enzymes during immobilization may also occur since the enzyme was immobilized in buffer solution. This ionization of reactive groups will improve its penetrability and thus it improves the stability of the immobilized CGTase [56]. Therefore, the immobilized CGTase has lower rate of denaturation compared to free CGTase especially at high temperature. 3.4. Storage stability and deactivation study of CGTase Fig. 5 shows the result of storage stability for free CGTase and all immobilized CGTases at 3 different storage temperatures, 4 ◦ C, 25 ◦ C and 60 ◦ C. From the figure, free CGTase and all the immobi-
lized CGTases show the best retention of enzyme activity at the lowest storage temperature, 4 ◦ C. However, as the storage temperature increases to 25 ◦ C and 60 ◦ C, the activity retention for both free CGTase and all the immobilized CGTases decreased. The storage stability decreased was due to the deactivation of enzyme caused by prolonged of storage time and exposure to high temperature. From the figure, it can also be observed that the performance of free CGTase is better than immobilized CGTase when they were stored at 4 ◦ C. The activity of free CGTase shows almost constant at 4 ◦ C and decreased about 80% at 25 ◦ C (at the end of day 15). However, at higher storage temperature (60 ◦ C), all the immobilized CGTases show better performance than free CGTase. Free CGTase denatured completely since day 11 while all the immobilized CGTases still retained their respective catalytic activity. This result shows that the decrease of activity in free CGTase is higher than any of the immobilized CGTase at higher temperature. The deactivation constant, kd at 4 ◦ C, 25 ◦ C and 60 ◦ C for free and all the immobilized CGTases were obtained from the slope of graphs (Eq. (3)) and tabulated in Table 1. Using kd , Fig. 6 was plotted based on Eq. (5); Ed and kd0 of free and immobilized CGTase can be estimated from the slope and the y-intercept of the lines,
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b) 70
0.30
60
0.25
Immobilized CGTase activity (U/mg fibre)
Protein loading (%)
a)
50 40 30 20 10
0.20 0.15 0.10 0.05 0.00
0 0
10
20
30
40
50
0
60
10
20
30
40
50
60
CGTase added (U/mL)
CGTase added (U/mL)
c) Specific activity of immoblized CGTase (U/mg protein)
35 30 25 20 15 10 5 0 0
10
20
30
40
50
60
CGTase added (U/mL) Fig. 3. Effect of amount of CGTase added during immobilization on a) protein loading, b) activity recovery, and c) specific activity of CGTase. Symbols: (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)-CGTase; (), (EDA-OPA)-CGTase. The immobilization procedure was conducted at pH 6.0 and 4 ◦ C.
120
Relative activity (%)
100 80 60 40 20 0
20
30
40
50
60
70
80
90
100
Temperature (oC) Fig. 4. Relative activity of free and immobilized CGTases at different temperatures. Symbols: (), Native CGTase; (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)-CGTase; (), (EDA-OPA)-CGTase. The assay was conducted at pH 6.0.
respectively. The values of Ed and kd0 for free and immobilized CGTases were also summarized in Table 1. It shows that free CGTase has better stability than the immobilized CGTase when stored at 4 ◦ C and 25 ◦ C. It can be seen that free CGTase has the lowest kd (0.0026 day−1 ) at 4 ◦ C. However, at 60 ◦ C, the kd of free CGTase is the highest (0.2929 day−1 ) compared to all the immobilized CGTases. Among the immobilized enzymes, CGTase immobilized with EDA-OPA shows the lowest kd at 25 ◦ C (0.0161 day−1 ) and 60 ◦ C (0.0361 day−1 ). The lower deactivation constant, results in the higher stability of the enzyme. It seems that the immobilization has altered its conformation structure, resulting in a gradual decrease its residual activity during the storage (even it was stored at 4 ◦ C). Thus, the CGTase immobilization is recommended to be prepared freshly before use. However, all the immobilized CGTases have better thermal stability than free CGTase when they were stored at 60 ◦ C. EDA-OPA was the best combination of spacer and ligand in maintaining the storage stability of the immobilized CGTase while HMDA-GA provides the
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b) 100
100
90
90
80
80
Relative activity (%)
Relative activity (%)
a)
7
70 60 50 40 30
70 60 50 40 30
20
20
10
10 0
0 0
2
4
6
8
10
12
14
0
16
2
6
4
8
10
12
14
16
Time (days)
Time (days)
c) 100
Relative activity (%)
90 80 70 60 50 40 30 20 10 0 0
2
4
6
8
10
12
14
16
Time (days) Fig. 5. Storage stability at different temperatures a) 4 ◦ C, b) 25 ◦ C, and c) 60 ◦ C. Symbols: (), Native CGTase; (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)-CGTase; (), (EDA-OPA)-CGTase. The assay was conducted at pH 6.0 and 60 ◦ C.
least storage stability to the immobilized CGTase. This is due to the influence of different coupling agents used. HMDA is a longer spacer arm and hence lower rigidity can be transmitted to the immobilized enzymes through multipoint covalent attachment compared to EDA [29]. For the ligands, GA has a linear structure while OPA is globular. Hence the distance between support and enzyme will be longer when GA is used as the ligand. Due to this, lesser protection could be provided by the support matrix to the enzyme when compared to using OPA as ligand. This study is very important for getting insight about operational stability especially when the immobilized enzyme was used for several times or/and certain period of time. Initial deactivation constant (kd0 ) for free CGTase is extremely higher (3.86 × 109 day−1 ) compared to the immobilized CGTases (15.32–148.62 day−1 ). This indicates that the activity of free CGTase is high sensitive by temperature change.
3.5. Production of ˛-CD Fig. 7 shows the production of ␣-CD for 24 h by free and immobilized CGTase. The ␣-CD production by all CGTases increased significantly during the first 30 min in which free CGTase, immobilized CGTase with HMDA-GA, HMDA-OPA, EDA-GA and EDA-OPA achieved approximately 77%, 72%, 51%, 74% and 72% of their maximum production, respectively. After that, the production of ␣-CD by all the CGTase slowed down and eventually reached their plateau. The time required to reach maximum production was about 14 h for free CGTase and approximately 19 h for immobilized CGTase. The reasons for the production to stop in the end include inhibition by its product and substrate limitation [57]. A substantial amount of ␣-CD may act as the inhibitor and occupy the active site, thus it will cause the conformation change and lead to the inactivation of CGTase molecule.
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0.0028 -0.5
0.003
0.0032
0.0034
0.0036
Relative production (%)
-1.5
-2.5
ln (kd)
100
0.0038
-3.5
-4.5
80
60
40
20
-5.5
0 -6.5
1
2
3
(1/T)
5
6
7
8
9
10
11
12
Number of cycles
Fig. 6. Determination of initial deactivation constant and deactivation energy of free and immobilized CGTases. Symbols: (),Free CGTase; (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)-CGTase; (), (EDA-OPA)-CGTase. The assay was conducted at pH 6.0 and 60 ◦ C.
Fig. 8. Reusability of CGTase immobilized with different coupling agents. Symbols: (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)- CGTase; (), (EDA-OPA)-CGTase. The reaction was conducted at pH 6.0 and 60 ◦ C using 5% starch.
temperature and 70 ◦ C–90 ◦ C for reaction temperature) when compared to free CGTase. This is due to the structure rigidification of the immobilized CGTase developed by the presence of covalent bonds and multipoint covalent attachments after immobilization. Besides that, when the same spacer arm is used, OPA acts as a better ligand when compared to GA in improving the thermal stability of the immobilized CGTase. In terms of enzyme activity and reusability, EDA-GA is the best coupling agent combinations which resulted the highest activity retention, specific activity and reusability (0.23 U/mg-fiber, 29.4 U/mg-protein and 72.72%, respectively).
100 90
Relative α-CD production (%)
4
80 70 60 50 40 30
Acknowledgements 20 10 0 0
2
4
6
8
10
12
14
16
18
20
22
24
Time (hours) Fig. 7. ␣-CD production by free and immobilized CGTases. Symbols: (), Free CGTase; (♦), (HMDA-GA)-CGTase; (䊐), (HMDA-OPA)-CGTase; (), (EDA-GA)CGTase; (), (EDA-OPA)-CGTase. The reaction was conducted at pH 6.0 and 60 ◦ C using 5% starch.
3.6. Reusability of immobilized CGTase In this study, the reusability of the immobilized CGTase was investigated by carrying out 12 cycles of batch reaction and the results are shown in Fig. 8. It was observed that after 12 cycles, CGTase immobilized on microfiber using EDA-GA retained the highest residual activity (72.72%) and followed by CGTase immobilized with HMDA-OPA (66.10%), EDA-OPA (58.35%) and HMDA-GA (52.20%). From the result, it can be observed that the residual activity of all the immobilized CGTase decreased with the number of cycle up to 10 cycles. The main reason that can be related to the reduction of CGTase residual activity includes the presence of irreversible product inhibitor [57]. Within the reaction period (total cycle times was less than 24 h), based on the thermal deactivation study, the loss of residual activity due to alteration on its conformation structure by immobilization is not significant. 4. Conclusions Bleached kenaf bast microfiber has potential to be used as a support for enzyme immobilization. Immobilized CGTase generally has improved stability at high temperature (60 ◦ C for storage
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Please cite this article in press as: N.L. Cieh, et al., Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.02.025