Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization

Journal Pre-proofs Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization Tanchanok Poonsin, Benjamin K. Simpson, Wonnop Visessanguan, Asami Yoshida, Sappasith Klomklao PII: DOI: Reference:

S0141-8130(19)32881-8 https://doi.org/10.1016/j.ijbiomac.2019.10.030 BIOMAC 13411

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

18 April 2019 2 October 2019 2 October 2019

Please cite this article as: T. Poonsin, B.K. Simpson, W. Visessanguan, A. Yoshida, S. Klomklao, Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.030

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1 Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization

To be submitted to International Journal of Biological Macromolecules

Tanchanok Poonsina, Benjamin K. Simpsonb, Wonnop Visessanguanc, Asami Yoshidad and Sappasith Klomklaoe,*

a Biotechnology

Program, , Faculty of Agro- and Bio-Industry, Thaksin University,

Phatthalung Campus, Phatthalung, 93210, Thailand b Department

of Food Science and Agricultural Chemistry, McGill University, Macdonald Campus, Ste. Anne de Bellevue, Que, Canada H9X 3V9

cNational

Center for Genetic Engineering and Biotechnology, National Science and

Technology Development Agency, 113 Paholayothin Road, Klong 1, Klong Luang, Pathumthani, 12120, Thailand d Graduate

School of Fisheries Science and Environmental Studies, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan

eDepartment

of Food Science and Technology, Faculty of Agro- and Bio-Industry,

Thaksin University, Phatthalung Campus, Phatthalung, 93210, Thailand

* Corresponding

authors: [email protected], Phone: 66-7460-9618, Fax: 66-7460-9618

2 Abstract Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5-4,4-dimethyltryptamine-thymidinesuccinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL4B were 40 mg/mL trypsin solution, pH 7 at 4o C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DLBAPNA) by the immobilized trypsin were 55o C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 M of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.

Keywords: Albacore tuna trypsin; immobilization; physicochemical properties

3 1. Introduction Trypsin (EC 3.4.21.4) is one of the major digestive enzymes, which acts on peptide bonds on the carboxyl side of lysine and arginine residues. Trypsin has a catalytic triad of serine, histidine and aspartate and plays a major role in the biological processes of digestio n and the activation of zymogens for chymotrypsin and other enzymes [1]. Trypsins are digestive enzymes that have many biochemical and industrial applications due to their high specific ity that allow for a controlled proteolysis [2]. Among all trypsins, fish trypsins are of imme nse interest because some of them are more active catalysts at relatively low temperatures compared with similar enzymes from thermophilic organisms from mammalian and plant sources, making them very suitable for a number of biotechnological and food processing applications [3]. Nevertheless, the enzymes have some limitations that may hinder their industria l applications. For example, widespread use of the free form of the enzyme in batch reactions is limited by stability, recovery, and reusability [4]. To enhance the convenience and costeffectiveness of enzymes in catalysis, immobilization, among other strategies, has been suggested [5-12]. Immobilization involves the attachment of the soluble/free enzyme onto inert solid support materials. This technique does not only fix enzyme molecules to make them reusable, but also has potential to modify the properties of enzymes, such as the stability and catalytic properties to enhance the feasibility of applications [13]. The main advantages of enzyme immobilization include reusability, ease of process control, reduced costs, and ease of separation of the enzyme from the reaction products after the desired transformation is achieved [7]. Many methods exist for the immobilization of enzymes but usually one of the following four methods is used: (i) physical adsorption; (ii) entrapment; (iii) co-polymerization; and (iv) covalent attachment. The methods and supports employed for enzyme immobilization are chosen to ensure the highest retention of enzyme activity and for their stability and durability.

4 The most important advantage of the former method is the reversible immobilization of enzyme on the support [11]. Among these immobilization methods, covalent attachment and physical adsorption are the two most widely used to immobilize enzymes. The covalent binding method gives the strongest link, thus providing the most stable support-enzyme conjugates without the release of the enzyme into the solution. However, this method of immobilization leads to greater strain on the enzyme and sometimes leads to drastic changes in the conformational and catalytic properties of the enzyme due to the harsh immobilization conditions. In comparison, the adsorption technique for the immobilization of an enzyme may be a good option because the adsorption is very simple and the method needs very little work and time. In addition, the supports can be reused after desorption of the inactivated enzyme and the final cost of the method can be reduced [14-15]. Sílica, Octyl Sepharose CL-4B, or 5-DMT-T-SUC-CPG are widely used for enzyme immobilization and have a plethora of applications in biocatalysis, biosensor developme nt, decontamination and energy storage. These methods for enzyme immobilization are simple, reproducible, and do not require sophisticated equipment. In this context, several supports can be adopted to perform enzyme immobilization. In general, the carrier structure needs enough mechanical strength, resistance to a chemical attack and microbial decomposition. Meanwhile, abundant reactive groups and a hydrophilic chain are necessary for the surface of the carriers. After immobilization, other phenomena (desired or undesired) may occur, but the researcher must detect these phenomena and develop tools to control them. That is, the first immobilization may be through a one-point or a multipoint interaction, and after this immobilization, the support may continue increasing the number (or even the quality) of the interactions or involve new groups as is the case for heterofunctional supports [16-19]. Certain trypsins have previously been immobilized

by cross-linking,

covalent

attachment, entrapment in mesoporous materials, and surface adsorption using various supports

5 [20] such as controlled pore glass [21], silica-based materials [20], polyamidoa mine dendrimers [22], chitosan [23], magnetite nanoparticles [24], alginate [25], lignocellulo ses [26], agarose activated with divinyl sulfone [27], electro-responsive graphene oxide hydrogels [28] and cashew gum polysaccharide/PVA films [29]. Recently, the immobilization of trypsin from Nile tilapia was reported by Azevedo et al. [30]. Nevertheless, the prospects of immobilizing alternative and underutilized sources of trypsin such as from fishery resources have received little attention. Based on our previous investigation, anionic trypsin from albacore tuna spleen was purified by chromatographic separations on Q-Sepharose, Superdex 75 and Arginine Sepharose 4B [31]. Purified trypsin showed high purity of about 313.54-fold with 477.51 units/mg protein specific activity and 10.33% yield. Therefore, the aims of this study were to investigate the reaction conditions for the immobilization and physicochemic a l properties of immobilized albacore tuna trypsin. To prepare the enzyme for possible commercial application, the advantages of increased operational stability as well as ease of enzyme recovery for repeated use via immobilization were also explored in this study. The studies reported here could provide useful information for using immobilized fish trypsin in industrial applications.

2. Materials and methods 2.1 Reagents Silicon dioxide, Octyl Sepharose CL-4B, glutaraldehyde, dimethyl sulfoxide and N-αbenzoyl-DL-arginine-p-nitroanilide Chemical

Co. (St. Louis,

(DL-BAPNA) were purchased from Sigma-Aldr ic h

MO, USA). 5-4,4-dimethyltryptamine-thymidine-succ inyl

controlled pore glass (5-DMT-T-SUC-CPG) was obtained from LGC Biosearch Technologies (Burlington, ON, Canada). The salts and other chemicals with the analytical grade were procured from Fisher Scientific (Whitby, ON, Canada).

6 2.2 Trypsin Purification Trypsin from the spleen of albacore tuna was purified by the procedure of Poonsin et al. [31]. The purified trypsin migrated as a single band on SDS-PAGE and native-PAGE, and was dialyzed against 10 volumes of 50 mM Tris-HCl (pH 7.5, containing 5 mM CaCl2 ) for 18 h with three changes of buffer in the first 3 h and five changes in the last 15 h and then concentrated by lyophilization. The powdered purified albacore tuna trypsin (8.86 mg protein/mL) thus obtained, was used for immobilization studies.

2.3 Effect of support on albacore tuna trypsin immobilization 2.3.1 Albacore tuna trypsin immobilization on glutaraldehyde activated silica The immobilized albacore tuna trypsin on glutaraldehyde activated silica was prepared based on the method of Daglioglu and Zihnioglu [32]. For this, silicon dioxide was suspended in 0.1 M acetate buffer (pH 4) containing 1% glutaraldehyde (1:10 w/v silicon dioxide to buffer). The slurry was rotated end over end at room temperature (25o C) for 1 h. The excess glutaraldehyde was removed by washing with distilled water, until the pH of the washing was neutral. The activated silica was added to a 10 mM sodium phosphate buffer with a pH of 7.5, (1:10 w/v activated silica to buffer) containing a 15 mg enzyme/mL buffer, and incubated on a multi-purpose rotator (Barnstead International, Iowa, USA) at 50 rpm and 4o C for 6 h. At the end of the incubation period, the immobilized enzyme preparation was vacuum-filtered on a Pyrex® Büchner funnel with a sintered (fritted) glass disk, and the filtrate was collected. The immobilized albacore tuna trypsin was washed with three changes of 10 mL of 10 mM sodium phosphate buffer (pH 7.5) to remove unbound and loosely bound trypsin. The immobilized trypsin from albacore tuna was collected and vacuum-dried in a desiccator over silica overnight. The dried immobilized enzyme was stored at -20°C. The filtrate in each washing step was collected for residual protein concentration and trypsin activity assays.

7 2.3.2 Albacore tuna trypsin immobilization on Octyl Sepharose CL-4B The albacore tuna trypsin immobilized onto Octyl Sepharose CL-4B was prepared according to the method of Aryee and Simpson [4]. The previously washed Octyl Sepharose CL-4B equilibrated with a 10 mM sodium phosphate buffer and a pH of 7.5 was added to the 15 mg/mL enzyme solution in a 10 mM sodium phosphate buffer with a pH of 7.5 (1:10 w/v resin to buffer), and incubated on multi-purpose rotator (Barnstead International, Iowa, USA) at 50 rpm and 4o C for 6 h. The trypsin-resin mixture was vacuum-filtered using a Pyrex® Büchner funnel with a sintered (fritted) glass disc, and the filtrate was collected. The immobilized albacore tuna trypsin was washed with three changes of 10 mL of 10 mM sodium phosphate buffer, pH 7.5. The immobilized trypsin was then filtered and vacuum-dried in a desiccator over silica overnight. The dried immobilized trypsin from albacore tuna was stored at -20°C. The filtrate in each washing step was collected for residual protein concentration and trypsin activity assays.

2.3.3 Albacore tuna trypsin immobilization on 5-DMT-T-SUC-CPG 5-DMT-T-SUC-CPG was immersed in a 15 mg/mL enzyme solution in a 10 mM sodium phosphate buffer with a pH of 7.5 (1:10 w/v support to buffer) and incubated on a multi-purpose rotator (Barnstead International, Iowa, USA) at 50 rpm and 4o C for 6 h. The immobilized trypsin from albacore tuna was vacuum-filtered using a Pyrex® Büchner funne l with a sintered (fritted) glass disk. The filtrate was collected and the immobilized enzyme was then washed with three changes of 10 mL of 10 mM sodium phosphate buffer (pH 7.5). The immobilized trypsin was dried using vacuum desiccator over silica overnight and stored at 20o C until used. The filtrate in each washing step was collected for residual protein concentration and trypsin activity assays.

8 Based on the activity of immobilized trypsin from albacore tuna, the appropriate support rendering the most effective immobilization was selected for further study.

2.4 Effect of pH on albacore tuna trypsin immobilization Albacore tuna trypsin immobilized on Octyl Sepharose CL-4B was used to study the effect of pH on trypsin immobilization by performing the immobilization under different pH conditions (pH 3-11). The buffer solutions used were 10 mM citric acid-NaOH buffer (pH 3 and pH 5); 10 mM potassium phosphate buffer (pH 7); 10 mM glycine-NaOH buffer (pH 9); and 10 mM disodium hydrogen phosphate-NaOH buffer (pH 11). Enzyme immobilization was performed as previously described. The systems with pH showing the highest amount of bound protein and activity of the immobilized trypsin were chosen for further study.

2.5 Effect of enzyme concentration on albacore tuna trypsin immobilization To determine the optimum conditions for albacore tuna trypsin immobilization onto Octyl Sepharose CL-4B, enzyme solution concentration was chosen as the one of factors. The albacore tuna trypsin solution concentration used were 5, 10, 15, 20, 25, 30, 40, 50 and 75 mg/mL. The reaction pH was pH 7. Enzyme immobilization was performed as previous ly described. The system showing the highest amount of bound protein and activity of the immobilized trypsin was selected for studies on the effect of incubation time.

2.6 Effect of incubation times on albacore tuna trypsin immobilization Albacore tuna trypsin solution concentration of 40 mg/mL and reaction pH of 7 were selected for the study on the effect of incubation times on immobilization trypsin onto Octyl Sepharose CL-4B. The incubation times used were 0.5, 1, 1.5, 3, 6, 9 and 12 h. Enzyme immobilization was performed as previously described. The extent of immobilization was

9 evaluated by amount of bound protein and the activity of the immobilized trypsin. A schematic summary of the immobilized trypsin process in this study is shown in Fig. 1.

2.7 Activity determination of free and immobilized trypsin The activities of the free trypsins were determined using DL-BAPNA as a substrate following the method of Erlanger et al. [33], as modified by Poonsin et al. [34]. An aliquot of the enzyme solution (200 μL) was added to 1000 μL of reaction mixtures containing 800 μL of 1 mM DL-BAPNA solution in 0.05 M Tris-HCl buffer (pH 8.2, containing 0.02 M CaCl2 ) and 200 μL of 0.05 M Tris-HCl buffer containing 0.02 M CaCl2 , pH 8.2. The mixture was incubated at room temperature (25o C) for precisely 10 min. The release of p-nitroaniline from DLBAPNA was measured at 410 nm using a DU® 800 UV-vis spectrophotometer (BackmanCoulter, Brea, Canada). For the immobilized trypsin, the method was modified as follows : about 10 mg of dry immobilized albacore tuna trypsin were added to 800 μL of 1 mM DL-BAPNA in 0.05 M TrisHCl buffer (pH 8.2, containing 0.02 M CaCl2 ) and 400 μL of 0.05 M Tris-HCl buffer containing 0.02 M CaCl2 , pH 8.2. After incubation at room temperature (25o C) for 10 min, the reaction mixture

was

centrifuged

at

12,000g

(accuSpin

Micro

17R,

Fisher

Scientific,

Massachusetts, USA) for 1 min at room temperature (25o C) and the absorbance of the supernatant was measured at 410 nm in the DU® 800 UV-vis spectrophotometer (BackmanCoulter, Brea, Canada). One unit of trypsin activity was defined as 1 μmol p-nitroaniline released/min, using 8800 M-1 cm-1 as the extinction coefficient of p-nitroaniline at 410 nm [33].

10 2.8 Protein content determination Protein content was determined by measuring their absorbances in a spectrophotometer (Beckman-Coulter DU® 800 UV-vis spectrophotometer, Brea, Canada) at both 260 nm and 280 nm according the method of [35]. The protein concentration was determined using Eqn. (1) Protein (mg/mL) = 1.55 A280 - 0.76 A260

(1)

The amount of bound protein was calculated

as the difference between the

concentration of protein in solution before and after immobilization.

2.9 Characterization of free and immobilized albacore tuna trypsins 2.9.1 Optimum pH of free and immobilized albacore tuna trypsins The optimal pH values of free and immobilized albacore tuna trypsins were determined by preparing 1 mM DL-BAPNA in different buffers ranging from pH 3-11. The pH 3-5 buffers were prepared using 10 mM citric acid-NaOH buffer; the pH 6-8.5 buffers were prepared with 10 mM potassium phosphate buffer; the pH 9-10 buffers were prepared with 10 mM glycine-NaOH buffer and pH 11 buffer was prepared from disodium hydrogen phosphateNaOH buffer. The relative trypsin activity (%) was calculated using the highest trypsin activity obtained as 100%.

2.9.2 Optimum temperature of free and immobilized albacore tuna trypsins The activities of free and immobilized albacore tuna trypsins were assayed at differe nt temperatures ranging from 20 to 70°C using DL-BAPNA as substrate. The assay was conducted at pH 8.2 using a 0.05 M Tris-HCl buffer containing 0.02 M CaCl2 for 10 min. The relative trypsin activity (%) was calculated using the highest trypsin activity obtained as 100%.

11 2.9.3 pH and thermal stability of free and immobilized albacore tuna trypsins To evaluate pH stability, free albacore tuna trypsin was incubated in various buffers (pHs 4, 5, 6, 7, 8, 9, 10 and 11) for 30 min at room temperature (25o C), and residual trypsin activity was measured as previously described. For the immobilized enzyme, 10 mg of the immobilized trypsin were incubated at the various pHs under study (4, 5, 6, 7, 8, 9, 10 and 11) for 30 min at room temperature (25o C). At the end of the incubation period, the sample was centrifuged at 3,000g (accuSpin Micro 17R, Fisher Scientific, Massachusetts, USA) at room temperature (25o C) for 5 min and the supernatant (buffer) was carefully removed. To the damp immobilized albacore tuna trypsin was added the substrate (1 mM DL-BAPNA in 0.05 M TrisHCI buffer, pH 8.2, containing 0.02 M CaCl2 ) and assayed as described above. The relative trypsin activity (%) was calculated using the highest trypsin activity obtained as 100%. For thermal stability studies, the free and immobilized albacore tuna trypsins were incubated at different temperatures (20, 30, 40, 50, 60, 70, and 80o C) for 30 min in a temperature controlled water bath (Shaking Water Bath 25, Precision Scientific, Chicago, USA). After incubation, the treated samples were taken out from the temperature controlled water bath and cooled in an ice bath. The residual activities of free and immobilized albacore tuna trypsin were assayed as described earlier using 1 mM DL-BAPNA (in 0.05 M Tris-HCI buffer, pH 8.2, containing 0.02 M CaCl2 ) as substrate at room temperature for 10 min. The relative trypsin activity (%) was calculated using the highest trypsin activity obtained as 100%.

2.9.4 Determination of kinetic parameters The kinetic parameters (Vmax and Km ) were determined by measuring the initial rates of the reaction of the free and immobilized albacore tuna trypsins with varying concentrations of DL-BAPNA ranging from 0.2 to 2 mM. The reactions were carried out with constant amounts of the free enzyme (0.02 mg/mL) and immobilized enzyme (1 mg) at room temperature. The

12 determinations were repeated in triplicate and the respective kinetic parameters were evaluated by plotting the data on a Lineweaver-Burk double-reciprocal graph [36].

2.9.5 Effect of soybean trypsin inhibitor on free and immobilized albacore tuna trypsins The effect of soybean trypsin inhibitor on the activities of the free and immobilized albacore tuna trypsin was studied. Soybean trypsin inhibitor was added into the standard reaction assay to obtain final concentrations of 0, 0.25, 0.50, 0.75 and 1 M. The remaining activities of free and immobilized albacore tuna trypsins were assayed using DL-BAPNA as substrate at room temperature (25o C) and pH 8.2 for 10 min, as mentioned above. The residual trypsin activity (%) was calculated using the trypsin activity without the inhibitor as 100%.

2.9.6 Stability of free and immobilized albacore tuna trypsins in organic solvents The stabilities of the free and immobilized albacore tuna trypsins in selected organic solvents were also assessed. An aliquot of 100 L of free albacore tuna trypsin was incubated in anhydrous acetone, ethanol, methanol, butanol, isopropanol, hexane, petroleum ether, and isooctane at the ratio of 1:1 (v/v) for 60 min at room temperature (25o C) and residual activities were measured as described earlier. For the immobilized enzyme, 10 mg of immobilized albacore tuna trypsin were incubated in various organic solvents at the ratio of 1:1 (w/v) (anhydrous acetone, ethanol, methanol, butanol, isopropanol, hexane, petroleum ether, and isooctane) for 60 min at room temperature (25o C). After the incubation period, the solvents were recovered and the remaining activities were measured using DL-BAPNA as a substrate at room temperature (25o C) and pH 8.2 for 10 min, as previously described. The control was conducted in the same manner except that distilled water was used instead of solvent. The residual trypsin activity (%) was calculated using the control as 100%.

13 2.9.7 Operational stability of immobilized albacore tuna trypsin The operational stability of immobilized albacore tuna trypsin was determined during six repeated batches (10 min/batch) of the hydrolysis of DL-BAPNA at room temperature (25o C). Each assay was carried out as mentioned above. Between consecutive batches, immobilized albacore tuna trypsin was separated and washed twice with 10 mM potassium phosphate buffer with a pH of 7. Residual activity was determined and compared with the activity of the first batch. The relative trypsin activity (%) was calculated using trypsin activity of the first batch as 100%.

2.10 Statistical analysis All experiments were run in triplicate. Data were subjected to analysis of variance (ANOVA). Comparison of means was carried out by Duncan’s multiple range tests. T-test was used for paired comparison [37]. Statistical analysis was performed using the Statistica l Package for Social Science (SPSS 11.0 for windows, SPSS Inc., Chicago, IL, USA).

3. Results and discussion 3.1 Optimum conditions for albacore tuna trypsin immobilization 3.1.1 Effect of support on albacore tuna trypsin immobilization The selection of a suitable support is an important consideration for immobilization due to the strong effect of the microenvironment of the support matrix on the stability and activity of the enzyme [4]. Moreover, the particle size is an important factor for support materials since smaller particles have a larger specific surface area. This property provides more enzymes on their surface and less restriction for the diffusion of the substrate and product. The immobilization of trypsin from albacore tuna was investigated using different supports, namely, Octyl Sepharose CL-4B, glutaraldehyde activated silica, and 5-DMT-T-SUC-CPG,

14 15 mg/mL trypsin solution in 10 mM sodium phosphate buffer, pH 7.5 at 4o C for 6 h. Among the different supports, the immobilization of albacore tuna trypsin using Octyl Sepharose CL4B showed the highest activity (6.26 ± 0.01 U/g support) and specific activity (1.45 ± 0.02 U/mg bound protein) (Table 1), even though it had the lowest amount of bound protein. In contrast, immobilizations using glutaraldehyde activated silica and 5-DMT-T-SUC-CPG as a support, achieved more than 90% of protein binding. However, trypsin immobilized to the two latter supports showed relatively little or no activity (Table 1). The data indicate that covalent immobilization of trypsin onto glutaraldehyde activated silica and 5-DMT-T-SUC-CPG could have resulted in a stronger multipoint enzyme-support complex which was irreversible with minimal enzyme loss by desorption or leaching from the support [38-39]. However, activities of covalently bonded enzymes were reduced due to conformational changes and decreased accessibility of active sites in the protein structure[18]. Comparative studies have shown that immobilizing the same enzyme on differe nt support matrices, or by different immobilization methods could result in immobilized enzymes with different characteristics [38]. The Octyl Sepharose CL-4B resin did not require any complex pre-treatment steps prior to use, and the relatively fast adsorption onto this resin strongly suggests a pronounced adsorption ability of the resin and high affinity of the enzyme to the resin [4]. Among the trypsin immobilization methods, surface adsorption has advantages because the active sites of the enzyme are more accessible. Moreover, this method avoids reactive chemicals, which are unfavorable toward the stability of trypsin [20]. The adsorption method has been widely employed for trypsin immobilization. Gómez et al. [40], immobilized trypsin via adsorption on silica gel. Sun et al. [20] developed a method for immobilizing trypsin by adsorption onto the surface of hydrophobic cellulose-coated silica nanoparticles. Therefore, Octyl Sepharose CL-4B was chosen to study the effect of pH on albacore tuna trypsin immobilization.

15 3.1.2 Effect of pH on albacore tuna trypsin immobilization The pH was also one of the most important factors in enzyme immobilization, as this parameter influences not only the bonding between the enzymes and matrices but also the molecular conformation and structure of the enzyme in the final catalyst. Additionally, the reactivity of the protein groups with the support depends on the pH, and that way the orientatio n of the enzyme molecules on the support may be altered by changing the immobilization pH. The final blocking of the support with nucleophiles (e.g., ethylenediamine) permitted to eliminate the chemical reactivity of the support avoiding undesired enzyme-support covalent bonds and being a useful endpoint reaction [27]. The effect of pH on the immobilization of albacore tuna trypsin onto Octyl Sepharose CL-4B is shown in Fig. 2a. The immobili zed trypsin shown the highest activity (7.23 ± 0.01 U/g support) and the amount of bound protein (4.376 ± 0.01 mg/g support) at pH 7. A similar result was observed with immobilized trypsin onto polyester [41]. Bayramoğlu et al. [42] also reported that the maximal adsorption of trypsin onto poly(GMA-MMA)-g-MAA beads occurred at pH 7, where the electrostatic interactio n between protein and ion exchange adsorbent should be predominant. The results indicated that the amine groups of trypsin molecules can undergo protonation to NH3 + and the extent of protonation will be dependent on the pH of the solution. The decrease in adsorption capacity of albacore tuna trypsin onto Octyl Sepharose CL-4B at acid and alkaline pHs could be attributed to electrostatic repulsion effects between the oppositely charged groups. Moreover, the free enzyme may experience protein aggregation (mainly near the isoelectric point). This may be caused by undesired enzyme-interactions where inactivation can stabilize incorrect enzyme structures. Therefore, pH 7 was chosen as the optimum immobilization condition for further study on the effect of albacore tuna trypsin concentration on immobilization onto Octyl Sepharose CL-4B.

16 3.1.3 Effect of enzyme concentration on albacore tuna trypsin immobilization The immobilization of enzymes has been shown to be affected by pH, temperature, enzyme concentration, and incubation time [4]. The results obtained using different albacore tuna trypsin concentrations to determine the optimum conditions for trypsin immobiliza tio n onto Octyl Sepharose CL-4B at pH 7 is shown in Fig. 2b. The amount of bound protein increased with increasing the initial concentrations of albacore tuna trypsin (P<0.05). When albacore tuna trypsin concentration was increased up to 40 mg/mL, the activity of immobilized albacore tuna trypsin increased (P<0.05). However, no significant increases in activity of immobilized enzyme were found with treatment of albacore tuna trypsin at concentratio ns above 40 mg/mL (P>0.05). A similar result was reported for the immobilization trypsin cellulose-coated

silica

nanoparticles

[20] and poly(GMA-MMA)-g-MAA

beads [42].

Fernandez-Lopez et al. [43] reported that enzyme concentration had an effect in the preparation of an immobilized enzyme. The enzyme crowding may reduce enzyme mobility because the chain of one enzyme will crash versus the protein molecules surrounding it. Moreover, at higher concentrations, there were protein-protein interactions and/or distortion of the protein molecules, which resulted in its denaturation. It is possible that such denatured protein molecules were simply absorbed onto Octyl Sepharose CL-4B and did not express any activity [21]. For commercial-scale application, higher conversion yields in relatively low enzyme concentration are preferred. Therefore, albacore tuna trypsin solution of 40 mg/mL was selected as the optimum immobilization condition for further study on the effect of incubatio n times on albacore tuna trypsin immobilization.

3.1.4 Effect of incubation times on albacore tuna trypsin immobilization Figure 2c shows the effect of incubation times on immobilized trypsin from albacore tuna using Octyl Sepharose CL-4B as a support and 40 mg/mL trypsin solution at pH 7. The

17 activity of immobilized trypsin and the amount of bound protein increased as the incubatio n time increased (Fig. 2c). The bound protein and immobilized albacore tuna trypsin activity increased rapidly in the first 6 h (P<0.05). Thereafter, no marked changes in the amount of bound protein were noticeable (P>0.05) and immobilized albacore tuna trypsin activity decreased after 6 h (P<0.05). A similar curve was reported for the immobilization of bovine trypsin onto controlled pore glass [9]. The longer incubation times caused the enzyme proteins to autolyze and denature. It is possible that such denatured protein molecules were absorbed onto the Octyl Sepharose CL-4B but did not exhibit any activity. Also the longer incubatio n times are not economical for industrial application. Therefore, the optimum conditions for immobilized albacore tuna onto Octyl Sepharose CL-4B were 40 mg/mL albacore tuna trypsin for 6 h, at pH 7 and 4o C.

3.2 Biochemical properties of free and immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B 3.2.1 Optimum pH for the hydrolyses of DL-BAPNA by free vs Octyl Sepharose CL-4B immobilized albacore tuna trypsins The effect of pH on the activity of free and immobilized albacore tuna trypsins on DLBAPNA hydrolysis is shown in Fig. 3a. Optimal hydrolyses were obtained at pHs 8 and 8.5 for the free enzyme and the immobilized enzyme, respectively. Thus, the optimum pH for the hydrolysis of the substrate by the immobilized trypsin shifted to a more alkaline pH compared to that of the free form. This shift toward a more alkaline region could be possibly due to secondary interactions (e.g., ionic and polar interactions, hydrogen bonding) between the enzyme and polymeric support [42]. When the enzyme was immobilized, the negative charge was carried on the surface of the support. After the formation of immobilized enzyme, the negatively charged support could increase the density of protons around the active site of the

18 immobilized enzyme. Therefore, the pH within the bulk solution was higher than that of immobilized enzyme. To compensate for this effect, the optimum pH of the immobilized enzyme had to shift to a higher pH [44-45]. Seabra and Gil [46] reported that the optimal pH of immobilized trypsin on sterilized cotton gauze bandage for the hydrolysis of BAPNA was 9.5, which was higher than those of the free form (pH 7.5). Bayramoğlu et al. [26] also reported that there was a shift in the optimal pH of the immobilized trypsin onto poly(GMA-MMA)- gMAA beads to a higher value (9.5) when compared to that of the free trypsin. Moreover, the pH activity profile of the immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B was broader than that of the free trypsin (Fig. 3a). This indicates that the free enzyme is more sensitive to pH changes, and immobilization can preserve the enzyme activity over a wider range of pH. Similar results have been reported for immobilized trypsin and other proteases onto polyaniline [47], sterilized cotton gauze bandage [46], poly(GMA-MMA)-g-MAA beads [42], and pore glass beads [48]. The possible cause of this phenomenon is that the interactio ns between the support and enzyme could stabilize the conformation of the active site of the enzyme. Therefore, the immobilized enzyme could keep relatively higher activity at a wider pH range. This improvement of property of immobilized trypsin could enhance the feasibility of its potential application in different industries, such as food processing, the pharmaceutic a l industry and biochemical fields.

3.2.2 Optimal temperatures for the hydrolyses of DL-BAPNA by free vs Octyl Sepharose CL4B immobilized albacore tuna trypsins Figure 3b shows the optimal temperatures for DL-BAPNA hydrolyses by the free form versus the Octyl Sepharose CL-4B immobilized albacore tuna trypsins. The free form of the enzyme had an optimal temperature of 50ºC, while its immobilized counterpart had an optimal temperature of 55ºC, using the same substrate for hydrolysis. Moreover, the immobilized

19 enzyme could keep relatively high activity over a wider temperature range than that of the free form. For instance, the immobilized enzyme exhibited higher relative activity (>70%) between 40 to 60o C compared with the free form that exhibited much lower relative activities, i.e., 60% relative activity at 40o C versus 50% at 60o C. The result suggests that the immobilization made the enzyme sturdier than its free form. Similar results have been observed for trypsin immobilized to other supports like poly(GMA-MMA)-g-MAA beads [42], polyaniline [47] and hydrophobic cellulose-coated silica nanoparticles [20]. The increase in optimum temperature of immobilized enzyme was caused by the changing physical and chemical properties of the enzyme. The non-covalent multipoint interactions via amino groups of the enzyme might also reduce the conformational flexibility for the binding to its substrate [49].

3.2.3 pH and thermal stabilities of the free versus the Octyl Sepharose CL-4B immobilized albacore tuna trypsins The pH stabilities of the free versus the Octyl Sepharose CL-4B immobilized albacore tuna trypsins are presented in Fig. 4a. Both the free and immobilized albacore tuna trypsins were stable over a broad pH range (pH 6-11). Nonetheless, the immobilized enzyme was less susceptible to activity loss at extreme pH values (both acid and alkaline) versus the free form. For example, after incubation at pH 4 for 30 min, the free trypsin lost more than 50% of its activity, while its Octyl Sepharose CL-4B immobilized counterpart lost only about 30% of its activity at this pH. The results indicate that the pH change in the system has a significant effect on the activity of the enzyme because the dissociation state and behavior of the enzyme are affected by the pH in the system. Moreover, the effect of the pH on the immobilized enzyme activity also depends on the strategy employed. Kang et al. [45], found that trypsin immobilized onto poly[(methyl methacrylate)-co-(ethyl acrylate)-co-(acrylic acid)] latex particles was more resistant to both acidic and alkaline media than the free form. The results presented above

20 suggest that immobilized trypsin was less sensitive to pH than its free form, and that the stability of the enzyme against pH was significantly improved by immobilization using Octyl Sepharose CL-4B as a support. Thermal stabilities of both the free and Octyl Sepharose CL-4B immobilized albacore tuna trypsins were also investigated using DL-BAPNA hydrolysis to measure residual activities (results summarized in Fig. 4b). After incubation of free and immobilized enzymes at different temperature from 20o C to 80o C, the results indicate that the inactivation rate of the Octyl Sepharose CL-4B immobilized albacore tuna trypsin was much slower than that free trypsin (Fig. 4b). According to Sun et al. [20], Bayramoğlu et al. [42], Kang et al. [45] and Pinheiro et al. [50], immobilized enzymes are more resistant to temperature inactiva tio n compared to their free forms. The thermal stability of trypsin on supports is one of the most important application criteria for different applications. This stability depends on the enzyme preparation strategy. It also depends on the stabilization of the enzyme [51-53]. The higher stability of immobilized enzyme could partly be caused by the limitation of autolysis [42]. Furthermore, the increased thermostability of albacore tuna trypsin immobilized onto Octyl Sepharose CL-4B can also be ascribed to improved conformational stabilization by a stronger degree of attachment of the enzyme on the support (Octyl Sepharose CL-4B) through hydrophobic interactions [18].

3.2.4 Kinetic parameters of the free versus the Octyl Sepharose CL-4B immobilized trypsins The kinetic constants (Km and Vmax) for DL-BAPNA hydrolysis by the free and the Octyl Sepharose CL-4B immobilized

albacore tuna trypsins were determined

using

Lineweaver-Burk plots (Table 2). The Km and Vmax values were determined as 0.18 mM and 7.14 µmol/min, respectively, for free albacore tuna trypsin; and as 0.67 mM and 6.25 µmol/min, respectively, for its Octyl Sepharose CL-4B immobilized counterpart. The Km of

21 free enzyme was lower than that of its Octyl Sepharose CL-4B immobilized derivative. This result suggests that free enzyme has higher affinity to DL-BAPNA, compared with the immobilized form possibly from reduced access of the substrate to the active site of the latter . It is also observed that the Vmax of immobilized albacore tuna trypsin was lower than that of the free form. Similar changes in the kinetic parameters of enzymes after immobilization have been reported by other researchers. Seabra and Gil [46], reported the Km values of the immobilized trypsin (onto cotton gauze bandage) and free trypsin to be 3.98 µM and 2.46 µM, respective ly; and the corresponding Vmax values to be 0.719 μmol/(min mg) and 2.89 μmol/(min. mg), respectively. And the study by Sun et al. [11], found that the Km values of immobilized trypsin (onto carboxymethyl

chitosan-functionalized

magnetic

nanoparticles cross-linked

with

glutaraldehyde) and the free form were 1.35 mM and 0.99 mM, respectively; and the corresponding Vmax values were 1378.74 μM/min and 1536.57 μM/min, respectively. As indicated above, this increase in Km of the immobilized trypsin could be due to a lower accessibility of the substrate to the active site of the immobilized enzyme, which was caused by several factors such as protein conformational changes induced by attachment to the support, steric hindrance and diffusion effects [46]. Possible reason for the lower Vmax of the immobilized enzyme versus the free enzyme can be due to conformational change of the immobilized enzyme when supported on a support. Moreover, immobilization can affect substrate diffusion to, as well as product diffusion from the catalytic sites of enzymes [54]. The ratio of Vmax/Km is another parameter used to measure the efficiency of catalysis. The catalytic efficiency (Vmax/Km ) of the Octyl Sepharose CL-4B immobilized albacore tuna trypsin was lower 4.25 fold than that of the free form of the enzyme when DL-BAPNA was used as a substrate. The higher catalytic efficiency, the more efficient the enzyme is in transforming the substrate to product [55]. This result suggests that free trypsin from albacore tuna was more effective in hydrolyzing DL-BAPNA than immobilized albacore tuna trypsin.

22 Nonetheless, the catalytic function of albacore tuna trypsin was not impaired significantly by its immobilization as judged by the degree of difference.

3.2.5 Effect of soybean trypsin inhibitor on free and immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B Soybean trypsin inhibitor (SBTI) is one of the well-known trypsin inhibitors. It is a plant protein which has inhibitory activity against serine proteinases such as trypsin. The residual activities of the free and the Octyl Sepharose CL-4B immobilized albacore tuna trypsins subjected to inhibition by SBTI are shown in Fig. 5a, which indicates that the free albacore tuna trypsin was more sensitive to the inhibitor than the immobilized albacore tuna trypsin. At a level of 0.25-1 M SBTI, the free albacore tuna trypsin lost more activity than the immobilized albacore tuna trypsin at the same concentration of inhibitor solution with the value ranging from 6.94% to 35.40%. The result suggests that the Octyl Sepharose CL-4B immobilized albacore tuna trypsin was inhibited to a lesser degree by soybean trypsin inhib itor than the free form. Shtelzer et al. [56] also reported that the sol-gel-entrapped trypsin activity was stable when they were incubated with soybean trypsin inhibitor. The less sensitivity of the immobilized enzyme to SBTI inhibition may be due to the active sites of free enzyme molecules were relatively more accessible to the inhibitor molecules, to enable more binding and more obstruction of substrate binding and its transformation to products. On the other hand, the active sites of the immobilized enzyme molecules were shielded by the support partially or completely, so the inhibitor molecules could not occupy the active sites of the enzyme as much to achieve the same degree of substrate binding and transformation. When mixed with the substrate molecules, the size of substrate molecules (BAPNA, molecular weight: 138 Da) was much smaller than that of soybean trypsin inhibitor (molecular weight : 20,100 Da) [57], thus the substrate molecules could easily diffuse and gain access to the active

23 site and cause the enzymatic reaction. From the above, it is obvious that the steric hindrance caused by the support material might protect the immobilized enzyme molecules from the inhibition. This feature of the immobilized enzyme could permit its use in certain applicatio ns such as modification of complex raw materials with naturally present inhibitors instead of the free form of the enzyme.

3.2.6 Stability of free and immobilized albacore tuna trypsins in organic solvents The effects of various organic solvents on the activity (and hence the stability) of the free versus the Octyl Sepharose CL-4B immobilized albacore tuna trypsins were determined and the results are shown in Fig. 5b. The order of decreasing stability of free albacore tuna trypsin after 60 min of incubation in the various solvents was: acetone > butanol > isopropanol > ethanol > methanol > isooctane, hexane > petroleum ether. The immobilized trypsin from albacore tuna also lost some activity in these solvents, but the degree of inactivation by the different solvents tested was lesser for the immobilized form versus the free form. Thus, the immobilized albacore tuna trypsin retains comparatively higher activity after the same solvent treatments than the free albacore tuna trypsin. Immobilization may have protected the enzyme from solvent denaturation. This indicates that solvent penetration and interaction with the enzyme were altered, and inactivation was reduced with immobilization [4]. These results further confirm that immobilization of albacore tuna trypsin on Octyl Sepharose CL-4B promoted a stronger adsorption interaction with the hydrophobic surface, thereby minimiz ing direct and full trypsin-solvent interaction, negative partitioning of the solvent from the enzyme environment, and possible unfolding [38]. The low stability of immobilized albacore tuna trypsin in methanol and ethanol may be due to the distortion and stripping of the essential ordered layer of water molecules that surrounds the enzyme needed for conformatio n a l integrity and stability, thereby fractionally inactivating the enzyme and leaving only a partially

24 active enzyme [58], [59]. Kang et al. [28] reported that immobilized trypsin onto poly[(me thyl methacrylate)-co-(ethyl acrylate)-co-(acrylic acid)] latex particles retained only 32% of its initial activity after incubating the immobilized enzyme in 60% ethanol solution for 30 min. However, immobilized albacore tuna trypsin on Octyl Sepharose CL-4B incubated in acetone, and hexane, retained more than 85% of its activity after 60 min of incubation. This may be ascribed to the additional reduction in the amount of available water, structural flexibility of the enzyme upon immobilization, and the effects of the organic solvents on enzyme [58]. The high stability of Octyl Sepharose CL-4B immobilized albacore tuna trypsin in various organic solvents could be a very important and useful characteristic for potential applications in nonaqueous media, a fast growing industry.

3.2.7 Reusability of immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B The reusability of immobilized enzymes is important for economical use of an enzyme. To demonstrate the advantage of immobilized trypsin on supports, a recyclability study was performed by repeatedly using the 10 mM potassium phosphate buffer with a pH of 7 and DLBAPNA as a washing solution and a substrate, respectively. As shown in Fig. 5c, the relative activity of immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B decreased gradually as the cycle number increased. The marked activity retention displayed by immobilized albacore tuna trypsin during the first three cycles implied that minimal desorption and activity loss of enzyme from the support (Octyl Sepharose CL-4B) occurred on repeated use. This can be attributed to the strong affinity between the support and the enzyme produced by the immobilization procedure. This also indicates that trypsin from albacore tuna remained adsorbed on the Octyl Sepharose CL-4B after incubation with the substrate during repeated use, while the free enzyme could not be reused after the first cycle because of difficulty in separation. The activity of immobilized trypsin decreased after reusability. This may have

25 occurred due to the protein diffusional limitations imposed by the considerably high amount of deposits that the reaction product accumulated on the matrix surfaces. As a consequence, it caused mass transfer restrictions to the protein molecules. However, these diffusion restrictio ns are not imposed on reactants and other products [60-61]. Aryee and Simpson [4] found that immobilized lipase from grey mullet (Mugil cephalus) viscera retained 88, 79, 62, 49, and 32% of its initial activity after the second, third, fourth, fifth, and sixth cycles. Immobilized lipase from Pseudomonas fluorescens on magnetic support proved to be a robust biocatalyst in the kinetic resolution, being reused five times without significant loss of activity [17]. The reusability of the immobilized enzyme is one of the most important advantages of enzyme immobilization. Immobilization was an effective way to produce a heterogeneous system which enabled easy separation and recovery from the reaction media for repetitive use. The capacity to reuse the enzyme will decrease the cost of a relatively expensive catalyst.

4. Conclusion Trypsin from albacore tuna was immobilized onto Octyl Sepharose CL-4B effective ly. The optimum immobilization conditions for albacore tuna trypsin were obtained with an initia l albacore tuna trypsin concentration of 40 mg/mL, incubation time of 6 h, at pH 7 and 4o C. Immobilized albacore tuna trypsin had different properties compared to its free form. The optimum pH and temperature of immobilized enzyme were higher than those of its free counterpart. The immobilized albacore tuna trypsin also showed higher pH and thermal stabilities than its free form. Additionally, immobilization reduced the sensitivity of the enzyme to inhibition by soybean trypsin inhibitor and inactivation by various organic solvents. This study demonstrated the simplicity of the separation and immobilization protocols together with the high activity of immobilized albacore tuna trypsin during repeated use. Therefore, immobilized albacore tuna trypsin could be used as an ideal choice for industrial applications.

26 Acknowledgements The authors acknowledge the financial support provided by Thailand Graduate Institute of Science and Technology (SCA-CO-2559-2285-TH). Thaksin University were also acknowledged.

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34 Table 1 Effect of support on immobilization of trypsin from albacore tuna Activity

Support

of

Protein bound

Percentage of immobilized

(mg/g

protein

albacore

tuna

support)

bound (%)

trypsin

(U/g

Specific activity (U/

mg

bound

protein)

support Octyl Sepharose

4.32a  0.02

8.73c  0.01

6.26c  0.01

1.45c  0.02

8.04b  0.01

90.74b  0.03

(8.17  0.01)10-4a

(1.02  0.01) 10-4a

8.73c  0.01

98.53c  0.19

(3.83  0.01)10-2b

(4.39  0.04)10-3b

CL-4B Glutaraldehyde activated silica 5-DMT-TSUC-CPG Note: 1. Reactions were carried out at 10 mM sodium phosphate buffer, pH 7.5 containing 15 enzyme mg/mL buffer (8.86 mg protein/g support) temperature at 4o C for 6 h. 2. Enzyme activities were measured at room temperature with 1mM BAPNA in 0.05M Tris-HCl buffer, pH 8.2 containing 0.02M CaCl2 as a substrate. 3. Each point on the graph is the average of three replicates. The error bar shows the standard deviation among the three replicates. 4. The different letters in the same column denote the significant differences (P<0.05).

35 Table 2 Kinetic properties of free and immobilized trypsin from albacore tuna Km

Vmax

Vmax/Km

(mM)

(µmol/min)

(µmol/min.mM)

Free albacore tuna trypsin

0.18 a  0.03

7.14 b  0.05

39.67b  0.04

Immobilized albacore tuna trypsin

0.67 b  0.01

6.25 a  0.03

9.33a  0.02

Note: 1. Km, Vmax values were determined using BAPNA as a substrate at room temperature and pH 8.2. 2. Values are mean  standard deviation (n=3).

36 Figure captions Fig. 1 Schematic overview of the immobilized trypsin process. Fig. 2 Effect of pH (a), enzyme concentration (b) and incubation times (c) on immobiliza tio n of albacore tuna trypsin onto Octyl Sepharose Cl-4B. Bars represent the standard deviation (n=3). Different letters within the same parameter indicate the signific a nt differences (P<0.05). Fig. 3 pH (a) and temperature (b) profiles of free and immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B. Bars represent the standard deviation (n=3). Fig. 4 pH (a) and thermal (b) stabilities of free and immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B. Bars represent the standard deviation (n=3). Fig. 5 Effect of soybean trypsin inhibitor (a) and organic solvents (b) of free and immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B. Operational stability (c) of immobilized albacore tuna trypsin onto Octyl Sepharose CL-4B. Activities are relative to the cycle showing the highest activity. Bars represent the standard deviation (n=3). Different lowercase letters on the bars within the same treatment indicate signific a nt differences (P<0.05). Different uppercase letters on the bars within the same sample indicate significant differences (P<0.05).

37 Highlights 

The immobilization of trypsin from albacore tuna was optimized

and

characterized. 

The immobilized albacore tuna trypsin showed better properties than free trypsin.



The immobilized trypsin was also stable in organic solvents and trypsin inhibitor.



The performance of immobilized trypsin demonstrates its potential applicatio ns.