Effect of enzymatic hydrolysis on developing support of polyamide woven fabric for enzyme immobilization

Abstract

Polyamide fiber has been considered as a suitable support for enzyme immobilization because of its low cost, chemical and mechanical properties and ready availability in a number of different forms. In particular, polyamide fabric has highly specific surface and good elasticity. The present study aims to develop an immobilization support from polyamide fabric and to establish the optimum immobilization conditions for laccase. For this, the enzymatic hydrolysis process was introduced to the hydrolysis of polyamide, creating amino groups that immobilize enzyme molecules. When polyamide fabric was hydrolyzed by bromelain during enzymatic hydrolysis, the highest immobilization yield (68 ± 0.7%) and relative activity (95 ± 0.52%) of immobilized laccase were obtained. For successful enzyme immobilization, the optimal glutaraldehyde crosslinking conditions were pH of 9.0 with 10% (v/v) of glutaraldehyde concentration for 240 min at 45℃. The most favorable immobilization conditions were as follows: pH of 6.0 with 35% (owf) of laccase concentration for 600 min at 4℃. Under the optimum treatment conditions, the pH and thermal stability of immobilized laccase were improved. After 20 days of storage, the immobilized laccase on enzymatic hydrolyzed polyamide fabric retained approximately 30% of its initial activity. Furthermore, the immobilized laccase indicated potential reuse over 10 use cycles. The structural changes of polyamide fabric according to treatment processes were demonstrated by Fourier transform infrared spectroscopy. The changes of surface morphology were measured by scanning electron microscopy according to the multi treatment steps.

Keywords

chemical modification chemistry enzymatic hydrolysis enzyme immobilization fabrication fiber fabric formation laccase nylon polyamide

For many years, the concept of immobilizing enzymes has been the subject of considerable research. Consequently, various methodologies and support materials have been suggested.¹ Many studies have reported the immobilization of a variety of enzymes, such as proteases,^2,3 pectinases,⁴ trypsin⁵ and laccases,^1,6,7 onto various support materials. The support itself may play on important role in enzyme immobilization by covalent bonding, since its interaction with the enzyme potentially affects enzyme stability and kinetics.⁵ It may be formed from either natural polymers, such as pulp fiber,⁴ chitosan⁸ and cellulose nanofibers,⁹ or from synthetic polymers, such as PLA (polylactic acid),⁵ polyester¹⁰ and polyamide fibers.^2,11,12 Synthetic polymers have advantages as a support material due to their high strength and durability.

In particular, polyamide fiber, which is composed of linear polymers containing amide bonds, is one of the strongest synthetic fibers because its amide functional groups (–CO–NH–) form hydrogen bonding between the polyamide chains, providing high strength, abrasion resistance and good chemical resistance. Moreover, polyamide fiber is a suitable support for enzyme immobilization because of its physical and chemical properties, such as non-porosity, mechanical strength, good flow-through properties and resistance to microbial attack. Therefore, polyamide fibers have been used in various forms, such as beads,¹³ nanofibers¹² and nonwoven fabric.¹⁴ However, polyamide woven fabric has been rarely employed as a support material.¹ Polyamide woven fabric possesses outstanding advantages, such as high structural and dimensional stabilities, a highly specific surface and good elasticity. In addition, the immobilization takes place only on the external surface of the woven support, allowing a better expression of the enzymatic activity.¹⁴ Nevertheless, several drawbacks restrict the extensive preparation of enzyme-immobilized polyamide surfaces: (1) the lack of strongly polar functional groups to react with proteins or/and enzymes and (2) the unfavorable interactions of the enzyme with the weakly polar surface.^15–17 Therefore, for further improvement in the performance of polyamide woven fabric, it is necessary to introduce specific functional groups onto its surface that can interact with the enzymes.^15–17 Several attempts have been made to improve the properties of polyamides fibers. Polyamide surfaces typically have been modified through activation by ultraviolet (UV) irradiation,¹⁷ plasma treatment,¹⁸ chemical partial hydrolysis¹⁹ and enzymatic modification.^15,20–23 Since the environmental issues have increased interest, the enzymatic hydrolysis on the polyamide surface has been focused upon in textile industries.^15–24

Enzymatic treatments are conducted under mild conditions and can produce similar or even better results for the surface modification of synthetic fibers.^25–28 Through enzymatic hydrolysis, the amino (NH₂) and carboxyl groups (COOH) are created on polyamide fiber by the cleavage of the amide bond (–CONH–).^15,20,29,30 The hydrolysis of polyamide fiber by protease introduces some reactive functional groups, such as primary amines (NH₂), and sulfhydryl onto polyamide fabric, without the loss of mechanical strength. The functional groups can interact with enzymes by using specific coupling reagents, such as glutaraldehyde (GA). When hydrolyzed polyamide fiber reacts with GA, a Schiff base reaction occurs between an aldehyde group (–CHO) of GA and the amine functional group generated on polyamide. Then, the second aldehyde group of GA reacts with an amine functional group of the enzyme; these processes are described in Figure 1.^{11,25–29,30–32} Consequently, the hydrolysis of polyamide woven fabric via enzymatic treatment can improve its capability as a support material for enzyme immobilization. Furthermore, to the best of our knowledge, comprehensive studies on the application of polyamide woven fabric as an immobilization support have rarely been reported.

Figure 1.

Scheme of enzymatic hydrolysis, crosslinking between polyamide fiber and glutaraldehyde, and covalent immobilization of laccase on polyamide fabric.

Therefore, in this work, polyamide woven fabric was used as an immobilization support material through the enzymatic hydrolysis process. To establish the optimum treatment conditions for enzymatic hydrolysis, polyamide woven fabric was treated with different types of commercial proteases, such as bromelain, alcalase and flavourzyme. After enzymatic hydrolysis, the change in polyamide woven fabric was observed by 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay and water contact angle (WCA) measurements. The effect of enzymatic hydrolyzed polyamide woven fabric on the immobilized enzyme was evaluated by measuring the percentage of immobilization yield and relative activity. Firstly, laccase (EC.1.10.3.2), which is one of the most practical and versatile enzymes, was employed as a model enzyme to be immobilized onto the polyamide woven fabric. Secondly, crosslinking was performed to ensure stable interaction between the enzyme and the support by the addition of GA, and the treatment conditions, such as pH value, temperature, GA concentration and crosslinking time, were optimized for the enzymatic hydrolyzed polyamide woven fabric. Thirdly, the optimum conditions for enzyme immobilization, such as pH value, temperature, enzyme concentration and immobilization time, were determined as well. The pH, thermal and storage stabilities and reusability of the laccase immobilized on the modified polyamide woven fabric were investigated. Lastly, after multiple treatment steps, such as enzymatic hydrolysis, GA crosslinking and laccase immobilization, the structural and morphological characteristics of the polyamide fabric were monitored by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM), respectively.

The present study exhibits the efficiency of the enzymatic hydrolyzed polyamide fabric and the optimum laccase immobilization conditions, thus demonstrating the utility of this strategy in enzyme immobilization.

Materials and methods

Materials

All the experiments were conducted using 100% commercial polyamide 6 woven fabric (Table 1). Before the enzymatic hydrolysis, all the woven samples were washed with 2 g/L Triton-X (Sigma Chemicals Co., USA) at 50℃ for 30 min and then rinsed with distilled water at 50℃ for 30 min for desizing.¹⁶ Laccase (EC 1.10.3.2) isolated from Aspergillus niger, bromelain (EC 3.4.22.32) isolated from pineapple stem, alcalase (EC 3.4.21.62) isolated from Bacillus licheniformis and flavourzyme (EC. 3.4.11.1) isolated from Aspergillus oryzae were used without further purification (Table 2). Sodium acetate and acetic acid (Sigma Chemicals Co., USA) were used as buffers for pH 4.0–5.0, sodium phosphate monobasic and sodium phosphate dibasic (Sigma Chemicals Co., USA) were used as buffers for pH 6.0–9.0 and sodium carbonate and sodium bicarbonate (Duksan Pure Chemicals Co., Korea) were used as buffers for pH 10.0–11.0. GA (Junsei Chemical Co., Japan) (25%) was used as the crosslinking reagent for crosslinking before enzyme immobilization. The optimum protease for the enzymatic hydrolysis of polyamide fabric was determined using TNBS (Sigma Chemicals Co., USA) and α-bromoacrylamide reactive dye (Lunasol Blue 3G, C.I. Reactive Blue 69; Huntsman Chemicals Co., USA). The optimum conditions for crosslinking with GA and immobilization of laccase on polyamide fabric were evaluated using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma Chemicals Co., USA) as a substrate for laccase.

Table 1.

Characteristics of polyamide fabric

Fiber (%)	Weave	Fabric count (yarns/inch)	Weight (g/m²)	Thickness (mm)
Polyamide 6 fabric	Plain	104 × 82	58 (±2)	0.11

Table 2.

Properties of enzymes

Enzyme	Source	Activity	Form	Manufacturer
Denilite IIS (EC 1.10.3.2)	Aspergillus	120 LAMU^a	Solid	Novozyme
Bromelain (EC 3.4.22.32)	Pineapple stem	3–7 Unit^b/mg	Lyophilzed Powder	Sigma Chemicals Co
Alcalase2.4L (EC. 3.4.21.62)	Bacillus lichenformis	2.4 AU^c/g	Liquid	Novozyme
Flavourzyme1000L (EC. 3.4.11.1)	Aspergillus oryzae	1000 (LAPU)^d/g	Liquid	Novozyme

LAMU: the amount of enzyme required to catalyze 1 µ mole mL⁻¹ of substrate per minute.

One unit will release 1.0 µ mole of p-nitrophenol from N a-Z-L-lysine p-nitrophenyl ester per min at pH 5.0 at 35℃.

One unit will release 1.0 µmole of p-nitrophenol from N α-Z-L-lysine p-nitrophenyl ester per min at pH 5.0 at 35℃.

LAPU: the amount of enzyme that hydrolyzes one micromole of leucine-p-nitroanilide per min at pH 5.0 at 35℃.

Methods

Enzymatic hydrolysis

The enzymatic hydrolysis of polyamide fabric was conducted according to the method reported by Kim and Seo¹⁶ and Kim et al.²³ The polyamide fabric (approximately 0.15 ± 0.02 g) was treated with 10% (owf) bromelain in phosphate buffer (50mM, pH 6.0) at 50℃ for 120 min. The liquor ratio was 50:1 (buffer solution:fabric), and the treatment was performed in a shaking water bath (BS-31; Jeio Tech, Korea) at 120 rpm. Enzymatic hydrolysis with alcalase and flavourzyme was conducted using 10% (owf) of enzymes in phosphate buffer (50mM, pH 7.0) at 60℃ and 50℃ for 60 and 120 min, respectively, in a shaking water bath at 120 rpm. After enzymatic hydrolysis, the enzymes were inactivated at 90℃ for 10 min. The samples were washed with 2 g/L sodium carbonate at 50℃ for 10 min, rinsed thoroughly, and dried at room temperature.¹⁶ After enzymatic hydrolysis of polyamide fabric, the hydrolytic activity was evaluated by TNBS assay. The difference of polyamide fabric hydrolyzed by enzymatic hydrolysis was determined by the K/S value after wool-reactive dyeing and WCA measurement.

Evaluation of the relative hydrolytic activity of enzymatic hydrolysis

The released hydrolysis products (free amino groups) in the treatment solution of enzymatic hydrolyzed polyamide fabrics were quantified using TNBS assay. Free amino groups were quantified by analyzing the color change due to the reaction between primary amines and TNBS (Figure 3). The TNBS assay is well suited for quantifying the degree of hydrolysis, regardless of the activity of the enzyme used.^15,16,23 Thus, the relative hydrolytic activity (RH) (%) of each protease on polyamide was evaluated by the TNBS assay. The TNBS assay of the sample test was implemented as follows: after enzymatic hydrolysis of polyamide fabric by bromelain, alcalase and flavourzyme solutions separately, the supernatant of each treatment solution was taken without polyamide. Then, each treatment solution was mixed with 2 ml of phosphate buffer (1 mM) and TNBS (30 mM). This mixture was incubated at 50℃ for 60 min at 80 rpm. After reaction, 4 mL of HCI (0.1 N) was added and the mixture was then cooled to room temperature for 30 min.^15,16,23 The formation of the amine-TNBS complex was monitored using an ultraviolet-visible (UV-Vis) spectrophotometer (S-3100; Scinco Co., Korea).^15,16,23 The TNBS assay of the control test was performed as a sample test. The control treatment solution was prepared after each protease treatment without polyamide fabric. RH (%) was calculated using the following equation^15,16,23

RH (%) = \frac{AB S_{S} - AB S_{C}}{AB S_{S}} \times 100

(1)

where ABS_S is the absorbance of the treatment solution of sample tests (proteases solution + polyamide fabric) at 340nm, and ABS_C is the absorbance of the treatment solution of the control test (proteases solution) at 340 nm.

Measurement of free amino groups generated on the polyamide fabric surface

The free amino groups generated on the polyamide surface were detected by the K/S value of the hydrolyzed polyamide fabric after α-bromoacrylamide reactive (wool-reactive) dyeing.^15,16,23 This measurement was based on the reaction α-bromoacrylamide groups with the free amino groups of the fiber surface. The polyamide fabric was dyed with wool-reactive dye (1% based on the weight of fiber (owf)) and 50 g/L of NaCI, using a liquor ratio of 50:1 at 80℃ for 90 min.^15,16,23 The reflectance and corresponding L*, a*, b* and color data for the dyed samples were determined using a computational color matching system (CM-2600d, KONICA MINOLTA.INC., Japan) and illuminant D65 with a 10° standard observer. The K/S value was calculated from the reflectance values using the Kubelka–Munk equation^15,16,23

K / S = \frac{(1 - R)^{2}}{2 R} \times 100

(2)

where R is the reflectance, K is the absorption coefficient and S is the light scattering coefficient. All measurements were performed at least five times.

The WCA of the enzymatic hydrolyzed polyamide was measured using a contact angle measurement system (DSA100, KRÜSS Inc., German).

Crosslinking by glutaraldehyde on polyamide fabric for enzyme immobilization

Crosslinking was conducted by immersing the pieces of enzymatic hydrolyzed polyamide fabric (approximately 0.15g) into each buffer solution with a specific GA concentration. The polyamide woven was then treated at different pH values (6.0–11.0), crosslinking times (30–1440 min), GA concentrations (1–20% v/v) and temperatures (20–65℃). Shaking was performed at 110 rpm using a shaking water bath.⁵ After treatment, the activated samples were washed thoroughly with both buffer solution and distilled water several times to remove any unreacted chemicals.¹⁸

Covalent laccase immobilization on enzymatic hydrolyzed polyamide fabric

Laccase was immobilized onto the surface of the modified polyamide woven. Covalent immobilization of laccase was carried out by immersing 0.15 g of the GA-activated polyamide woven in 25 mM buffer of varying pH values (4.0–9.0) with varying concentrations of laccase (15–100% owf). The mixture was agitated in a water bath for different durations (min) at different temperatures (4–50℃). Next, the immobilized laccase mixtures were washed several times with distilled water. The amount of immobilized laccase was determined by measuring the absorbance at 420 nm using a UV-Vis spectrophotometer.^19,20,27 After the immobilization process, the immobilization yield was calculated by measuring the difference between the enzyme activities of the supernatant before (A₀) and after (A_t) immobilization. The immobilization yield (IY) was calculated by the following equation²⁹

IY (%) = \frac{A_{0} - A_{t}}{A_{0}} \times 100

(3)

Laccase assay

The protein concentration of the free and immobilized enzyme was determined according to the method of Bradford using bovine serum albumin (BSA) as a standard material.^5,32–34 The amount of immobilized enzyme, in terms of protein loading (mg/g), was calculated using the following equation^5,33–35

Q = \frac{(Ci - Cf)}{mV}

(4)

where Q is the protein loading (mg enzyme/g support), Ci and Cf are the initial and final enzyme concentrations in the solution, respectively (mg/mL), V is the volume of the solution (mL) and m is the mass of the support (g).

Activity assays of free and immobilized laccase

The enzymatic activities of free and immobilized laccase were determined by the oxidation of ABTS. As shown in Figure 2, the nonphenolic heterocyclic compound ABTS is oxidized by laccase to the more stable and preferred state of the cation radical (ABTS^• +) and ABTS dication (ABTS²⁺).³⁶ ABTS is colorless, while ABTS^• + colors and presents absorption in the visible region. The concentration of the cation radical responsible for the intense blue-green color can be corrected to enzyme activity and is read at 420 nm.^18,19,27,30 The reaction mixture contained 1 mmol/L ABTS in acetate buffer (pH 5.0) and a suitable amount of free or immobilized enzyme. The reaction time was 5 min. During the process, the changes in the absorbance at 420 nm were measured using a UV-Vis spectrophotometer. These activity assays were carried out in different treatment conditions. The activity was converted to relative activity (percentage of the maximum activity obtained in that series) for both the free and immobilized laccase as follows^21,22

Relativeactivity (%) = \frac{ABTS 420}{MaximumABTS 420} \times 100

(5)

Figure 2.

Scheme of the oxidation reaction between 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and laccase.

Figure 3.

Scheme of the reaction between 2,4,6-trinitrobenzene sulfonic acid and amine functional groups generated on enzymatic hydrolyzed polyamide fabric.

Therefore, the highest activity is regarded as 100% for the free and immobilized enzyme.

Stability assessment

In order to assess the stability depending on the value of pH, free and immobilized laccase were incubated in solutions with pH values ranging from 3.0 to 8.0 at 4℃ for 60 min. Thermal stability assays were performed by incubating the free and immobilized enzyme in phosphate buffers at a pH of 6.0 at 40℃ and 50℃ for different incubation periods. After incubation, the remaining activities of the free and immobilized laccase were measured at room temperature using ABTS as a substrate.^20,27,30

In order to determine the storage stability, free and immobilized laccase were stored at 4℃ for 20 days. The storage stability of the enzyme was determined by measuring the relative activity of the samples taken at regular time intervals using the method described above.^37,38

To assess the reusability, after each reaction run, the laccase-immobilized sample was removed and washed with 50 mM sodium acetate buffer to remove any residual substrate. It was then reintroduced into a fresh reaction medium to determine the enzyme activity.⁷ The experiment was performed 10 times and the activity of immobilized laccase was measured each time using the method described above.³⁷

Surface characterization

The FT-IR spectra of polyamide woven after enzymatic hydrolysis, GA crosslinking and enzyme immobilization were recorded using a FT-IR spectroscope (FT-IR 670 Plus, Jasco, Easton, MD) in the region of 4000–400 cm⁻¹.

The surface morphology of polyamide woven after enzymatic hydrolysis, GA crosslinking and enzyme immobilization was analyzed using a scanning electron microscope (JSM-7600F, JEOL Ltd, Japan).

Results and discussion

Enzymatic hydrolysis

After hydrolysis by three different proteases, namely bromelain, alcalase and flavourzyme, the amide bonds (–CONH–) of polyamide fabric were cleaved, resulting in the generation of amino and carboxyl groups. TNBS, which is a nitroaryl oxidizing acid, is a highly sensitive and rapid chemical used to quantitate the free amino groups. As shown in Figure 3, the reaction between TNBS and primary amines generates highly chromogenic product sulfite complexes ( ${SO}_{3}^{2 -}$ ) that can be readily measured at 340 nm.^{16,21,22,30,39} Figures 4(A)–(C) show the hydrolytic activity of each protease on polyamide fabric as assessed through RH (%), the K/S value after wool-reactive dyeing and the WCA (°), respectively. As shown in Figure 4(A), the highest RH (%) of about 73% was obtained from bromelain hydrolysis. The free amino groups released into the treatment solution formed complexes with TNBS.^{16,21,22,30,39} When the number complexes between free amino groups and TNBS increased in the treatment solution after bromelain hydrolysis, the absorbance also increased.^16,17 Thus, the highest value of RH (%) demonstrated that bromelain was capable of hydrolyzing amide bonds in polyamide fabric. The effectiveness of bromelain was also evaluated by detecting amino groups generated on the fabric surface. This measurement was based on the K/S value of polyamide fabric after wool-reactive dyeing. As shown in Figure 4(B), the K/S value was increased by enzymatic hydrolysis on polyamide fabric. The highest K/S value was also observed for polyamide fabric hydrolyzed by bromelain. The increased K/S value was proportional to the increased free amino groups generated on the fabric surface.^16,17 The measured values for RH (%) and the K/S value of polyamide fabric exhibited the same trends, indicating that bromelain generated the cleavage of the amide bonds in the fiber, releasing free amino groups into the treatment solution and forming them on the surface of the fabric. The formation of amino groups on the fabric surface are also detected by enhanced hydrophilicity due to newly formed amino and carboxyl groups.^16,17 The values of the contact angle (WCA) of polyamide fabric, depending on the different enzymatic hydrolysis conditions, are shown in Figure 4(C). The WCA of polyamide fabric was changed after enzymatic hydrolysis. In particular, after bromelain treatment, the WCA of polyamide fabric significantly decreased from 100° to approximately 60°. The lower WCA implies that a large number of hydrophilic molecules formed onto the polyamide surface through the enzymatic hydrolysis with bromelain.¹⁶

Figure 4.

(A) Relative hydrolytic activity (RH) (%) of enzymatic hydrolysis, (B) K/S value (640 nm) and (C) water contact angle of polyamide fabric subjected to different treatments ((a) native; (b) bromelain; (c) alcalase; (d) flavourzyme).

Laccase immobilization on polyamide fabric

Partial enzymatic hydrolysis of the polyamide surface generates both amino (–NH₂) groups, exploited here in the subsequent coupling process, and –COOH groups. Both these groups could bind with the protein molecules, resulting in the highest immobilization yield and enzymatic activity.²⁹ To evaluate the effects of enzymatic hydrolysis on the polyamide fabric, laccase was immobilized on the enzymatic hydrolyzed polyamide fabric with different proteases. Table 3 shows that the value of protein loading (mg/g), immobilization yield and relative activity (%) were dependent on the type of protease that was used for polyamide fabric. A large amount of protein was loaded on hydrolyzed polyamide by bromelain, approximately 0.22 mg/g. Also, the highest immobilization yield (68 ± 0.7%) and relative activity (95 ± 0.52%) for the immobilized laccase were achieved when the support was hydrolyzed using bromelain. This may be attributed to the generation of abundant amino groups for enzyme binding because of the bromelain treatment. Based on these results and considering the immobilization yield and relative activity, the bromelain treatment was selected as a suitable modification method for polyamide fabric to be developed as a support.

Table 3.

The value of protein loading, immobilization yield and relative activity of immobilized laccase on (a) native polyamide fabric and treated by (b) bromelain, (c) alcalase and (d) flavourzyme

Sample conditions	Protein loading(mg/g)	Immobilization yield (%)	Relative activity (%)
(a)	0.1	58 ± 0.5	56 ± 0.4
(b)	0.217	68 ± 0.7	95 ± 0.52
(c)	0.111	58 ± 0.2	71 ± 0.2
(d)	0.172	65 ± 0.5	78 ± 0.5

GA crosslinking on polyamide fabric

Effect of pH on GA crosslinking

To determine the effect of pH of the GA solution, changes in the relative activity of the immobilized laccase were evaluated for pH ranging from 6.0 to 11.0. As shown in Figure 5, the activity of immobilized laccase gradually increased with the shift of pH from basic to weakly alkaline conditions. The maximum activity was observed at pH 9.0, whereas for pH values over 9.0, the enzymatic activity of laccase sharply declined. Generally, for high pH values, particularly pH 9.0, the number of aldehyde groups bound on the support rapidly increases due to the polymerization of GA molecules resulting from aldol condensation.¹⁴ However, the Schiff base, which is formed by the aldehyde groups, is more stable in basic pH; hence, its reaction with the enzyme under strong alkaline conditions, such as pH 11.0, is not favorable. Thus, it decreases the catalytic activity of the enzyme.²⁰

Figure 5.

Effect of various pH values of glutaraldehyde (GA) solutions on polyamide fabric during GA crosslinking (GA crosslinking conditions: different pH values, GA 3% (v/v), 25℃ and 120 min).

Therefore, pH 9.0 was chosen as the optimal value of pH for the immobilization of laccase on the enzymatic hydrolyzed polyamide fabric.

Effect of temperature on GA crosslinking

Figure 6 shows the enzymatic activity of the immobilized laccase by different crosslinking temperatures, ranging from 20℃ to 65℃. As shown in Figure 6, with rising temperature, the activity of immobilized laccase sharply increased during 20–25℃, and the highest activity was obtained at 45℃. In contrast, the activity gradually decreased over 45℃. This is because of the increase in the amount of bound enzyme with increasing reaction temperature during the initial stage, leading to relatively high catalytic activity.¹⁶ However, the aldehyde groups of GA molecules bind rapidly with the enzyme under a high temperature, leading to the steric hindrance of enzymes, thereby reducing the enzyme activity.

Figure 6.

Effect of various treatment temperatures on polyamide fabric during glutaraldehyde (GA) crosslinking (GA crosslinking conditions: different temperatures, pH 9.0, GA 3% (v/v) and 120 min).

Therefore, 45℃ was considered the optimum crosslinking temperature for laccase immobilization.

Effect of GA concentration on GA crosslinking

Figure 7 shows the effects of GA concentration ranging from 1% to 20% (v/v) on laccase immobilization. As shown in Figure 7, the activity of immobilized laccase gradually increased with increasing GA concentration, and the highest activity was obtained for 10% GA concentration. Subsequently, as the GA concentration increased above 10%, the activity significantly decreased. In particular, when the GA concentration was 20%, the activity of immobilized laccase decreased to almost 50% compared with the highest activity. This indicates that 10% of GA provided enough aldehyde groups on the support that were capable of binding with the enzyme molecules.¹⁷ Generally, lower concentrations of GA are not enough to form sufficient cross linkages with enzymes. With increasing concentration of GA, the amino groups of laccase were activated; thus, the amount of immobilized enzyme also increased. However, higher amounts of GA may cause the denaturation of the enzyme due to the saturation of the support with excessive GA molecules. Consequently, it reduces the accessibility of the enzyme to the substrates, thereby decreasing the catalytic activity of enzyme.

Figure 7.

Effect of various concentrations of glutaraldehyde (GA) on polyamide fabric during GA crosslinking (GA crosslinking conditions: different GA concentrations, pH 9.0, 45℃ and 120 min).

Therefore, 10% GA concentration, which showed the highest activity, was selected as the optimum concentration for the immobilization of laccase.

Effect of treatment time on GA crosslinking

Figure 8 shows the effect of crosslinking time on laccase immobilization. It shows the changes in relative activity with various crosslinking times ranging from 30 to 1440 min. As seen in Figure 8, the relative activity of immobilized laccase increased with increasing reaction time. The maximum activity was observed at 240 min of crosslinking. However, the relative activity of immobilized laccase sharply decreased with prolonged crosslinking for over 240 min. The loss of activity resulted from extensive GA polymerization on the support at prolonged reaction times.¹⁷ This also leads to the configuration of immobilized enzyme, causing denaturation of the enzyme protein.³⁰ In addition, at initial reaction times, the GA molecules tend to rapidly react with the amino groups of the enzyme; hence, the enzymatic activity of laccase increased.¹⁴ However, after 240 min, the number of amino groups reacting with the aldehyde groups of GA became limited; thus, longer reaction times did not result in an additional increase in the activity of the immobilized laccase. Therefore, the optimum crosslinking time for the immobilization of laccase was selected as 240 min.

Figure 8.

Effect of various glutaraldehyde (GA) crosslinking times on polyamide fabric during GA crosslinking (GA crosslinking conditions: different treatment times, pH 9.0, GA 10% (v/v) and 45℃).

Covalent immobilization of laccase

Effect of pH on enzyme activity for laccase immobilization

The pH of the buffer solution is one of most important factors in enzyme immobilization, since it influences not only the properties of the amino acid side groups of the enzyme but also the active sites of the functional groups on the support.³⁸ It is important to establish the suitable pH conditions for the immobilization of laccase on the enzymatic hydrolyzed polyamide fabric. To optimize the pH condition for laccase immobilization, the pH was varied from 4.0 to 9.0. Figure 9 demonstrates the effects of pH on the immobilization of laccase. The relative activity of immobilized laccase increased gradually and attained the maximum at pH 6.0. In contrast, when the pH values were higher than 6.0, the activity of immobilized laccase drastically decreased following the change to alkaline conditions. This resulted from the denaturation of the enzyme in highly acidic or alkaline conditions.^6,40 This damage generally results in a decrease in binding sites between the enzyme and the support, thereby leading to a reduction in the quantity of immobilized laccase.⁴⁰

Figure 9.

Effect of various pH values of laccase solutions on polyamide fabric during laccase immobilization (laccase immobilization conditions: 4℃, 600 min, 10% (owf) of laccase).

Therefore, pH of 6.0, which yielded the highest activity, was chosen as the optimum pH for the immobilization of laccase on the enzymatic hydrolyzed polyamide fabric.

Effect of temperature on immobilization of laccase

Figure 10 shows the change in the relative activities of immobilized laccase on the enzymatic hydrolyzed polyamide fabric with varying temperature from 4℃ to 50℃. As shown in Figure 10, the maximum activity of immobilized laccase was observed at 4℃; thereafter, the enzyme activity reduced with increasing temperature. In particular, from 40℃ to 50℃, the enzyme activity drastically reduced. This is because the higher reaction temperatures do not effectively facilitate the crosslinking between the enzyme molecules and the active groups on the support.⁴¹ Moreover, the loss of activity at temperatures higher than 4℃ is because of the thermal denaturation of the enzyme.²⁵ Thus, a lower immobilization temperature was preferable to achieve a high activity of the immobilized laccase. Therefore, the optimum temperature for the immobilization of laccase was found to be 4℃.

Figure 10.

Effect of various temperatures on polyamide fabric during laccase immobilization (laccase immobilization conditions: pH 6.0, 600 min, 10% (owf) of laccase).

Effect of concentration on immobilization of laccase

In order to determine the optimum laccase concentration for immobilization, the enzyme concentrations were varied from 15% to 100% (owf) at 4℃. Figure 11 shows the influence of laccase concentration on enzymatic activity during the immobilization process. Two slopes are seen in Figure 11; the first slope, corresponding to lower laccase concentrations until 35% (owf) of used laccase, was steeper than the second slope.²⁷ When the first slope shifted to the second slope at 35% of used laccase, an optimum level of activity was observed. However, above 35% of enzyme, the relative activities remained constant. At initial concentrations, the enzyme molecules were covalently immobilized covering the entire support surface, thereby preventing enzyme inactivation.²⁶ However, at the saturation point, all the substrates were bound to the enzyme and, thus, excessive enzyme molecules could not react with any substrate.⁷

Figure 11.

The slopes of relative activity (%) of immobilized laccase at various enzyme concentrations during laccase immobilization (laccase immobilization conditions: pH 6.0, 600 min and 4℃).

Therefore, excessive laccase adsorption would result in the saturation and agglomeration of enzyme at other active sites of the support.²⁶ That is, the relative activity reached an equilibrium state over 35% of laccase concentration, causing no further increase in activity. Hence, 35% (owf) laccase concentration was selected as the optimum concentration for immobilization. Subsequent studies were carried out using this concentration.

Effect of immobilization time

The effect of immobilization time on the activity of immobilized laccase was studied in the range from 30 to 1440 min. As seen in Figure 12, the relative activity of immobilized laccase increased with prolonged immobilization time and reached the maximum value at 600 min. However, when the reaction time increased over 600 min, the activity sharply declined. This decrease in activity resulted from the exhaustion of the available aldehyde groups reacting with the enzyme molecules during the initial period. Moreover, prolonged reaction times result in the formation of multiple linkages between the enzyme molecules and the support, which leads to the distortion and denaturation of the enzyme. In addition, it causes conformational changes in the support by rendering the apertures of the support relatively thin, eventually resulting in the lower accessibility of the substrate to the active sites.⁶ Consequently, the enzyme activity decreased with prolonged immobilization time.²⁷ Therefore, the optimal immobilization time for laccase was concluded to be 600 min, during which the highest relative activity was observed.

Figure 12.

Effect of various immobilization times on polyamide fabric during laccase immobilization (laccase immobilization conditions: pH 6.0, 35% (owf) and 4℃).

pH stability of free and immobilized laccase

The stability of both free and immobilized laccase on the enzymatic hydrolyzed polyamide fabric was compared in pH values ranging between 3.0 and 8.0 at 4℃ during 60 min of incubation. As shown in Figure 13, free laccase demonstrated maximum activity in the pH range of 3.0–4.0, while the immobilized laccase was the most active in the pH range of 5.0–8.0. These results were expected because the number of positively charged groups of the enzyme linked with the amino groups of the support decreases after immobilization. Thus, the enzyme exhibits a more polyanionic character.³⁸ The activity of the free enzyme significantly changed depending on pH variation, while the activity of the immobilized enzyme remained constant at over 40% during pH change. Thus, the activity of the free enzyme readily decreased because of denaturation due to a change in the value of pH. However, in the case of immobilized laccase, the denaturation of laccase molecules would considerably reduce due to immobilization. The stabilization of laccase activity is most probably the result of multi-point linkages of laccase and aldehyde molecules on the support, which prevented not only autolysis but also laccase denaturation.^23,38,42

Figure 13.

pH stability of the free and immobilized laccase (○: free laccase; •: immobilized laccase).

Therefore, the loss of enzyme activity greatly reduced under changing in the value of pH via its immobilization strategy on the enzymatic hydrolyzed polyamide fabric compared to free laccase.

Thermal stability of free and immobilized laccase

The thermal stability profiles of free and immobilized laccase were recorded at 40℃ and 50℃ and are shown in Figures 14(a) and (b), respectively. As seen in Figure 14(a), while free laccase retained 32% of its initial activity at the end of 120 min of incubation at 40℃, the immobilized laccase retained approximately 60% of its initial activity. The same finding also was monitored at 50℃, as shown in Figure 14(b); immobilized laccase demonstrated 40% of its initial activity at the end of 120 min of incubation, while free laccase retained only 20% of its initial activity. This result clearly demonstrates the efficiency of the immobilization method in protecting the enzyme against heat inactivation.²³ This could be the result of conformational limitations of the enzyme due to multi-point attachment. Hence, the limitation of conformational degeneration might allow laccase to retain its activity when the immobilized laccase is exposed to heat for a long time.¹⁹ In addition, the covalently immobilized enzyme on the support is more capable of resisting denaturation and conformational changes caused by heat.³⁷

Figure 14.

Thermal stability profile of the free and immobilized laccase at 40℃ (a) and 50℃ (b) (○: free laccase, •: immobilized laccase).

These results are in agreement with other reported works.^10,23,37 The better thermal stability of the immobilized laccase will extend the potential application of the enzyme as a biocatalyst.²³

Storage stability of free and immobilized laccase

The storage stability of immobilized laccase is crucial for practical applications.²⁷ The stability of free and immobilized laccase on the enzymatic hydrolyzed polyamide fabric was evaluated for 20 days of storage at 4℃. As can be seen in Figure 15, after 20 days of storage, the relative activity of free laccase was less than 10%. In contrast, the immobilized laccase retained over 60% of its initial activity until 10 days of storage and its activity of over 30% remained after 20 days. The retention of stability despite the long duration of storage is probably the result of the prevention of autolysis and unfolding of laccase molecules due to the covalent bonding between the enzyme molecules and the fiber surface that enhanced the enzyme's resistance to denaturation.^4,27

Figure 15.

Storage stability of the free and immobilized laccase (○: free laccase, •: immobilized laccase).

As a result, the covalently immobilized laccase maintained its activity during prolonged storage. Generally, the catalytic activity of the enzyme in soluble state rapidly reduces during long periods of storage and is difficult to recover. Further, the study of laccase leakage from the polyamide fabric demonstrated that laccase immobilization on the enzymatic hydrolyzed polyamide fabric accords significant advantages in terms of retention of stability of the enzyme over long storage times.

Reusability of immobilized laccase

The reusability of immobilized enzyme is of particular importance for the economical use of an enzyme. As shown in Figure 16, the immobilized laccase retained 60% of its original activity after three use cycles. After five use cycles, it retained approximately 12% of its initial activity. The retained enzymatic activity after repetitive usage indicated that the enzyme protein molecule immobilized on the support was quite stable under the optimal treatment conditions.⁴²

Figure 16.

Reusability of immobilized laccase on the enzymatic hydrolyzed polyamide fabric.

Therefore, the immobilized laccase on the enzymatic hydrolyzed polyamide fabric can be efficiently used as a biocatalyst for continuous industrial application.⁹

Surface characterization

FT-IR analysis

The structural changes of polyamide fabric were determined by FT-IR analysis. As shown in Figure 17(a), characteristic absorption peaks of polyamide fiber appeared around 1350–1630, 2850–2930 and 3292.45 cm⁻¹. These are attributed to the C = O stretching vibration of the amide groups, the N–H in-plane bending vibration of the secondary amines and –CONH groups, respectively.^30,43,44 After enzymatic hydrolysis by bromelain, the spectrum between 1200 and 1350.7 cm⁻¹, which were attributed to C–N stretching vibration, intensified, confirming the hydrolysis of polyamide chains (Figure 17(b)). Moreover, the absorption band at 2931.29 and 2857.69 cm⁻¹ became stronger compared to the band of N–H bending for native polyamide fabric due to the cleavage of peptide groups.^30,43,44 In addition, a new absorption peak at 3083.84 cm⁻¹ was observed that corresponded to the C-H group. Its intensity was enhanced after the GA crosslinking reaction since the CH₂ groups of GA were introduced on the polyamide surface (Figure 17(c)).^27,30,43,44 These results indicated that the GA molecules, which are available to react with enzymes, were attached on the polyamide surface. After enzyme immobilization (Figure 17(d)), the band associated with the vibrations of the –CONH group broadened and the –NH group was significantly decreased. This may be due to the establishment of hydrogen bounds and/or interactions between the enzyme molecular–amine functional groups, reducing -NH groups on polyamide.^27,30,43,44 Thereby, no new bands appeared, thus indicating that laccase was immobilized by crosslinking with amine groups generated on polyamide fabric.

Figure 17.

Fourier transform infrared spectra of polyamide fabric subjected to different treatments: (a) native; (b) enzymatic hydrolysis by bromelain; (c) crosslinking with glutaraldehyde; (d) laccase immobilization.

Morphology by scanning electron micrography

Figure 18 shows the morphologies of native and modified polyamide woven after various treatments, as observed by SEM. Compared with native polyamide fabric (Figure 18(a)), no substantial change was observed on the enzymatic hydrolyzed polyamide after enzymatic hydrolysis (Figure 18(b)). This suggests that the intrinsic morphology of the polyamide fabric remained unchanged under these reactions without any distortion of the woven. However, as shown in Figure 18(c), irregular ripple marks of rounded structures were observed on the surface of the polyamide after GA crosslinking. After laccase immobilization, the surface morphology of polyamide fabric was significantly changed. Figure 18(d) shows some irregular particles attached to the fiber surfaces after laccase immobilization, which could be small enzyme aggregates formed through molecular interactions.²⁷ The flat surface of polyamide fabric seems to provide a large contact area for multi-point attachment with the enzymes.⁸ Xu et al.²⁷ and Silva and Cavaco-Paulo⁴³ reported similar results in the study of laccase immobilization.

Figure 18.

Scanning electron micrographs of different conditions polyamide fabric at 1500×: (a) native; (b) enzymatic hydrolysis; (c) crosslinking with glutaraldehyde; (d) laccase immobilization.

Overall, laccase could be successfully immobilized on enzymatic hydrolyzed polyamide fabric.

Conclusion

The purpose of this study was to suggest an enzymatic hydrolyzed polyamide fabric for enzyme immobilization support and to evaluate the optimum conditions for laccase immobilization onto the enzymatic modified polyamide woven. The polyamide fabric was first activated via enzymatic hydrolysis in order to generate amino groups onto its surface. Next, laccase was covalently immobilized on the enzymatic hydrolyzed polyamide fabric by GA crosslinking. Several parameters potentially influencing the enzyme activity during immobilization, such as the pH value of solution, treatment temperature, time and concentrations of both GA and laccase, were evaluated and optimal treatment conditions were established. After the enzymatic hydrolysis of polyamide fabric, a large number of amino groups were generated on its surface due to the cleavage of its amide bonds.

In particular, the enzymatic hydrolysis using bromelain showed the highest hydrolytic activity as determined by TNBS assay and the highest K/S value after wool-reactive dyeing due to amino groups generated on the polyamide surface. Moreover, the highest immobilization yield and relative activity of immobilized laccase were also obtained when the polyamide fabric was hydrolyzed using bromelain.

For successful hydrolysis of polyamide fabric, the optimal crosslinking conditions were pH of 9.0 with 10% (v/v) GA concentration for 240 min at 45℃. The most favorable immobilization conditions were as follows: pH of 6.0 with 35% (owf) laccase concentration for 600 min at 4℃. The pH and thermal stability of immobilized laccase improved under the optimal immobilization conditions. In addition, after 20 days of storage, the immobilized laccase retained approximately 30% of its initial activity. Furthermore, the immobilized laccase indicated potential reuse over 10 use cycles. The FT-IR results confirmed the structural changes in the surface of the polyamide fabric owing to the multi-step treatments. Further, the surface morphologies were analyzed by SEM. The surface of polyamide fabric was changed according to the multi-steps treatments. In particular, after laccase immobilization, the roughness of polyamide fabric was increased due to the immobilized enzyme clusters. This result indicated that laccase was successfully immobilized onto the enzymatic hydrolyzed polyamide fabric. In conclusion, the enzymatic hydrolysis process for polyamide fabric has an effect on high efficiency as a support material for laccase immobilization.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2014R1A1A1005314).

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