Covalent immobilization of enzyme on aminated woven poly (lactic acid) via ammonia plasma: evaluation of the optimum immobilization conditions

Abstract

The present study aims to develop an immobilization support from woven poly (lactic acid) (PLA) and establish the optimum immobilization conditions for trypsin. Woven PLA was modified by ammonia-based plasma treatment in order to incorporate amine groups on its surface. X-ray photoelectron spectroscopy analysis showed that the N1s composition of PLA increased significantly, from 0.66% to 5.92%, after ammonia-based plasma processing. Trypsin from porcine pancreas was immobilized onto modified woven PLA by covalent binding after activating PLA with glutaraldehyde (GA). The results indicated that the optimal GA treatment conditions were as follows: pH of 10.0, 2% GA (v/v), and 180 min crosslinking time. In addition, the optimum immobilization conditions were as follows: pH of 8.5, 10% (owf) of trypsin concentration, 30 min, and 25℃. Under the optimum conditions, the amount of immobilized enzyme on woven PLA was 0.28 mg/mg and specific activity was 3.763 U/mg. In addition, the pH and thermal stabilities of the immobilized trypsin were improved. The immobilized trypsin retained approximately 55% of its initial activity after 20 days of storage and exhibited the potential for repetitive use through approximately 15 cycles. GA crosslinking and trypsin immobilization were found to improve the roughness of the PLA surface and increase its hydrophobicity. The data indicate that modified woven PLA, used as an immobilization support, shows suitable properties for use as a biocatalytic material in enzymatic applications.

Keywords

poly (lactic acid)glutaraldehyde cross-linking immobilization plasma trypsin

Enzyme immobilization has been proved to be effective for the improvement of enzyme stability and durability during storage and operational processes.^1–3 Furthermore, it facilitates an enzyme’s catalytic activity to retain and improve enzyme stability against denaturation due to the enhanced affinity between biomolecules and their receptors, thereby allowing the enzyme to be reused.^4,5 Covalent binding-based enzyme mobilization, which utilizes bifunctional crosslinking reagents, provides a powerful linkage between the enzyme and its support matrix, resulting in highly stable preparations compared with the other immobilization procedures. Consequently, the immobilized enzyme does not separate easily during the reaction, and may be reused more often than in the other available immobilization methods.^6–8

Proteases have been widely used in industrial and biomedical applications, among which trypsin has been extensively concerned. It is highly efficient as a specific for catalyzing the breakdown of peptide linkages. In terms of cleavage reaction, trypsin is very specific. For this reason, trypsin has been used not only as medical material,⁹ but also in various textile fields.^10–13 For the further application of trypsin at an effective industrial level, trypsin could be immobilized onto heterogeneous derivatives by a suitable immobilization method.

Glutaraldehyde (GA) is an excellent protein crosslinking reagent that is widely employed in the field of enzyme immobilization and microbiology. GA, which is a protein crosslinking reagent, is more efficient than other aldehydes under a broad range of reaction conditions, as it forms a strong bond via an intense multi-point crosslink between the enzyme and the support, enabling an increase in enzyme stability.^8,14,15

The support itself (i.e., beads, fibers, sheets, etc.) may play an important role in enzyme immobilization by covalent binding, since its interaction with the enzyme potentially affects enzyme stability and kinetics.^7,16 An appropriate support material must fulfill the following requirements: high affinity for proteins, availability of reactive functional groups, mechanical stability, non-toxicity, and biodegradability.⁸ It may be formed from either natural polymers, such as various chitosan matrices and cellulose, or from synthetic polymers such as polypropylene, polyacrylonitrile, polyamide, and poly (lactic acid) (PLA). In particular, immobilization supports constructed from synthetic polymers are known to promote enzyme stability and reusability due to their mechanical stability and binding force.⁷

PLA is an abundantly available renewable and degradable material derived from corn or rice.¹⁷ This linear aliphatic thermoplastic polyester is non-toxic and possesses useful mechanical properties, such as density and tensile strength.¹⁸ In addition, the PLA woven matrix possesses outstanding advantages, such as high structural stability, mechanical intensity, and a highly specific surface.¹⁹ However, as PLA lacks functional groups required for interaction between the enzyme and support, surface modifications must be applied for this material to be used effectively for this purpose.^17–20 Plasma treatment, which is one of the most effective techniques for the introduction of functional groups onto the surface of materials, has been shown to be able to control surface structure and energy, and to uniformly modify the surface without affecting bulk properties.^15,17 Moreover, plasma treatment is capable of introducing a large variety of reactive functionalities depending on the gases included in the plasma, for example, oxygen, helium, nitrogen, and ammonia. Plasma surface treatment with a variety of reactive functionalities has been utilized to synthesize PLA surfaces with various functional groups on the top layer. Ammonia-containing plasma is known to be an effective precursor for the introduction of a large quantity of amine functionalities onto other materials, thereby enhancing their hydrophilicity and biocompatibility in comparison with those of n-butylamine or arylamine mixtures.⁶ Supports constructed by this method have been reported to show close affinity with enzyme molecules, forming strong bonds between aldehyde groups and amino groups via ammonia plasma processing.^14,21 Therefore, the application of ammonia gas in plasma treatment is expected to effectively modify the surface of woven PLA.

The purpose of this study was to develop a novel method of enzyme immobilization on aminated woven PLA using ammonia-based plasma treatment. Firstly, trypsin was employed as a model enzyme to be immobilized onto the PLA surface in the present work. In order to modify the surface of woven PLA, the amine groups that were to interact with the enzyme were incorporated on the surface using ammonia gas in the plasma medium. Next, the effects of crosslinking conditions (GA concentration, crosslinking time, and pH value) on modified woven PLA were investigated. In addition, the optimum enzyme conditions, in terms of pH value, immobilization time, temperature, and enzyme concentration, for immobilization were determined. Next, the pH, thermal stability, storage stability, and reusability of the immobilized enzyme on aminated woven PLA were investigated. Finally, the characteristics of aminated, crosslinked, and immobilized woven PLA were examined by X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), and scanning electron microscopy (SEM). The present study demonstrates the construction of woven PLA aminated by ammonia-based plasma treatment and the optimum immobilization method of trypsin, thereby demonstrating the utility of this strategy for providing support in enzyme immobilization.

Materials and methods

Materials

In this study, 100% PLA yarn of 84 dtex with 72 DTY-type filaments was supplied by Huvis. Woven PLA was to the specification listed in Table 1. Commercial trypsin isolated from porcine pancreas (EC 3.4.21.4, powder, Sigma) was used without further purification (Table 2). Sodium acetate and acetic acid from Sigma Chemicals Co., USA, were used as a buffer of pH 4.0–5.0. Tris buffer from Sigma Chemicals Co., USA, was used as a buffer of pH 6.0–9.0. Sodium phosphate monobasic, sodium phosphate dibasic (Sigma Chemicals Co., USA), sodium carbonate, and sodium bicarbonate (Duksan Pure Chemicals Co., Korea) were used as a buffer of pH 10.0–11.0. A buffer of pH 12.0–13.0 was obtained by using commercial buffer solution from Samchun Chemicals Co., Ltd, Korea. GA (Junsei Chemical Co., Japan) of 25% was used as the crosslinking reagent. Optimum crosslinked conditions for woven PLA were evaluated using N_a-benzoyl-L-arginine 4-nitroanilide hydrochloride (L-BAPNA, Sigma Chemicals Co., USA) as a substrate of trypsin. Activity of immobilized trypsin was measured using dimethyl sulfoxide (DMSO, Sigma Chemicals Co., USA). Bovine serum albumins (BSA), Bradford Reagent from Sigma Chemicals Co., USA, were used to determine the concentration of trypsin.
Table 1.
Characteristics of woven poly (lactic acid) (PLA)

Composition (%) Yarn count (denier) Weight (g/m²) Density (yarn/inch) Thickness (mm)

PLA 100 75 56.83 90 × 68 0.132

Table 2.
Enzyme characteristics

Enzyme Source Activity Form Manufacturer

Trypsin (EC 3.4.21.4) Porcine pancreas 1000–2000 BAEE units^a/mg solid Lyophilized powder Sigma Chemicals Co.

a
One BAEE unit produces a △A₂₅₃ of 0.001 per min at pH 7.6 at 25℃ using Na-Benzoyl-L-Arginine Ethyl Ester (BAEE) as a substrate.

Methods

Ammonia-based plasma treatment

The woven PLA surface was modified using ammonia-based plasma techniques (CD 400 PC®, Euro Plasma, Belgium). Gas flow, operation pressure, and plasma power were set at 100 sccm, 30 mTorr, and 220 W, respectively. Treatment was carried out for 10 minutes.

Crosslinking of woven PLA by GA

GA treatment was carried out by immersing pieces of aminated woven PLA weighing approximately 0.1 g into each buffer solution containing specific GA concentrations. The woven PLA was then treated using different pH values (7.0–11.0), crosslinking times (30–1440 min), and GA concentrations (0.1–15% v/v). Crosslinking was performed at 110 rpm using a shaking water bath at 25℃.⁵ After treatment, the activated samples were washed thoroughly with each buffer and distilled water several times to remove any unreacted chemicals.²²

Covalent immobilization of trypsin

In order to preserve trypsin activity and to achieve high immobilization efficiency, immobilization conditions should be optimized. Covalent immobilization of trypsin was carried out by immersing 0.1 g GA-activated woven PLA in 25 mM buffer of varying pH (7.0–10.0) with varying concentrations of trypsin (2, 4, 6, 8, 10, and 14% owf). The mixture was agitated in a water bath for various time periods (15–240 min) at different temperatures (20–45℃). Next, the immobilized trypsin mixtures were washed several times with distilled water. The amount of immobilized trypsin was determined by measuring absorbance at 410 nm with L-BAPNA as the substrate.^7,22,23 After the immobilization process, the recovered activity of the immobilized trypsin was calculated from the following equation:^7,24–28
$Ra (%) = \frac{A_{i}}{A_{f}}$
(1)
where Ra is the activity recovery of the immobilized enzyme (%), A_i is the activity of the immobilized enzyme (U), and A_f is the activity of the same amount of free enzyme in solution as that immobilized on the support (U).

Trypsin assay

The protein concentration of free and immobilized enzyme was determined according to the method of Bradford using BSA as a standard material.^29,30 The amount of immobilized trypsin was calculated using the following:³¹
$Q = \frac{(Ci - Cf)}{mV}$
(2)
where Q is the immobilized enzyme (mg enzyme/g support), Ci and Cf are the initial and final enzyme concentrations in the solution (mg/mL), V is the volume of the solution (mL), and m is the mass of the support (g).³⁰

Trypsin activity assay

Trypsin activity was measured with L-BAPNA. A total of 35 µL of L-BAPNA (50 mM in 50 mM phosphate, pH 8.5) was incubated with enzyme extract at 25℃ and change of absorbance was recorded at 410 nm for fixed time intervals. One unit of trypsin activity was defined as the amount of enzyme required to release 1 µmol of p-nitroaniline per min by use the molar extinction coefficient of p-nitroaniline at 410 nm of 8800 M^–1cm^–1. The specific activity was defined as the number of enzyme units per milligram of protein.³¹ Trypsin activity unit was expressed as change in absorbance per min per mg protein of the enzyme used in the assay. Activity units were calculated by the following equation:^32,33

The activity was converted to relative activity (percentage of the maximum activity obtained in that series) of both free and immobilized trypsin was determined by the following equation:^34,35
$Relativeactivity (%) = \frac{ABS 410}{MaximumABS 410} \times 100$
(4)
Therefore, the highest activity is regarded as 100% for the free and immobilized enzyme.

Stability assessment

pH and thermal stability

In order to assess the effects of pH levels, free and immobilized trypsin was incubated in solutions of pH ranging from 4.0 to 13.0 after 60, 120, and 240 min, respectively, at 25℃.³⁶ The effects of different temperature were assessed by measuring the activity of free and immobilized trypsin in tris-HCI buffer (25 mM, pH 8.5) at a broad range of temperature (25–65℃) for 1 h. After incubation, the remaining activity of the free and immobilized trypsin was measured at room temperature using L-BAPNA as a substrate.^7,15,16 Thermal stability of free and immobilized trypsin was measured at 25℃ and 50℃ for 240 min, respectively.^37,38

Storage stability and reusability of immobilized trypsin

In order to investigate storage stability, immobilized trypsin was stored at 4℃ without buffer. The activity of immobilized trypsin was monitored for 20 days. The reusability of the immobilized trypsin in woven PLA was evaluated by repeating the activity assay 15 times using the same support. At the end of each cycle, the immobilized trypsin was washed with tris-HCI buffer (25 mM, pH 8.5), and a fresh aliquot of substrate was added. The activity of immobilized trypsin was measured each time using the method described above.^22,23,34

X-ray photoelectron spectroscopy analysis

Ammonia-based plasma treatment-induced changes in the surface chemistry of the woven PLA were determined by XPS using an AXIS NOVA (Kratos Analytical, Ltd, UK) instrument equipped with a monochromated AI K_∞ source (1486 eV). A narrow scan was completed at pass energy of 40 eV. This was followed by high-resolution scans of C 1s (275–300 eV), N 1s (388–408 eV), and O 1s (520–540 eV). For quantitative analysis, a subtraction method in the linear line background was applied to all the main spectral bands and the respective areas were calculated. These data and the respective X-ray cross-sectional values were used to calculate the percentage atomic concentration for each element present. Component analysis of the spectral regions was carried out by peak fitting using pure Gaussian line shapes.³⁹

Analysis of hydrophilic properties

The WCA was used to assess surface wettability of woven PLA, which is indicative of changes in hydrophilicity. The WCA was determined using the contact angle measurement system (DSA100, KRÜSS Inc., Germany). The apparatus was used in conjunction with specialized software for determination of surface tension. The Girifalco–Good–Fowkes–Young model was used to calculate the average surface energy under various conditions.⁴⁰

Surface characterization

The surface morphology of the woven PLA was analyzed by SEM (JSM-7600 F, JEOL Ltd, Japan).

Results and discussion

XPS analysis

XPS measurements were performed in order to investigate the changes in chemical bonds in modified woven PLA due to ammonia-based plasma processing. In Figures 1(a)–(c), typical high-resolution O1s, C1s, and N1s spectra of woven PLA, before and after ammonia-based plasma treatment, are shown. The modification of woven PLA by ammonia-based plasma treatment does not lead to large changes in the C1s and O1s spectra; however, changes in the N1s spectra are clearly observed. The atomic compositions of untreated woven PLA are 26.4% O, 72.94% C, and 0.66% N. After plasma treatment, the O content was slightly increased to 28.54%, C content was decreased to 65.54%, and N content was enhanced to 5.92%. In Figure 1(a), the typical high-resolution O1s spectrums of untreated and ammonia-based plasma-modified woven PLA are shown. The O1s spectrum was deconvolved into two component Gaussian peaks: peak (1) at 530.91 eV, attributed to C-O, and peak (2) at 529.54 eV, considered to correspond to the C=O group.⁶ Quantitative analysis indicated that ammonia-based plasma modification leads to an increase in the C=O component, from 59.3% in the untreated woven PLA to 80.39% in the ammonia-based plasma-modified woven PLA.⁴¹ It is well known that oxygen incorporation during ammonia-based plasma treatment results from radical formation on the substrate surface and subsequent reaction with oxygen on exposure to the atmosphere.⁴²
Figure 1.
X-ray photoelectron spectra of untreated woven poly (lactic acid) (PLA) and plasma- treated woven PLA: (a) O1s peak; (b) C1s peak; (c) N1s peak (—untreated woven PLA, ··· plasma-treated woven PLA).

Figure 1(b) shows the C1s spectra of untreated and ammonia-based plasma-modified woven PLA. The C1s spectrums were deconvolved into three component Gaussian peaks: peak (1) at 286.32 eV, attributed to the C-N group; peak (2) at 284.29 eV, considered as corresponding to C=O; and peak (3) at 282.44 eV, attributed to the C-H group.^21,43 Following ammonia-based plasma treatment, the C-N group at 286.32 eV was increased from 16.06% to 21.17%.⁴³ The N1s spectrums of untreated and ammonia-based plasma-modified woven PLA are shown in Figure 1(c). When PLA was treated with ammonia-based plasma, the N1s level showed a significant increase due to the binding of nitrogen to the woven PLA surface, as a result of which its atomic composition increased from 0.66% to 5.92%. Following treatment, nitrogen was incorporated into the surface, whereas the relative percentage of oxygen increased slightly and the relative percentage of carbon decreased by 7.4%. A moderate increase of O1s and a significant increase of N1s were observed due to a reaction between free radicals generated by plasma treatment and ammonia in the air.^41,43 The likely source of this was the ionization of oxygen in the vacuum, which may have led to a slight decrease in the effectiveness of surface activity for carbon.⁶

The data indicate that ammonia-based plasma treatment is effective for the introduction of amino groups onto the woven PLA surface, which enables incorporation of new nitrogen functional groups with greater effectiveness. Consequently, the incorporation of nitrogen onto the woven PLA surface was obtained by plasma treatment, which directly resulted in the modification of the surface.

Crosslinking of woven PLA by GA

The reactivity of GA is affected by pH value, with most GA-mediated crosslinking occurring within a slightly alkaline pH range.⁹ In order to determine the effect of pH of the GA solution, change in trypsin activity at different pH values (ranging from pH 7.0 to 11.0) of GA was evaluated. Figure 2(a) shows the relative and recovered activity of immobilized trypsin depending on the pH of GA solution during crosslinking. As illustrated in Figure 2(a), the relative activity of trypsin was gradually increased as the pH value changed from neutral to alkaline conditions, and then the highest activity was observed at pH 10.0. Also, the activity recovered was increased with a shift of pH value at the same time. However, when the pH of GA solutions was shifted to a strong alkaline condition, such as pH 11.0, the activity of immobilized trypsin was sharply declined. Generally, for higher pH values, the amount of aldehyde groups bound to the support rapidly increases as a result of polymerization of GA due to aldol condensation.⁴⁴ In particular, during the reaction between the aldehyde groups of GA and enzyme, a Schiff base on the support would be formed, which facilitates stable binding of the protein on the support. However, the Schiff base is unstable under acidic conditions or strong alkaline conditions, thus resulting in the decrease of enzymatic activity.
Figure 2.
Effect of various (a) pH values, (b) concentrations of glutaraldehyde (GA), and (c) treatment times on trypsin immobilization (GA crosslinking conditions: (a) different pH values, GA 3%(v/v), 25℃, and 120 min; (b) different GA concentrations, pH 10.0, 25℃, and 120 min; (c) different treatment times, pH 10.0, GA 2% (v/v), and 25℃).

That is, the immobilized trypsin showed approximately 98% of relative activity and 70% of recovered activity at pH 10.0 of GA solution used. Therefore, a pH of 10.0 was chosen as the optimal pH value for the crosslinking with trypsin on modified woven PA. Since GA is a highly reactive substance, it is important to determine suitable GA concentration for immobilization trypsin. Figure 2(b) shows the effects of GA concentration on immobilization of trypsin, ranging from 0.1% to 15% of GA concentration. As described in Figure 2(b), the relative activity of immobilized trypsin was slightly increased by increasing GA conditions, and then the highest relative activity of 95% was observed at 5% (v/v) of GA. However, the relative activity was retained at equilibrium state for above 5% of GA with no further increase in activity. In particular, when 2% GA concentration was used, a significant increase to approximately 92% in the relative activity was exhibited, which showed higher recovered activity than the sample with 5% GA. These results are because low concentrations of GA are unable to form sufficient crosslinkages with the enzyme, whereas higher concentrations may result in a denaturation of the enzyme structure. Thus, extensive crosslinking has occurred, reducing the accessibility of the substrate and the catalytic activity of the enzyme.⁹ That is, the conditions of 2% GA concentration, which showed approximately 92% of the relative activity, has a slight difference of less than 0.5%, compared with 5% GA. Taking into account both the highest increases of activity and chemical efficiency of treatment, 2% GA concentration was considered to be the optimum condition for immobilization of trypsin.

Figure 2(c) shows the effect of crosslinking time on the trypsin immobilization. Data demonstrated changes in enzymatic activity with various times of crosslinking, ranging from 30 to 1440 min. As seen in Figure 2(c), the relative activity of immobilized trypsin was found to increase with increasing crosslinking time. In particular, the maximum relative activity was observed for 180 min, which shows that approximately 100% of the activity was recovered also. However, the activity declined gradually for prolonged times longer than 180 min. This decrease in activity resulted from the extensive aggregation of the support due to increased GA polymerization during excessive crosslinking times.⁹ Moreover, the loss of activity may be attributable to diffusion limitation of the reaction within the amine groups of the enzyme.⁴⁵ For any given treatment time, the number of enzymatic amine groups reacting with GA molecules were limited.⁴⁶ Consequently, the activity of immobilized trypsin has been decreased despite the longer crosslinking times.

Covalent immobilization of trypsin

Effect of pH on the activity of the immobilized enzyme

The pH of the buffer solution is a significant factor during enzyme immobilization. The pH of the solution affects the active sites of functional groups on the surface of the support, thereby influencing the stable activity of the immobilized enzyme.⁴⁴ Therefore, it is important to establish suitable pH conditions for immobilization of trypsin on modified woven PLA.⁴⁴ Figure 3 shows the effects of pH conditions on the immobilization of trypsin. In order to optimize the pH conditions for trypsin immobilization, the enzymatic activities of immobilized trypsin were evaluated at different pH values, ranging from 7.0 to 10.0. The activity of immobilized trypsin on modified woven PLA was found to increase gradually with increasing pH value. However, over a pH of 8.5 (alkaline conditions), the activity was found to decline drastically. This may be because trypsin remains stably anchored to the support under a pH of 8.5 as compared with alkaline pH conditions.¹⁶ In addition, it has been reported that trypsin molecules may restrict the activation of the support at high pH values, such as pH 10.0.⁴⁷
Figure 3.
Effect of pH on the immobilization of trypsin onto glutaraldehyde-crosslinked woven poly (lactic acid) (trypsin immobilization conditions: different pH values, 25℃, 30 min, trypsin concentration 8% (owf)).

Therefore, a pH of 8.5 was found to result in the highest activity, and this was therefore chosen as the optimal pH value for the immobilization of trypsin on modified woven PLA.

Effects of temperature during immobilization

Figure 4 shows the influence of temperature on the trypsin immobilization process. Data indicated that changes in enzymatic activities of immobilized trypsin on modified woven PLA were observed at different temperatures, ranging from 20℃ to 45℃. The maximum activities of immobilized trypsin were observed at temperatures between 20℃ and 25℃; practically approximately 100% of relative activity was shown at 20℃, whereas at above 25℃ the activity of immobilized trypsin sharply declined. When the trypsin was immobilized at 45℃, the activity was recovered at less than 5%. This is attributable to the occurrence of a rapid reaction between the enzyme molecules and aldehyde groups, resulting in the formation of multiple linkages at higher temperatures.⁴⁸ These multiple linkages result in the distortion and denaturation of the enzyme, thereby decreasing its relative activity.
Figure 4.
Effect of temperature on immobilization of trypsin onto glutaraldehyde -crosslinked woven poly (lactic acid) (trypsin immobilization conditions: different temperature, pH 8.5, trypsin concentration 8% (owf), 30 min).

Taking into account both the maximum recovered and relative activities of immobilized trypsin and energy efficiency of treatment, 25℃ was considered the optimum temperature for immobilization of trypsin.

Effect of concentration of trypsin during immobilization

The changes in enzyme activity with different trypsin concentrations are shown in Figure 5. The effect of trypsin concentration (ranging from 2% to 14% (owf)) on enzyme activities was investigated. The enzymatic activities of immobilized trypsin were increased linearly until 10% (owf) of trypsin was used. In particular, the recovered activity was significantly increased from 14.5% to 55%. On the other hand, the relative activity then showed equilibrium for trypsin concentrations above 10% (owf). The increase in enzymatic activity of trypsin at concentrations below 10% was mainly due to the availability of large quantities of aldehyde groups on the PLA surface for the attachment of enzyme molecules.⁷ Moreover, at the initial state, enzyme molecules formed earlier tend to act as a spacer-arm on the surface of the support, enabling an increase in relative activity without inactivation.⁷
Figure 5.
Effect of trypsin concentration on the immobilization of trypsin onto glutaraldehyde-crosslinked woven poly (lactic acid) (trypsin immobilization conditions: different trypsin concentration, pH 8.5, 25℃, 30 min).

In addition, the formation of the spacer-arm decreases the steric hindrance; hence, enzyme activity was found to increase.⁴⁹ However, at trypsin concentrations above 10% (owf), aldehyde groups on the surface are fully bound to the enzyme molecules on the surface layer. As a consequence of this exhaustion of binding sites on the support, the bound trypsin molecules reach their saturation level. As a result, the relative activity reaches equilibrium state over 10% (owf) of concentration.

Therefore, a trypsin concentration of 10% (owf) was selected as the optimum concentration for immobilization of the enzyme.

Effect of immobilization time

Figure 6 presents the effects of immobilization time on the enzymatic activities of immobilized trypsin on modified woven PLA. The activity of immobilized trypsin was found to increase with increasing immobilization time, particularly for the first 30 min, for which the highest recovered activity was observed. However, the activity was found to decline slightly for immobilization times longer than 30 min. This decrease in enzymatic activity of immobilized trypsin resulted from the exhaustion of available aldehyde groups during this initial period.⁴⁸ Moreover, excessive reaction times resulted in the formation of multiple linkages between the enzyme and support, leading to distortion and denaturation of the enzyme structure.⁴⁹ Consequently, the relative and recovered activities of immobilized enzyme were reduced with increasing immobilization time.⁴⁹
Figure 6.
Effect of treatment time on immobilization of trypsin onto glutaraldehyde-crosslinked woven poly (lactic acid) (trypsin immobilization conditions: different treatment times, pH 8.5, trypsin concentration 8% (owf), 25℃).

Thus, it was concluded that the desired immobilization time for trypsin is 30 min, at which the highest recovered activity and relative activity were observed to be approximately 98%. Therefore, the optimum immobilization conditions are determined as 30 min at 25℃, pH of 8.5, and concentration of 10% (owf) of trypsin.

Under the optimum conditions, the amount of immobilized trypsin on woven PLA and specific activity was observed to be approximately 0.28 mg/mg and 3.763 U/mg, respectively (Table 3).
Table 3.
The amount of immobilized trypsin and specific activity of the free and the immobilized trypsin

Enzyme Amount of immobilized trypsin (mg/mg) Specific activity (U/mg trypsin)

Free – 5.375

Immobilized 0.28 3.763

Stability assessment

pH stability

The pH profiles of the free and the immobilized trypsin are shown in Figures 7(a) and (b), respectively. The effect of pH level on the free and immobilized trypsin stability was investigated at different incubation times of 60, 120, and 240 min, at 25℃. The free trypsin (Figure 7(a)) was observed with an optimum pH of 9.0 and the immobilized trypsin (Figure 7(b)), with an optimum pH of 10.0. This pH shift may be caused by the attraction of hydroxyl ions by carbonyl groups that provided a local hydroxyl ions aggregation, leading to higher pH of the enzyme microenvironment than that of the bulk solution and the shift of the optimal pH toward alkaline conditions.⁵⁰ Moreover, the immobilization methods preserved the enzyme activity in a wider pH range and presented higher pH stability than the free ones. For example, at pH 4.0–6.0 and 12.0–13.0, which are strong acid and alkaline conditions, the free trypsin was almost inactivated.
Figure 7.
Effect of pH on the stability profile of the free and immobilized trypsin at 25℃: (a) free trypsin; (b) immobilized trypsin (▪: 60 min, •: 120 min, ▴: 240 min).

On the other hand, the immobilized trypsin was still retained at approximately 35% at pH 6.0, and up to 20% at pH 12.0–13.0, despite the prolonged incubation time. In addition, accordingly over time, the activity of free trypsin showed substantial reduction 240 min later. However, the immobilized trypsin showed a small loss of its activity after a long incubation. These data indicated that immobilization of the enzyme resulted in the protection of its active sites against denaturation due to changes in pH value.

Therefore, the optimum pH range of the immobilized trypsin was significantly broader with a long period incubation, indicating that the stability of trypsin was improved under various pH conditions by immobilization on modified woven PLA in comparison with free trypsin.

Thermal stability

Figure 8 indicates the thermal stability of free and immobilized trypsin at different temperature conditions, evaluated by incubating the enzyme in wide temperatures ranging from 25℃ to 65℃ for 60 min. As shown in Figure 8, the immobilized trypsin was more stable than the free enzyme, particularly when the temperature exceeded 40℃. At temperatures over 45℃, the free enzyme lost more than 65% of the initial activity, whereas the immobilized enzyme retained over 60% of its initial activity. A similar effect was observed at 55℃, in that immobilized trypsin retained 15% of its initial activity after 60 min incubation, whereas the free enzyme showed less than 10% of its initial activity. The increased stability of immobilized trypsin over 40℃ was due to the restricted conformational flexibility of the enzyme molecules following immobilization with multi-point attachments on the support.^5,51 In addition, the covalently immobilized enzyme on the support was protected against denaturation and conformational changes due to changes in temperature.^45,51 Therefore, it may be concluded that the immobilization process enhanced the thermal stability of trypsin.
Figure 8.
Thermal stability of free and immobilized trypsin (•: immobilized trypsin, ○: free trypsin).

The changes of thermal stability profiles of the free and immobilized trypsin were investigated at 25℃ (Figure 9(a)) and 50℃ (Figure 9(b)) for 120 min. As shown in Figures 9(a) and (b), the relative activity of both free and immobilized trypsin were gradually decreased with the prolongation time. At 25℃ (Figure 9(a)), both free and immobilized trypsin retained over 70% of its initial activity until 60 min of incubation. However, at the incubation time of 120 min, the relative activity of free trypsin sharply decreased to 50% of its initial activity, whereas the activity of immobilized trypsin remained at approximately 70%. At 50℃ (Figure 9(b)), the immobilized trypsin retained approximately 50% of its initial activity at the end of 120 min incubation, while the activity of free trypsin was significantly reduced to just 10% of initial activity.
Figure 9.
Thermal stability profile of the free and immobilized trypsin at 25℃ (a) and 50℃ (b) (•: immobilized trypsin, ○: free trypsin).

From these results, it can be concluded that thermal stability and resistance to high heat during a long period of trypsin were enhanced by the immobilization process.^44,52 This is because the immobilization strengthens the enzyme molecules’ structural rigidity; hence, the limitation of conformational degeneration might allow trypsin activity to be retained when the immobilized trypsin is exposed to heat with prolongation time.³⁹ In addition, the covalently immobilized enzyme on the support is more capable of resisting against denaturation and conformational changes during extended exposure from heat.⁵²

According to the above results, the immobilization process used in this study was efficient for enhancing the thermal stability of trypsin.

Storage stability

One of the most important factors to be considered in enzyme immobilization is storage stability, as this greatly affects activity retention during long periods of storage.^33,49 The stability of free and immobilized trypsin on modified woven PLA was evaluated during 20 days of storage at 4℃. As seen in Figure 10, the free trypsin lost its activity with prolonged storage times. After 20 days, the relative activity of free trypsin was less than 40%, while the immobilized trypsin retained over 55% of its initial activity. The retention of stable activity despite the long duration of storage is attributed to covalent binding between the trypsin molecules and fiber surfaces, which protects the enzyme from autolysis and structural denaturation.⁵¹ As a result, the covalently immobilized enzyme was shown to maintain its activity during prolonged storage, without large reduction in residual activity.^52,53 Generally, the catalytic activity of the soluble state of the enzyme is rapidly reduced during long periods of storage and it is difficult to recover.
Figure 10.
Storage stability of free and immobilized trypsin (•: immobilized trypsin, ○: free trypsin).

The above data demonstrate that immobilization on modified woven PLA confers significant advantages in terms of retention of stability of the enzyme over longer storage times.

Reusability

The durability of the immobilized enzyme after repeated catalysis is important to enable continuous use of the enzyme in industrial applications.⁵⁴ In this experiment, the reusability of immobilized trypsin on modified PLA was examined by repeated catalysis of the substrate, followed by washing with buffer solution (pH 8.5). Figure 11 shows the effect of repetitive catalysis on the activity of the immobilized trypsin. As seen in Figure 11, immobilized trypsin was found to retain residual activity of 40% and 30% after 5 and 15 reaction cycles, respectively. The loss of activity may be attributed to the inactivation of the enzyme caused by the denaturation of protein and desorption of protein molecules from the surface of the support during repetitive catalysis.^54,55
Figure 11.
Reusability of immobilized trypsin on woven poly (lactic acid).

These data demonstrate that the immobilized trypsin on modified PLA retained over 20% residual activity after 15 consecutive reactions, thereby confirming the reusability of trypsin. Therefore, immobilized trypsin may be valuable for the set-up of a biocatalyst for continuous industrial application.⁵¹

Surface characterization

Hydrophilic properties

The hydrophilic properties were evaluated by assessing surface wettability. To this end, WCA and surface tension were measured. The WCA and the surface tension of modified PLA samples with different treatments are indicated in Figure 12 and Table 4, respectively. As shown Figure 12, when the WCA of woven PLA was changed from 120° to 85° by ammonia-based plasma treatment, the surface tension of PLA was increased to 49.225 mN/m (Table. 4). The lower WCA indicated that a large number of hydrophilic molecules were grafted onto the PLA surface as a result of ammonia-based plasma processing. The processing of the polymer surface by plasma treatment generally results in the simultaneous introduction of predominantly nitrogen-containing moieties, such as amine and amide groups, as well as oxygen moieties, which are exposed to the air.⁶ In contrast, when PLA was crosslinked with GA, its WCA was increased to 141°, with no change after trypsin immobilization. In addition, the surface tension declined to below 1 mN/m as a result of GA crosslinking and trypsin immobilization. This may result from the increased hydrophobicity of the PLA surface due to crosslinking with GA, as the more hydrophobic amino groups become blocked with aliphatic chains on treatment with GA.^8,55 Moreover, the increased surface roughness of PLA (Figure 13(d)) due to immobilized trypsin increased the hydrophobicity of the PLA surface.
Figure 12.
Water contact angle (WCA) of (a) poly (lactic acid) (PLA), (b) plasma-treated PLA, (c) glutaraldehyde-crosslinked PLA, and (d) trypsin-immobilized PLA.

Figure 13.
Scanning electron micrographs of (a) poly (lactic acid) (PLA), (b) plasma-treated PLA, (c) glutaraldehyde-crosslinked, and (d) trypsin-immobilized woven PLA.

Table 4.
Changes in surface tension on woven PLA surface

Sample Surface tension (mN/m)

PLA 22.85

NH₃ plasma-treated PLA 49.335

GA-crosslinked PLA 0.11

Trypsin-immobilized PLA 0.1

GA: glutaraldehyde.

Therefore, plasma treatment of PLA was found to affect its hydrophilic properties by increasing the hydrophobicity of the PLA surface upon GA crosslinking and trypsin immobilization.

Morphology by scanning electron microscopy

Figure 13 shows the morphology of modified woven PLA under various treatment conditions. The morphologies of the woven PLA after ammonia-based plasma treatment, GA crosslinking, and trypsin immobilization were examined. As shown in Figure 13, compared with Figure 13(a), no surface change was observed following ammonia-based plasma treatment (Figure 13(b)).

Thus, the PLA surface was maintained under the same conditions as the smooth surface after plasma treatment. However, as indicated in Figure 13(c), irregular ripple marks of a round shape were produced after GA crosslinking. The roughness of the PLA surface was significantly increased following trypsin immobilization, as observed in Figure 13(d). Rounded structures and irregular small cracks, which were likely to be protein aggregates, were observed.⁸ These may have resulted from the recrystallization of trypsin molecules during immobilization.

Therefore, the PLA surface has been shown to provide a large contact area for the immobilization of large amounts of enzyme.⁸ These results confirm that the woven PLA surface was modified by GA crosslinking and trypsin immobilization, thereby enhancing its roughness.

Conclusion

The purpose of this study was to develop a woven PLA-based immobilization support and to establish the optimum immobilization conditions. To this end, ammonia-based plasma processing was performed on woven PLA in order to introduce amine groups, and trypsin was covalently immobilized on the modified PLA surface by GA crosslinking. Several parameters potentially influencing enzyme activity during immobilization, such as pH, treatment temperature, time, and concentrations of both GA and enzyme, were investigated and an optimum range was established.

XPS analysis showed that the proportion of N1s in PLA increased from 0.66% to 5.92% following ammonia-based plasma treatment, demonstrating that an abundance of amine groups had been introduced onto the PLA surface. For successful amination of woven PLA, the optimal crosslinking conditions were found to be as follows: pH of 10.0, with 2% (v/v) of GA concentration, for 180 min. The most favorable immobilization conditions were as follows: 30 min at 25℃, pH of 8.5, and concentration of 10% (owf) of trypsin. Under the optimum treatment conditions, the amount of bound trypsin on woven PLA was observed as approximately 0.28 mg/mg and the specific activity was 3.763 U/mg. The pH and thermal stability of immobilized trypsin were found to show improvement under these optimum immobilization conditions. In addition, after 20 days of storage, the immobilized trypsin was found to have retained about 55% of the initial activity. Furthermore, the immobilized trypsin showed the potential for reuse over 15 cycles. The results of WCA showed that plasma treatment affected the hydrophilic properties of woven PLA; however, it was found that the PLA surface was more hydrophobic after treatment due to GA-induced crosslinking and trypsin immobilization. SEM analysis showed improved roughness of woven PLA after GA-induced crosslinking and trypsin immobilization.

In conclusion, woven PLA shows high potential as a support material for enzyme immobilization. This work may provide a basis for the development of woven PLA-based immobilization supports by plasma processing.

Composition (%)	Yarn count (denier)	Weight (g/m²)	Density (yarn/inch)	Thickness (mm)
PLA 100	75	56.83	90 × 68	0.132

Enzyme	Source	Activity	Form	Manufacturer
Trypsin (EC 3.4.21.4)	Porcine pancreas	1000–2000 BAEE units^a/mg solid	Lyophilized powder	Sigma Chemicals Co.

Enzyme	Amount of immobilized trypsin (mg/mg)	Specific activity (U/mg trypsin)
Free	–	5.375
Immobilized	0.28	3.763

Sample	Surface tension (mN/m)
PLA	22.85
NH₃ plasma-treated PLA	49.335
GA-crosslinked PLA	0.11
Trypsin-immobilized PLA	0.1

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-2013R1A6A3A01025361).

Abbreviations

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