polyaniline composite

Materials Science and Engineering C 31 (2011) 252–257

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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c

Trypsin immobilization on discs of polyvinyl alcohol glutaraldehyde/polyaniline composite Samantha Salomão Caramori a,b,⁎, Flaviana Naves de Faria b, Miriam Pereira Viana b, Kátia Flávia Fernandes c, Luiz Bezerra Carvalho Jr. a a b c

Laboratório de Imunopatologia Keizo Asami, UFPE, 50670-901, Recife-PE, Brazil Unidade Universitária de Ciências Exatas e Tecnológicas, UEG, Rodovia BR 153, km 98, 75001-970, Anápolis-GO, Brazil Laboratório de Química de Proteínas, ICB 2, UFG, 74001-970, Goiânia-GO, Brazil

a r t i c l e

i n f o

Article history: Received 4 March 2010 Received in revised form 10 June 2010 Accepted 6 September 2010 Available online 15 September 2010 Keywords: Enzyme immobilization Casein hydrolysis Trypsin polyaniline Polyvinyl alcohol

a b s t r a c t Discs of polyvinyl alcohol cross-linked with glutaraldehyde (PVAG) were synthesized and covered with polyaniline activated with glutaraldehyde (PANIG). Trypsin was covalently immobilized on this composite yielding a preparation containing 21.1 units per disc. The FT-IR spectra of the discs showed bands of PVA (3300 cm−1, 2930 cm−1 and 1440 cm−1) and PANI (1594 cm−1 and 1100 cm−1). The best immobilization conditions were: trypsin concentration at 0.2 mg mL−1, pH 7.6 and 60 min of incubation, similar to polyaniline–trypsin systems reported in the literature. The PVAG–PANIG–trypsin derivative showed an optimal pH and an optimal temperature of 7.0 and 35 °C, respectively. Hydrolysis of casein showed variations in the size of the products, revealing differences between the immobilized enzyme and the mechanism catalyzed by the native enzyme. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The use of immobilized trypsin (E.C. 3.4.21.4.) for cleavage of proteins has several advantages compared to its soluble form. For instance, immobilized trypsin reactors have been integrated into separation systems such as reversed-phase liquid chromatography or capillary electrophoresis, prior to mass spectrometry analysis, for proteome studies [1]. Furthermore, immobilized trypsin derivatives have been used for continuous hydrolysis of casein [2], affinity chromatography [3], cutaneous dressing [4] and many different applications. In general, immobilized protein systems have the advantage of protein structural stabilization and active site preservation, allowing access of the substrate to the catalysis environment. The chemical nature of the support material must be further investigated, and the choice of this material should take into consideration factors such as hydrophobicity/hydrophilicity, surface area, pore diameter, reactive superficial groups, synthesis route, costs and ease of handling. Additionally, when considering trypsin as a target enzyme for immobilization, the choice of the support material must take into account the voluminous substrate (proteins), and the hydrophilicity of the catalysis environment, and hence, the microenvironment of the immobilized enzyme.

⁎ Corresponding author. Laboratório de Imunopatologia Keizo Asami, UFPE, 50670-901, Recife-PE, Brazil. Tel.: +55 62 3328 1160; fax: +55 62 3328 1177. E-mail address: [email protected] (S.S. Caramori). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.09.005

In our laboratory, trypsin was directly immobilized on the surface of PANI previously treated with glutaraldehyde [5]. However, this enzymatic derivative was a powder and presented difficulties in separating it from the reaction mixture. Attempts to overcome the separation step were also conducted in our research group, culminating in the preparation of PET–PANIG composite as strips with immobilized trypsin, which could be manually removed from the reaction medium. However, this material presented low protein loading [6]. PVA (polyvinyl alcohol) is a highly hydrophilic, biocompatible material that has been used in our laboratory for protein immobilization. Discs of PVA cross-linked with glutaraldehyde (PVAG) were synthesized under acid catalysis (H2SO4). The antigen F1 purified from Yersinia pestis and soluble adult Schistosoma mansoni antigen preparation (SWAP) were then covalently linked on this modified polymer [7,8]. An enzymelinked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human and human schistosomiasis based on these derivatives [8,9]. Filter paper discs (5-mm diameter) were also plasticized with PVAG and used for antigen immobilization [9]. In spite of the bio-friendly properties of PVA, the amounts of protein effectively immobilized in this support alone were quite low. On the other hand, polyaniline (PANI) has been used for the immobilization of different proteins and enzymes, such as antigen [10], peroxidase [11,12], glucoamylase [13] and β-galactosidase [14], with high loading capacity. Recently, Purcena et al. [5] showed that it is possible to apply polyaniline–trypsin on proteomic applications, since the authors obtained a stable and reusable preparation and could preserve the original trypsin catalyses characteristics. Despite

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their advantages, chemically synthesized polyaniline preparations result in powder materials and require centrifugation and/or filtration steps to be separated. So, combining the properties of PVA and PANI to produce a composite could result in a support material for trypsin immobilization with properties advantageous to the modern applications of this enzyme. In this work discs of PVAG were covered with polyaniline and activated with glutaraldehyde (PANIG). This composite (PVAG– PANIG) was employed as a matrix for trypsin immobilization and some properties of the derivative were investigated.

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producing 1 μmol of p-nitrophenol per minute and using 9.1 as the p-nitroaniline molar absorption coefficient. 2.6. PVAG–PANIG–trypsin characterization 2.6.1. pH profile The effect of the pH on the activity of the native and the immobilized trypsin on BApNA prepared in 0.1 M of sodium phosphate buffer at pH varying from 6.0 to 8.6 was established as in 2.5.

2.1. Materials

2.6.2. Temperature The effect of temperature on the activity of the native and the immobilized trypsin on BApNA was established in 0.1 M sodium phosphate buffer, pH 7.0, at temperatures ranging from 30 to 60 °C.

Trypsin (EC 3.4.21.4), BApNA (Nα-benzoyl-DL-arginine-p-nitroanilide), Casein and Sephadex G-50 (medium) were purchased from Sigma-Aldrich (St. Louis, EUA). PVA, dimethyl sulphoxide and ammonium persulphate were obtained from Vetec Química Fina Ltda. (Rio de Janeiro, Brazil). Aniline (previously distilled) was obtained from Merck (Germany) and all other chemicals were of analytical grade.

2.6.3. Thermal stability The native (0.1 mL, 9 U) and the PVAG–PANIG–trypsin discs were incubated in a water bath at temperatures ranging from 30 °C to 70 °C during time intervals from 25 to 125 min. The enzymes were then left at 25 °C for 30 min, before their activities were determined according to item 2.5.

2.2. Synthesis of PVAG–PANIG

2.6.4. Shelf life The PVAG–PANIG–trypsin discs were stored in three stabilizer solutions, at 4 °C: 1) 0.1 mM glycine pH 3.6; 2) 0.1 mM glycine pH 3.6 containing 0.6 mM CaCl2 and 3) 1% (w/v) poly (ethylene glycol) in 0.1 mM sodium phosphate buffer, pH 7.6. The remaining activity of immobilized trypsin was evaluated during 46 days, by the measurement of the PVAG–PANIG–trypsin activity, followed by washing with 0.1 mol L−1 phosphate buffer pH 7.6 and storage again in the stabilizer solution at 4 °C.

2. Experimental

Discs of PVAG were synthesized by heating an aqueous solution of 2% (w/v) of PVA (12 mL) up to 65 °C and 25% (v/v) of glutaraldehyde (2 mL) was added. This mixture was vigorously stirred for 50 min and aliquots (20 μL) were introduced into microplate wells containing 3 M HCl (120 μL) and kept for 24 h at 25 °C to allow polymerization. A number of 240 discs (15 mg each) in three microplates were synthesized by using the 14.0 mL of mixture. The discs of PVAG were treated with 0.61 M ammonium persulphate prepared in 2.0 M HCl during 30 min. The discs were then immersed in 0.44 M aniline and kept for 60 min (PANI covering). PVAG–PANI discs were washed extensively with 2.0 M HCl. The activation step was carried out by incubating 1.0 g of PVAG–PANI discs at 60 °C with 2.5% (v/v) glutaraldehyde aqueous solution [11]. Finally, PVAG–PANIG discs were washed five times with 0.1 M sodium phosphate buffer, pH 7.6, and stored in the same buffer at 4 °C until use. 2.3. PVAG–PANIG characterization PVAG–PANIG was analyzed by FT-IR spectra (Hartman & Braun— MB Series—Michelson), as KBr pellets, between 500 and 4000 cm−1. 2.4. The best conditions for the trypsin immobilization on PVAG–PANIG One PVAG–PANIG disc was incubated with 1.0 mL of trypsin solution in concentrations varying from 0.05 to 0.5 mg mL−1 during 60 min, under orbital agitation, at 4 °C. The PVAG–PANIG–trypsin disc was then incubated with 0.1 M glycine to block any remaining reactive groups, and finally the derivative was washed extensively with 1 M NaCl. The following two additional experiments were carried out: 1) one PVAG–PANIG disc was incubated with 1.0 mL of trypsin solution (0.1 mg mL−1) at a time interval varying from 30 to 120 min and 2) with 1.0 mL of trypsin solution (0.1 mg mL−1) prepared in different solutions of 0.1 M sodium phosphate buffer at pH ranging from 6.0 to 8.6 for 60 min. Blocking and washings were carried out as described above. The enzyme activity of all preparations was then determined. 2.5. Measurement of enzyme activity Trypsin activity was measured (triplicate) according to Alencar et al. [15]. One enzymatic unit was defined as the amount capable of

2.6.5. Casein hydrolysis The soluble (9 U) and immobilized trypsin (one disc) preparations were incubated with casein (1 mg) prepared in a 0.1 M phosphate buffer, pH 7.0, containing 0.6 M CaCl2 at 37 °C (final volume of 1 mL) while stirred. Aliquots were withdrawn at time intervals and immediately added to trichloroacetic acid to precipitate the unhydrolyzed casein. The soluble aromatic aminoacids in the supernatants were then measured at 280 nm. Aliquots were also withdrawn before at the 8th hour of incubation and introduced into a Sephadex G-50 column (2× 10 cm). Gel filtration was then carried out and fractions were collected for the absorbance determination at 280 nm. Previously, the column was calibrated by using the following markers: chymotrypsinogen (25 kDa), insulin (5.6 kDa), ribonuclease A (3.7 kDa) and aspartame (294.31 Da). 3. Results and discussion To overcome the low protein loading of the last composites we produced before [6], discs of PVAG covered with PANI were proposed as a matrix for trypsin immobilization. The analysis of this material showed its macroporous nature and hence, higher surface area compared to the PET–PANIG composite. Fig. 1A shows typical discs of PVAG (yellow, transparent) and covered with PANI (dark green, opaque) and also their probable chemical structures. Fig. 1B presents scanning electronic microscopy of PVAG–PANIG, showing some aspects of its morphology and macroporous structure. Trypsin was covalently fixed onto PANI via glutaraldehyde [5,6] and also onto the PVAG through the free carbonyl groups [16]. The disc shape of the PVAG was provided by microplate flat wells. However, other shapes can be attained, e.g., surface coating such as filter paper [9]. The infrared spectrum of PVA displayed band at 3400 cm−1 relative to (–OH) stretching, the typical PVA band at 2900 cm−1

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A

Polyvinyl alcohol glutaraldehyde network

B

Fig. 1. Structure of PVAG–PANIG discs. Discs of PVAG (yellow) and PVAG–PANIG (dark green) and their chemical structures (A) and scanning electron microscopy (B) of PVAG–PANIG. Magnifications: 20× and 8000×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

relative to –CH stretching, and the band at 800 cm−1 related to high sindiotacticity. The spectrum of PVAG–PANIG composite presented the same characteristic bands of PVA and those related to PANI, mainly the bands at 1593 cm−1 and 1500 cm−1 relative to the benzoid/quinoid structures and that at 1107 cm−1 relative to doping level of PANI (Table 1). The best immobilization parameters are presented in Fig. 2. These results show the best protein concentration, incubation time and pH value: 0.2 mg mL−1 of trypsin (Fig. 2A), 60 min (Fig. 2B) and 7.6 (Fig. 2C), respectively. There is a limit to the protein load per disc (0.04 mg). As previously reported [5,6], a longer incubation time yielded a preparation with a less active enzyme preparation, probably, due to trypsin being hydrolyzed by other soluble trypsin molecules and inactive enzyme (peptides) being fixed onto the composite. Anything beyond the optimum immobilization pH, either in the acidic

or in the basic range, resulted in less efficiency. In such optimized conditions the load obtained was 57 U of trypsin per cm2, which represents double that obtained with PET-PANIG–trypsin (25 U/cm2), using the same method [15]. In other words, the higher hydrophilic

Table 1 Chemical groups identified in PVA and PVAG–PANIG infrared spectra. Wavenumber (cm−1)

Chemical group PVA

PVAG–PANIG

3400 2900 1593/1500 1107 800

–OH stretching –CH stretching – – Sindiotacticity

–OH stretching –CH stretching Benzoid quinoid structures of PANI PANI doping level –

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A Fixed protein [mg]; Retained activity U/10; Specific activity U/mg protein/200

0.15

(3) (1) 0.10

0.05

(2)

0 0.05

0

0.1

0.2

0.15

Offered trypsin [mg]

C 12

10

Activity (U/disc)

Activity (U/disc)

B 12 8 6 4 2 0

10 8 6 4 2 0

0

25

50

75

100

125

6,0

6,5

7,0

Immobilization incubation time [min]

7,5

8,0

8,5

9,0

pH

Fig. 2. Best immobilization conditions. A—Offered trypsin versus (1) fixed protein (mg), (2) retained activity (U/10) and (3) specific activity (U/mg protein/200). Calculations of the parameters in panel A were made to adjust the values to the same scale. B—immobilization time and C—immobilization pH.

A 100 80

Activity (%)

60 40 20 0 5,5

6

6,5

7

7,5

8

8,5

9

pH

B 100 80

Activity (%)

nature of PVAG–PANIG compared to PET-PANIG combined with the macroporous morphology of this material can promote a better microenvironment for these enzyme molecules, yielding to higher activity on the surface of this material. Fig. 3 shows the effect of the pH and temperature on the BApNA hydrolysis catalyzed by the PVAG–PANIG–trypsin disc. The best pH (7.6) was the same as that found for the native trypsin (Fig. 3A), and that found by Purcena et al. [5] and Caramori et al. [6], in which trypsin was immobilized on PANI and PET–PANI, respectively. However, in this work the immobilized trypsin showed higher activity in both the acidic and basic range compared with the native trypsin. This phenomenon is frequently associated with proton donator or acceptor groups in the support material. These groups act by adjusting the pH in the surrounding environment of the immobilized enzyme. In the present case, hydroxyl groups from PVAG and amino groups from PANIG provide an environment able to simultaneously donate and accept protons from bulk solution. The optimum temperature (35 °C) for the activity of the PVAG–PANIG– trypsin disc was also equal to that of the native enzyme (Fig. 3B). Fig. 4 shows the thermal stability and shelf life of the native enzyme and the immobilized enzyme. The PVAG–PANIG–trypsin incubated at 40 °C retained the initial activity for 50 min whereas the soluble enzyme lost half of its initial activity (Fig. 4A). Furthermore, incubation at 70 °C inactivated the soluble enzyme after 50 min but the PVAG–PANIG–trypsin retained 20% of its initial activity (Fig. 4A). Increased thermal stability of immobilized trypsin preparations has been reported in the literature [17–19]. Fig. 4B shows the residual activity measured when PVAG–PANIG–trypsin was stored in the presence of three stabilizer solutions at 4 °C (shelf life). The best result was obtained when 0.1 mM Glycine containing 0.6 mM CaCl2 was used, resulting in 100% activity during 37 days. This result presented

60 40 20 0 25

35

45

55

65

˚

Temperature C Fig. 3. Influence of pH (A) and temperature (B) on the soluble (○) and PVAG–PANIG– trypsin (●).

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A

A 120 (1)

100

Absorbance [280 nm]

Retention of activity (%)

(1) 0.3

80 60

(2)

40

(3) 0.1

(3)

(4)

20

(2)

0.2

0

0 0

20

40

60

80

100

120

0

140

60

120

180

Time (min)

B

300

360

420

480

B 100

0.08

(1)

80 60

(2)

40 20

(3) 0 0

10

20

30

40

50

Absorbance [280 nm]

Residual activity (%)

240

Time [min]

0.06

0.04

0.02

Time (days) Fig. 4. A: Thermal stability the soluble and PVAG–PANIG–trypsin. Curve (1)—PVAG– PANIG–trypsin at 40 °C; curve (2)—soluble trypsin; curve (3)—PVAG–PANIG–trypsin; and curve (4)—soluble trypsin. B: Shelf life of PVAG–PANIG–trypsin at 4 °C stored in glycine+ CaCl2 (1), glycine (2) and polyethyleneglycol (3).

better performance if compared to the systems PANI–glutaraldehyde– trypsin and PET–PANIG–trypsin [5,6]. Finally, Fig. 5 presents the action of the soluble and the PVAG– PANIG–trypsin on casein. Full hydrolysis was almost achieved at the 1st hour by the soluble trypsin catalysis (Fig. 5A). Casein hydrolysis also occurred in the immobilized enzyme but at a slower rate, and the full hydrolysis occurred only after about 8 h. The profile of the products released was also different: the maximum absorbance values for the soluble and immobilized enzyme were 0.3 and 0.2, respectively. Fig. 5B presents the molecular gel filtration of the hydrolisates obtained from the action of the soluble and immobilized preparations for 8 h compared with the casein (column fractionation range of 1.5 kDa–30 kDa). Three observations should be addressed: 1) casein showed peptides ranging from the 1st to 43rd fractions; 2) the hydrolisate obtained by the soluble trypsin action presented peptides from the 1st to the 60th fractions and 3) the hydrolisate obtained by the PVAG–PANIG–trypsin action was obtained from the 10th to the 60th fractions. Smaller peptides were produced by the soluble and immobilized trypsin from the 44th fraction. Under the PVAG–PANIG– trypsin action peptides from casein sizing 1st–10th fractions disappeared whereas higher amounts of small peptides appeared (45th–60th fractions; 100–800 kDa). The soluble enzyme action also released peptides in this range but in lower amounts. These findings suggest a different mechanism of action for the two enzyme forms. Probably, the degree of freedom reduction of the immobilized trypsin acting on the macromolecule substrate (casein) and the microenvironment influence provided by the matrix (PVAG–PANIG) allowed a different action mechanism compared to the soluble enzyme. Considering the proteolysis mechanism occurring in this microenvironment (PVAG–PANIG–trypsin), hydrophilic groups from PVA

0 0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

Fraction Fig. 5. A: Action of the soluble (1) and PVAG–PANIG–trypsin (2 and 3) on casein. Curve (3) refers to casein hydrolysis by reused PVAG–PANIG–trypsin. B: Gel filtration (B) of casein (black line) and the hydrolisates produced by the soluble (red line) and PVAG– PANIG–trypsin (blue line) after 8 h of hydrolysis.

can attract the oligopeptides that resulted from casein hydrolysis and can keep them within the immobilized enzyme longer. This phenomenon can cause a delay in the trypsin catalysis, allowing it to produce smaller peptides. Moreover, with trypsin molecules immobilized on the same surface casein hydrolysis can be facilitated, because the casein oligopeptides will be accessible to many trypsin molecules, resulting in more fragmentation and consequently, in the disappearance of the high molecular mass peptides (1st–10th fractions).

4. Conclusion From these results one can conclude that discs of PVAG-PANI can be used for covalent trypsin immobilization. This derivative showed an optimal pH and an optimal temperature similar to those reported for the native enzyme. On the other hand, PVAG–PANIG–trypsin presented superior characteristics compared to similar materials synthesized for the same purpose, such as active enzyme loaded, pH profile, thermal stability and storage stability. Finally, this system showed the capability to catalyze casein hydrolysis, differentially to the native trypsin. Because of these characteristics, PVAG–PANIG– trypsin can be used for peptide production studies and especially for proteomic applications, considering the possibility of total trypsin removing from the bulk and the immobilized trypsin’s shelf life presented in this work.

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Acknowledgements The Pró-Reitoria de Pesquisa of the Universidade Estadual de Goiás and the Brazilian Agency CNPq financially supported this work. References [1] [2] [3] [4]

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