Horseradish peroxidase and chitosan: Activation, immobilization and comparative results

International Journal of Biological Macromolecules 60 (2013) 295–300

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Horseradish peroxidase and chitosan: Activation, immobilization and comparative results Saleh A. Mohamed a,b,∗ , Abdulrahman L. Al-Malki a , Taha A. Kumosani a , Reda M. El-Shishtawy c,d a

Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia Molecular Biology Department, National Research Center, Dokki, Cairo, Egypt c Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia d Dyeing, Printing and Textile Auxiliaries Department, Textile Research Division, National Research Center, Dokki, Cairo, Egypt b

a r t i c l e

i n f o

Article history: Received 3 May 2013 Received in revised form 4 June 2013 Accepted 6 June 2013 Available online 12 June 2013 Keywords: Peroxidase Chitosan Immobilization

a b s t r a c t Recently, horseradish peroxidase (HRP) was immobilized on activated wool and we envisioned that the use of chitosan would be interesting instead of wool owing to its simple chemical structure, abundant nature and biodegradability. In this work, HRP was immobilized on chitosan crosslinked with cyanuric chloride. FT-IR spectroscopy and scanning electron microscopy were used to characterize immobilized HRP. The number of ten reuses of immobilized HRP has been detected. The pH was shifted from 5.5 for soluble HRP to 5.0 for immobilized enzyme. The soluble HRP had an optimum temperature of 30 ◦ C, which was shifted to 35 ◦ C for immobilized enzyme. The soluble HRP and immobilized HRP were thermal stable up to 35 and 45 ◦ C, respectively. The apparent kinetic constant values (Km ) of soluble HRP and chitosan–HRP were 35 mM and 40 mM for guaiacol and 2.73 mM and 5.7 mM for H2 O2 , respectively. Immobilization of HRP partially protected them from metal ions compared to soluble enzyme. The chitosan–HRP was remarkably more stable against urea, Triton X-100 and organic solvents. Chitosan–HRP exhibited large number of reuses and more resistance to harmful compounds compared with wool–HRP. On the basis of results obtained in the present study, chitosan–HRP could be employed in bioremediation application. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Biocatalysts are increasingly being employed because of their high selectivity and potential as a greener alternative to chemical catalysts. Therefore, interest for enzymatic processes is over growing. The soluble enzymes makes their uses for large-scale relatively costly and their recoveries are difficult [1]. Numerous efforts have been devoted to the development of insoluble immobilized enzymes for various applications. There are several benefits of using immobilized enzymes as the reusability of enzyme with the reducing of the production cost, stable and reusable analytical devices for analytical and medical applications, purification of proteins and enzymes and as effective microdevices for controlled release of protein drugs [2–5]. Natural polymers, alginate, agarose, chitin, and chitosan are used as carrier materials in immobilization of enzymes. Chitosan

∗ Corresponding author at: Biochemistry Department, Faculty of Science, King Abdulaziz University, Box 80203, Jeddah 21589, Saudi Arabia. Tel.: +966 543395119; fax: +966 26952288. E-mail address: [email protected] (S.A. Mohamed). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.06.003

and chitin possesses distinct chemical and biological properties. In its linear polyglucosamine chains of high molecular weight, chitosan has reactive amino and hydroxyl groups, which are potentially capable of being crosslinked with different substances [6,7]. Along with unique biological properties include biocompatibility, biodegradability to harmless products, nontoxicity, remarkable affinity to proteins. Crucially, bio- and biodegradable polymers chitosan materials are eco-friendly, safe for humans and the natural environment [8,9]. Chitosan has been used as a support for immobilization of various enzymes [10–12]. Horseradish peroxidase (HRP) is one of the most extensively studied enzymes because of its growing number of applications. HRP has been used for removal of phenols from wastewater [13], organic syntheses [14], and applications for analytical purposes [15]. Applications of HRP have been developed because of its high activity, simple detection of products, relative stability, ease of immobilization and the stability of the immobilized preparations [15]. HRP has been immobilized with chitosan using distinct methods: crosslinking with gels [16], ␤-cyclodextrin [17], metallic nanoparticles [18] and polyethyl acrylate [19]. Cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) has been used as a coupling reagent to cross link enzymes to supports [20,21].

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The chlorine atoms in the molecule react with nucleophillic groups (thiol, amino, imino and hydroxyl functions) to form stable linkages. The first chlorine reacts readily at low temperatures and pH, the second at 25–40 ◦ C in alkaline pH and the third will react mainly with thiol groups at high temperature and pH [22]. The variations in the reactivity of the 1st and 2nd chlorine atoms and near inactivity of the 3rd atom make cyanuric chloride an efficient heterobifunctional coupling reagent for linking hydroxyl, amino and thiol groups. Recently, we immobilized HRP on wool activated with cyanuric chloride [23]. In continuation of our interest for enzyme immobilization it was necessary to select chitosan instead of wool. It is known that this typology of solids is not easy to recover. However, chitosan has reactive amino and hydroxyl groups, which are potentially capable of being crosslinked with different substances. In this study, an effort has been made to immobilize HRP on chitosan crosslinked with cyanuric chloride. The number of enzyme reuse and the effect of some compounds on chitosan–HRP were compared with wool–HRP [23]. 2. Materials and methods 2.1. Materials Chitosan (with specification: 85% deacetylated, medium molecular weight and medium viscosity), cyanuric chloride and all other reagent grade chemicals were purchased from Sigma Aldrich and were used as received.

units which represented 26,805 units/mg protein) made in 50 mM sodium acetate buffer, pH 5.5 or Tris–HCl buffer, pH 7.0 at room temperature during overnight. The solid modified chitosan beads immobilized with HRP were filtered and washed. The immobilization efficiency% was calculated from the following formula Immobilization efficiency% =

activity of immobilized enzyme initial activity of soluble enzyme ×100

The activity of enzyme has been also measured in the washing buffer. 2.6. ATR-FTIR analysis The attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra for chitosan samples were performed on a PerkinElmer spectrum 100 FT-IR spectrometer. 2.7. SEM analysis Scanning electron microscopy (SEM) images of chitosan samples were examined with a scanning electron microscope Quanta FEG 450, FEI, Amsterdam, Netherlands. The microscope was operated at an accelerating voltage of 10, 20 kV. The samples were placed on the double side carbon tape on Al-Stub and sputtered with a 20 nm thick gold layer (JEOL JFC-1600 Auto Fine Coater).

2.2. Horseradish peroxidase

2.8. Reusability of immobilized HRP

Horseradish peroxidase (HRP) was previously purified from horseradish cv. Balady with specific activity 26,805 units/mg protein [24].

After each assay the immobilized HRP preparation was taken out, washed with 50 mM sodium acetate buffer, pH 5.5 and stored overnight at 4 ◦ C. The immobilized enzyme recovered by this procedure was used repeatedly. The activity determined for the first time was considered as control (100%) for the calculation of remaining percentage activity after each use.

2.3. Peroxidase assay Peroxidase activity was assayed according to [25]. The reaction mixture contains in 1 ml: 8 mM H2 O2 , 40 mM guaiacol, 50 mM sodium acetate buffer, pH 5.5 and soluble HRP or immobilized HRP. The change of absorbance at 470 nm due to guaiacol oxidation was followed at 30 s intervals. One unit of peroxidase activity is defined as the amount of enzyme which increases the O.D. 1.0 per min under standard assay conditions. The relative activity% was Relative activity (%) =

activity × 100 maximum activity

2.9. Enzyme characterization Estimates of optimal temperature and pH for soluble HRP and immobilized HRP were made by using a temperature ranged from 10 ◦ C to 70 ◦ C and a pH ranged from 4.0 to 8.5. The thermal stability was investigated by measuring the residual activity of soluble HRP and immobilized HRP after 15 min of incubation at different temperatures. The Km values were determined from Lineweaver–Burk plots by using different concentrations of guaiacol and H2 O2 as substrates.

2.4. Activation of chitosan with cyanuric chloride An ice-cooled solution of cyanuric chloride (2–8%, w/v) in 100 ml of acetone–water mixture (1:1) was prepared. Chitosan (2 g) was added into this solution and left with shaking for 30 min at 0 ◦ C. Sodium bicarbonate solution (10%, w/v; 100 ml) was drop wisely added to the above reaction mixture while shaking within 30 min at 0 ◦ C. The reaction mixture was further kept under shaking and at 0 ◦ C overnight. The chitosan sample was removed from the shaker bath and washed several times with acetone, water and acetone, and dried in ventilated hood and kept in stored in a plastic bag under refrigeration until enzyme immobilization [22]. 2.5. Immobilization procedure Enzyme immobilization was performed by end over end at 90 rpm onto the activated chitosan using a solution of HRP (2000

2.10. Effect of metal ions The effects of various metal ions on enzyme activity of soluble HRP and immobilized HRP were determined by pre-incubating the enzyme with 2 mM metal ions for 15 min and then assaying the enzyme activity. The activity in absence of metal ions is taken as 100%. 2.11. Effect of urea, organic solvents and Triton X-100 The soluble HRP and immobilized HRP were incubated with urea or Triton X-100 or isopropanol or butanol or dioxan for 1 h at 37 ◦ C. The enzyme activity of soluble HRP and immobilized HRP was determined by assaying the enzyme in the presence of these compounds. Activity of enzyme without exposures to these compounds was taken as control (100%).

S.A. Mohamed et al. / International Journal of Biological Macromolecules 60 (2013) 295–300 Table 1 The effect of cyanuric chloride percentage and pH on the immobilization efficiency of HRP. Cyanuric chloride (%)

Immobilization efficiency (%) pH 5.5

2 4 6 8

15 25 60 33

± ± ± ±

0.7 1.8 2.7 2.1

pH 7.0 8 20 42 18

± ± ± ±

0.3 0.8 2.1 1.1

Each value represents the mean of three experiments ±S.E.

3. Results and discussion The success of the immobilization of enzymes on chitosan crosslinked with cyanuric chloride may be attributed to: (a) the chlorine atoms in the cyanuric chloride react with nucleophillic groups (thiol, amino, imino and hydroxyl functions) to form stable linkages, and (b) chitosan has reactive amino and hydroxyl groups, which are potentially capable of being crosslinked with different substances. De Lime et al. [21] reported that, in polyphenol oxidase immobilization, the enzyme is entrapped within the interstitial space of the chitosan–cyanuric chloride. Biosensors based on Ag or Au nanoparticles dispersed in ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and laccase from Aspergillus oryzae immobilized in chitosan chemically crosslinked with cyanuric chloride were also constructed [26]. In the present study, the immobilization of HRP on chitosan treated with different concentrations of cyanuric chloride at pH 5.5 or 7.5 was performed. An increase of cyanuric chloride concentration led to an increase in the immobilization efficiency. The maximum immobilization efficiency (60%) was detected at 6% cyanuric chloride and pH 5.5 (Table 1). Twelve percent of initial activity has been detected in washing buffer. The decrease in immobilization efficiency accompanied with increasing cyanuric chloride concentration. The lowering in the retained activity by increasing the cyanuric chloride value could be attributed to the presence of multipoint attachments of the enzyme to the modified chitosan support which of course lead to change in the structure of the enzyme. Similar observations were reported [5,27]. The ATR-FTIR spectra of the chitosan, activated chitosan and chitosan–HRP samples are shown in Fig. 1. All samples exhibit similar absorption bands with observed differences. Typical chitosan characteristic bands at 1543 cm−1 and 1644 cm−1 , corresponding

Fig. 1. FT-IR spectra of the chitosan, the activated chitosan and the chitosan–HRP samples.

297

to the asymmetric bending of NH2 band and the C O stretching (amide I band) were observed. The broad band appearing at 3320 cm−1 is due the overlapped stretching vibration of OH and NH2 together with both inter and intramolecular hydrogen bonding of chitosan macromolecules. Additionally, typical C O C glucoside linkage and C H stretching bands were clearly seen at 1024 cm−1 and 2882 cm−1 , respectively [28,29]. Comparing the spectra of chitosan with those obtained for the activated chitosan and the chitosan–HRP samples, a clear evidence of modification could be observed. These spectra showed a displacement and enlargement of the original chitosan band around 1543 cm−1 related to the N H deformation band, which has become at 1580 cm−1 and 1568 cm−1 for the activated chitosan and the chitosan–HRP, respectively. The enlargement of both bands could also be attributed to the vibration of the triazinyl ring which usually appears at 1540 cm−1 [30,31]. The surface characteristics of the chitosan, the activated chitosan and the immobilized chitosan–HRP samples are shown in Fig. 2. The SEM images show that the apparent compactness of chitosan has become less compact with clear cracks upon activation with cyanuric chloride. Upon enzyme immobilization (chitosan–HRP), some foreign materials together with small fragments dislodged from the cracked surface of chitosan were clearly observed. Due to the high cost of soluble enzymes, the immobilized enzymes should be reused several times. The activity loss during repeated use might be due to the inhibition of enzyme by leaching of enzyme during the repeated washing, a long time contact with high concentrations of H2 O2 and damage of the supports [32–34]. Fig. 3 shows the number of 10 reuses of the immobilized HRP, keeping the enzyme activity at 50%. This value is somewhat better than that reported for cashew gum polysaccharide–horseradish peroxidase, 9 reuses with 50% initial activity [35]. The immobilized chitosan–horseradish peroxidase retained 65.8% residual activity after 6 consecutive operations [19]. Generally, it is possible to change the optimal pH, temperature and kinetic parameters for an immobilized enzyme as a result of immobilized method, support structure and conformation change of enzyme after bounded, making the catalytic site more or less accessible to substrate [36]. The effect of pH on activity of soluble HRP and immobilized HRP was evaluated by incubating these preparations in the buffers of varying pH values ranged from 4.0 to 8.5 (Fig. 4a). The soluble HRP and immobilized HRP exhibited maximum activity at pH values 5.5 and 5.0, respectively, with progressive loss of activity recorded in acidic and alkaline sides for soluble HRP and in alkaline side for the immobilized enzyme. The immobilized HRP retained the most of its activity at acidic pH tested. Similar results were reported, where the pH was shifted from 5.0 for soluble gourd peroxidase to 4.0 for the immobilized enzyme [37]. On the contrary, the immobilized gourd peroxidase preparations showed same pH optima as their soluble counterpart, pH 5.0 [38]. Fig. 4b demonstrates the effect of temperature on the activity of soluble HRP and immobilized HRP. The soluble HRP had an optimum temperature of approximately 30 ◦ C, whereas this temperature was slightly shifted to 35 ◦ C for the immobilized enzyme. The immobilized enzyme retained remarkably higher fraction of catalytic activity at temperature above the temperature optimum as compared to the soluble enzyme. The immobilized HRP exhibited 65% of its activity at 65 ◦ C, while the soluble enzyme retained 35% of its activity at the same temperature. The same temperature optima (40 ◦ C) were detected for the soluble and the immobilized gourd peroxidase preparations [38]. It is well established that thermal exposure initiate unfolding of protein molecules which is followed by irreversible changes due to aggregation and formation of scrambled structures which takes place more in the soluble form as compared to the immobilized enzyme [39]. The thermal stability of

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Fig. 2. SEM images of the chitosan sample (10,000×), the activated chitosan sample (10,000×) and the chitosan–HRP samples with different magnifications (∼10,000× and ∼25,000×).

the soluble HRP and the immobilized HRP is shown in Fig. 4c. The results showed that the soluble HRP and the immobilized HRP were thermally stable up to 35 ◦ C and 45 ◦ C, and retained 45% and 75% of its activity at 65 ◦ C, respectively. Improvement in the thermal stability of the complex may come from a multipoint complexation of peroxidase with the support [17]. A similar suggestion was made to explain why lectin-bound enzymes were more stable than the soluble form [38]. The greatly improved thermal stability of the immobilized enzymes is very important for the practical industrial applications. In which usually high temperatures are needed to enhance the reaction rate, conversion and the solubility of some reactants [34]. The reduction in the affinity of immobilized enzyme for the substrate compared to soluble form could be due to the uneven surface of the support, the high concentration of protein that was immobilized, generating diffusion effects and the change of active

Residual activity %

100 80

site of enzyme after contact with the solid surface of the support [40]. In the present study, compared with the soluble HRP, the affinity of immobilized HRP to the substrates decreased. Fig. 5 shows Km values of the soluble HRP and the chitosan–HRP were 35 mM and 40 mM for guaiacol and 2.73 mM and 5.7 mM for H2 O2 , respectively. Similarly, the Km of modified chitosan–horseradish peroxidase was higher than that of soluble enzyme [19]. Metals induce conformational changes in enzymes, where soluble horseradish peroxidase was remarkably inhibited by heavy metal ions [41,42]. The inhibition of the immobilized HRP by metal ions was low as compared to the soluble enzyme (Table 2). Although Hg2+ caused strong inhibition for the activity of the soluble HRP, the immobilization of enzyme partially protected them from this harmful ion. The stability of the immobilized peroxidases against several metal compounds showed that such preparations could be exploited to treat aromatic pollutants even in the presence of metal ions [43]. The protein unfolding by direct interaction of urea molecule with a peptide backbone via non-covalent interactions contributes to maintenance of protein conformation [44]. The effect of increasTable 2 The effect of metal ions on the soluble HRP and the chitosan–HRP.

60

Metal ion

40 20 0

0

2

4

6

8

Number of repeated reaction Fig. 3. The reusability of the chitosan–HRP.

10

Control Cu2+ Ni2+ Ca2+ Zn2+ Hg2+ Co2+ Pb2+

Relative activity (%) Soluble HRP

Chitosan–HRP

100 100 56 44 68 20 75 62

100 117 64 62 70 74 89 76

S.A. Mohamed et al. / International Journal of Biological Macromolecules 60 (2013) 295–300

(a)

1/units per assay

% Relative activity

Soluble Immobilized 5

Soluble Immobilized

100 80 60 40 20

299

(a)

4 3 2

0

1

2

3

4

5

6

7

8

9

1

pH

(b) Soluble Immobilized

-0.04

-0.02

0.00

0.04

0.06

-1

60

Soluble Immobilized

40 20 0

0

10

20

30

40

50

60

70

Temperature

(c) 100

% Relative activity

0.02

[1/Guaiacol] mM

80

1/units per assay

% Relative activity

100

(b) 3

2

Soluble Immobilized

1

80 60 40

-0.6

-0.3

0

0.0

0.3

0.6

-1

20

[1/H2O2] mM 30

40

50

60

70

Temperature Fig. 4. Optimum pH (a), optimum temperature (b), thermal stability (c) of the soluble HRP and the chitosan–HRP. Each point represents the average of two experiments.

ing concentration of urea (2 and 4 M) on the activity of the soluble HRP and the immobilized HRP is shown in Table 3. The immobilized HRP retained 85% and 65% of its activity at 2 and 4 M urea, while soluble enzyme retained 65% and 56%, respectively. This is in agreement with the resistance of Concanavalin A-pointed gourd peroxidase [37] and chitosan–horseradish peroxidase [45] against 4 M urea. Wastewater from various sites including detergents that can be inhibited the proxidase can be used for treatment of aromatic compounds exist in polluted wastewater. Therefore, the use of the immobilized HRP for the removal of aromatic pollutants from wastewater it becomes necessary to evaluate the stability of enzyme in presence of detergents. The activity of the soluble HRP and the immobilized HRP was experimented with increasing concentrations of Triton X-100 (5% and 10%) (Table 3). The immobilized HRP exhibited 77% and 72% of its activity at 5% and 10% Triton X-100, where 52% and 39% of its activity were detected for soluble enzyme, respectively. Therefore, the chitosan–HRP was markedly more stable against the exposure caused by Triton X-100 compared to the soluble HRP. The immobilized peroxidases

Fig. 5. Lineweaver–Burk plot relating the soluble HRP and the chitosan–HRP reaction velocity to guaiacol (a) and H2 O2 (b) concentrations. Each point represents the average of two experiments.

were reported to be significantly stabilized against denaturation induced by some commonly used detergents [33,37,44]. The activity of the soluble HRP and the immobilized HRP was monitored with increasing isopropanol, dioxan and butanol (5% and 10%) (Table 3). The activity of immobilized HRP enhanced by iso-

Table 3 The effect of chemical compounds on soluble and chitosan–HRP. Chemical

Concentration

Control Urea

– 2M 4M

Relative activity (%) Soluble HRP

Chitosan–HRP

100 65 56

100 85 65

Triton X-100

5% 10%

52 39

77 72

Isopropanol

5% 10%

90 84

130 119

Dioxan

5% 10%

85 76

100 99

Butanol

5% 10%

70 55

90 85

300

S.A. Mohamed et al. / International Journal of Biological Macromolecules 60 (2013) 295–300

Table 4 The number of enzyme reuse and the effect of some compounds on chitosan–HRP and wool–HRP [23]. Parameter

Chitosan–HRP

Wool–HRP

Number of reuse, 50% remaining activity Effect of metals Effect of urea (4 mM) Effect of Triton X-100 (10%) Effect of isopropanol (10%) Effect of dioxan (10%) Effect of butanol (10%)

10

7

Highly resistance 65% remaining activity 72% remaining activity

Highly resistance 60% remaining activity 66% remaining activity

119% remaining activity

100% remaining activity

99% remaining activity 85% remaining activity

62% remaining activity 78% remaining activity

propanol (130% and 119%), where it caused slightly inhibition effect on the activity of soluble HRP. Dioxan had no effect on immobilized HRP and caused slightly inhibition effect on soluble enzyme. Butanol caused inhibitory effect toward immobilized enzyme less than soluble enzyme. The investigation of the stability of enzymes in organic solvents is very important due to the presence of organic solvents in wastewater. It has been reported that several immobilized peroxidases exhibited more resistance to organic solvents [37,45,46]. Concerning the economic of the immobilization methods, Qiu et al. [47] reported that enzyme immobilization is very important for practical applications due to the easy separation of enzymes from its reaction mixture and reusability. The solid support should be biodegradable and renders the enzyme accessible to its cofactors and substrates when used for biosensing and biotransformation. All these advantages allow the economical enhancement of enzymatic processes. This was agreement with the preparation and characterization of HRP immobilized on chitosan crosslinked with cyanuric chloride in the present study. The number of enzyme reuse and the effect of some compounds on chitosan–HRP were compared with wool–HRP, which recently published [23]. Table 4 shows that the number of reuse of chitosan–HRP was 10 compared to 7 for wool–HRP. The two immobilized enzymes appeared highly resistance toward metals. chitosan–HRP exhibited more resistance to urea, Triton X-100 and organic solvents compared with HRP–wool. 4. Conclusion On the basis of results obtained in the present study, it can be concluded that the chitosan–HRP improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent and water–miscible organic solvent, which made it employed in several applications especially bioremediation. Acknowledgments This paper was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. 147/130/1432. The authors, therefore, acknowledge with thanks DSR technical and financial support. References [1] [2] [3] [4]

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