Surface functionalized natural inorganic nanorod for highly efficient cellulase immobilization

Abstract

An attapulgite@chitosan (ATP@CS) nanocomposite was prepared by coating chitosan onto naturally needle-like nanoscale attapulgite clay. The nanocomposite was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis. Cellulase was covalently immobilized on this ATP@CS support using glutaraldehyde activation method and compared to (3-aminopropyl) triethoxysilane modified ATP (ATP–APTES) support. The amount of cellulase on the ATP@CS and ATP–APTES was 88.3 and 76.5 mg g⁻¹, respectively. The reusability and effects of pH and temperature on activity were studied. The ATP@CS immobilized cellulase exhibited higher pH and thermal stability as well as good reusability compared to the ATP–APTES immobilized cellulase and the free cellulase. The immobilized cellulase samples were also utilized to hydrolyze wheat straw to evaluate their potentially practical applications.

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1. Introduction

Enzymes are biocatalysts with the ability to effectively catalyze specific reactions under mild conditions, which have been widely used in the biofuel industry, medicine, food industry, environmental protection and industrial synthesis. Given the relatively high cost, difficulty in recovery and reuse, short shelf-life time and low thermal stability, the immobilization of enzymes on supports is an ideal solution to the large-scale industrial application of enzymes.^1–7 Clays with nanoscale possess unique physicochemical properties, such as large specific surface area, reactive –OH groups on their surface, non-toxicity, high thermal stability, low cost, good mechanical strength and antimicrobial properties, which make them emerge as preferable supports for enzyme immobilization.^8–10 Attapulgite (ATP, also known as palygorskite) is a naturally needle-like one-dimensional nanoscale clay composed of magnesium–aluminum silicate. ATP has a unique fibrous morphology, easy availability and high surface area, which is a suitable support to immobilize a variety of biomolecules including drugs, enzymes, and DNA.¹¹ However, the lack of functional groups and inorganic property of ATP generally result in low loading amount and weak bonding between biomolecule and ATP.¹² So surface modification of ATP is particularly important in improving enzyme immobilization. Nevertheless, the small size and fine hydrophilicity make it be easily dispersed in aqueous under modest conditions such as mechanical stirring or ultrasonic,¹³ which is prone to modification by biocompatible material with abundant functional groups.

Many researchers have chosen chitosan as a modifier to enhance immobilization capability of clay for the biomolecules since its numerous functional groups (amino, hydroxyl and hydroxymethyl groups) in addition to biodegradable and biocompatible.^14–20 Especially, the pK_a value of amino group in chitosan is ∼6.5, meaning that chitosan is soluble and protonated in an acid solution, which is favorable to combine positively charged chitosan with negatively charged clays via hydrogen bonding and electrostatic interactions.^19,21,22 Considering the effectiveness and facility of the modification, the ATP@CS nanocomposite was employed as a support for enzyme immobilization in this work.

Not only the support but also the immobilization technique plays an important role in the properties of immobilized enzyme.²³ Covalent bonding was selected to immobilize enzyme taking into account the prevention of enzyme loss from the support.²⁴ Biomass is widely available carbon source, and great efforts have been made to produce of ethanol from biomass, which is recognized as a “green” alternative to diminishing fossil fuels.^25–27 Hydrolysis of cellulose in biomass to fermentable reducing sugars which are further fermented to produce ethanol, is a critical step. Cellulase, composing of three components, is a key enzyme to effectively hydrolyze cellulose to produce glucose, which was studied as a model enzyme to investigate the enzyme-immobilizing properties of ATP@CS nanocomposite. The ATP without modification by chitosan was also used as a support to immobilize cellulase via covalent bonding for comparison purposes.

2. Experimental

2.1 Materials

Acremonium cellulase was purchased from Meiji Seika Pharma Co., Ltd. (Tokyo, Japan). ATP nanoclay was provided by BASF (Florham Park, New Jersey). Chitosan, carboxy methyl cellulose sodium salt (CMC), acetic acid, and sodium hydroxide (NaOH) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). (3-Aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GDA, 50%, v/v) were bought from Aladdin Industrial Corporation (Shanghai, China).

2.2 Immobilization of cellulase

0.25 g chitosan was completely dissolved in 50 mL of acetic acid solution (1%, v/v) under continuous stirring. 2 g ATP nanoclay was dispersed for 1 h in 50 mL of distilled water using an ultrasonic bath. Then the prepared chitosan solution was added to ATP suspension and the mixture was stirred for 30 min to ensure homogeneous dispersion of ATP in chitosan solution. After that, 100 mL of NaOH solution (1 M) was added to the mixture and ATP@CS nanocomposites were obtained. The obtained product was separated by centrifugation and washed by distilled water until the pH of the wash was neutral to immobilize cellulase via reported GDA activation method with some modifications.²⁸ Typically, the obtained support was added to 60 mL of 2.5% GDA and the reaction was performed at 30 °C for 2 h so as to activate the support by offering –CHO groups. The GDA activated support was separated by centrifugation and washed by distilled water three times to remove unreacted GDA. Afterwards the support was mixed with 100 mL of 6 mg mL⁻¹ cellulase solution (dissolved in 0.1 M acetate buffer, pH 5.0) with gentle stirring at 30 °C for 2 h. The final immobilized cellulase was collected by centrifugal separation and washed by acetate buffer to remove free cellulase.

ATP without coating by chitosan which was served as a support for cellulase immobilization was compared to the ATP@CS support. In order to covalently immobilize cellulase via GDA activation method as described above, ATP was firstly functionalized with –NH₂ groups by silanization. A mixture of 20 g ATP, 20 mL of APTES and 200 mL of toluene was refluxed for 24 h. The resulting product was washed with ethanol and dried under vacuum at 60 °C for 48 h to obtain –NH₂ functionalized ATP (ATP–APTES). 5.0 g ATP–APTES support was added to 141 mL of water and dispersed under ultrasonic for 1 h. Subsequently, 9 mL of 50% GDA solution was added and the reaction was performed at 30 °C for 10 h. The following cellulase immobilization were carried out according to the above method.

2.3 Characterization

The morphologies of the nanoparticles were characterized by scanning electron microscope (SEM, Hitachi S-4300, Japan). Fourier transform infrared (FT-IR, Impact 400, Nicolet, USA) spectroscopy was performed to confirm the chemical structures of the nanoparticles. Thermogravimetric analysis (TGA) was carried out with a TA Instrument Q500 (USA) at a heating rate of 10 °C min⁻¹ in N₂ atmosphere.

2.4 Determination of cellulase loading and activity

Bradford protein assay method was applied to determine the cellulase concentration,²⁹ and the cellulase loading on support was calculated from the following equation:

Cellulase loading (mg g⁻¹) = [(C₀V₀ − C_iV_i)/C₀V₀] × 100%

where C₀ and C_i is the protein concentration before and after immobilization, respectively. V is the volume of the cellulase solution. W is the weight of added support.

The activity of cellulase was measured according to the method of IUPAC with some modifications.³⁰ 0.5 mL of 1% CMC solution (dissolved in 0.1 M acetate buffer, pH 5.0) was used as substrate and hydrolyzed by 0.5 mL of cellulase solution at the appropriate dilution. The hydrolysis process was carried out at 50 °C for 30 min. The amount of glucose produced during the process was measured in a UV/vis spectrophotometer at 540 nm using DNS as reagent and used to evaluate the enzyme activity. One International Unit (IU) of cellulase activity is defined as the amount of cellulase, which produces 1 μmol glucose per minute at 50 °C and pH 5.0.

2.5 Reusability assay

The immobilized cellulase was subjected to a hydrolysis reaction with 1% CMC for 0.5 h. After separation by centrifugation, the immobilized cellulase was introduced into solution containing fresh substrate. The activity was determined following each 0.5 h recycle for 10 cycles and used to evaluate the reusability.

3. Results and discussion

3.1 Characterization of supports

The photographs and SEM micrographs of natural ATP clay and ATP@CS support are shown in Fig. 1. The fibrous ATP clay with an average diameter of 20 nm and length of 0.5–1 μm which showed smooth surface could be uniformly dispersed in water and form a stable colloidal network under ultrasonic treatment. Chitosan containing a large number of functional groups was selected as a modifier for functionalization of ATP clay. In an acid solution, chitosan was dissolved and positively charged which showed strong interactions with negatively charged ATP via electrostatic interactions and hydrogen bonding. Flocculation occurred when the pH of the suspension was adjusted to alkaline, and consequently chitosan coated ATP (ATP@CS) which had rough surface was formed. The flocculent structure of ATP@CS support is particularly suitable for enzyme immobilization since its large surface area, plenty of functional groups and open channels. For –NH₂ groups functionalized ATP by silanization (ATP–APTES), the morphology did not change a lot compared with original ATP clay.

Fig. 1 Photographs (a) of natural ATP clay and ATP@CS support and SEM micrographs (b) of natural ATP clay, ATP@CS and ATP–APTES.

TGA of natural ATP clay, ATP@CS, chitosan and silanized ATP–APTES were performed to calculate the content of chitosan coated on the surface of ATP. The ATP clay showed four decomposition stages over the entire testing range, corresponding to the loss of adsorbed water, loss of hydration water, loss of coordination water, and dihydroxylation, respectively.³¹ The overall weight loss of chitosan was 68.4% resulting by the release of adsorbed water and decomposition of polysaccharide units.³² The amount of chitosan in the ATP@CS composite estimated from the TGA curves of natural ATP clay, ATP@CS and chitosan was ∼11.6%, which was consistent with the mass feeding ratio of chitosan to ATP (m_cs : m_ATP = 0.25 : 2). The content of organic matter in silanized ATP–APTES was about 8.0% (Fig. 2).

Fig. 2 TGA curves of ATP, ATP@CS, chitosan and ATP–APTES.

The FT-IR spectra of ATP, ATP@CS and ATP–APTES are given in Fig. 3. The sharp peak at 3614 and 910 cm⁻¹ in the spectrum of ATP clay was associated with the –OH stretching vibration and deformation in Al–Al–OH groups, respectively. The peaks at 1032 and 983 cm⁻¹ were attributed to stretching vibration of the Si–O bond. The peak at 1192 cm⁻¹ was reported as the characteristic of ATP that does not appear in any other clay silicate.³³ For ATP@CS sample, the peaks at 2921 and 2856 cm⁻¹ for C–H stretching vibrations, 1384 cm⁻¹ for C–H bending vibration, 1640 cm⁻¹ for amide I, 1259 cm⁻¹ for C–O group, and 1035 cm⁻¹ for saccharide structure (overlapping the peak for ATP at 1032 cm⁻¹) appeared.^34,35 For APTES silanized ATP, the characteristic C–H stretching vibrations at 2930 and 2870 cm⁻¹, C–H bending vibration at 1384 cm⁻¹, and –NH₂ bending vibration peak at 1565 cm⁻¹ arose, indicating the ATP was successfully functionalized with –NH₂ groups.

Fig. 3 FT-IR spectra of ATP, ATP@CS and ATP–APTES.

3.2 Immobilization of cellulase

Positively charged chitosan was coated on the surface of negatively charged ATP clay via electrostatic interactions and hydrogen bonding. Then cellulase was immobilized onto the resulting ATP@CS supports via covalent bonding. More specifically, the amino groups of chitosan were converted to aldehyde groups via GDA to covalently bond cellulase by Schiff base linkage. The schematic representation for the preparation of ATP@CS support and cellulase immobilization is shown in Fig. 4(a). To compare the immobilization efficiency of ATP@CS supports, ATP was also modified by APTES to functionalize with amino groups to immobilize cellulase using the same GDA activation method. The process is given in Fig. 4(b). The amount of cellulase immobilized onto ATP@CS supports was found to be 88.3 mg g⁻¹ with an activity of 25.7 IU mg⁻¹ cellulase; whereas the value for ATP–APTES supports was 76.5 mg g⁻¹ with an activity of 18.8 IU mg⁻¹ cellulase. The loading capacity was comparable with other reported works that also used nanomaterials as solid support. G. Bayramoglu et al. prepared clay–poly(glycidyl methacrylate) composite support for immobilization of cellulase and the maximum immobilization capacity was found to be 43.4 mg g⁻¹ K⁻¹.³⁶ Khoshnevisan reported that the adsorption capacity of cellulase onto commercial superparamagnetic nanoparticles reached 31 mg g⁻¹.³⁷ The difference in loading capacity for these two kinds of support was caused by the different morphologies. ATP@CS support that presented flocculent structure had large surface area and open channels, which was conducive to the diffusion of cellulase. Meanwhile, the plenty of functional groups attached with ATP@CS support, especially for aldehyde groups, could improve the immobilization capacity. Conversely, APTES modified ATP showed dense surface morphology which may prevent the diffusion of cellulase, and only the aldehyde groups presented on the surface of ATP–APTES support could react with cellulase.

Fig. 4 Schematic representation for the immobilization of cellulase on ATP@CS support (a) and ATP–APTES support (b).

3.3 Properties of the immobilized cellulase

Effect of pH and temperature on cellulase activity. Since pH and temperature could influence the hydrolysis performance of cellulase, the activity under different pH and temperature were tested. Fig. 5(a) reflects the effect of pH on the activity in the pH range of 3–6 at 50 °C. The optimum pH was found to be 4 for ATP@CS immobilized cellulase and ATP–APTES immobilized cellulase, which was the same as free cellulase. ATP@CS immobilized cellulase exhibited more excellent activity in the pH range of 3–5 in comparison with free cellulase and higher activity over the entire pH range than ATP–APTES immobilized cellulase, which benefited from the chitosan coating. As mentioned above, the flocculent structure of ATP@CS could provide large surface area to contact with substrate CMC and, more importantly, a high number of active sites of cellulase immobilized onto the ATP@CS may expose to the substrate. Moreover, in an acid medium chitosan coated on the surface of ATP was positively charged. This not only can improve dispersion stability due to the electrostatic repulsive force, but also can expand open channels to promote the diffusion of substrate CMC. In addition, numerous functional groups such as amino, hydroxymethyl and hydroxyl groups of chitosan had strong interactions with functional groups of CMC (carboxymethyl and hydroxyl groups), leading to higher concentration of CMC at the surface of ATP@CS support than its concentration in the bulk solution. For these reasons, the ATP@CS immobilized cellulase prepared in this work showed higher activity.

Fig. 5 Effect of pH (a) and temperature (b) on activity.

The activity of free and the immobilized cellulase samples was studied from 40 to 70 °C at pH 4 (Fig. 5(b)). We can know from the test results that the maximum activity of free and both immobilized cellulase samples were found to be at 60 °C. It is worth noting that both immobilized cellulase samples had greater activity over the entire testing temperature range than free cellulase. In particular, ATP@CS immobilized cellulase showed the best heat resistance due to the protective chitosan.

Reusability. The immobilized cellulase samples were recycled 10 times and activity assay was carried out after each recycle. The activity of the first cycle was assumed as 100% and the change in residual activity was shown in Fig. 6. The activity of both immobilized cellulase samples decreased with number of times of reuse. However, ATP@CS immobilized cellulase exhibited more remarkable reusability than ATP–APTES immobilized cellulase. The activity of ATP@CS immobilized cellulase kept above 47% of the original after 10 cycles, which was double that of ATP–APTES immobilized cellulase (21%). The decrease in activity may be caused by end-product inhibition, protein denaturation and loss of constituents of the cellulase complex.³⁸

Fig. 6 Reusability of immobilized cellulase.

Application. From the viewpoint of practical applications, both immobilized cellulase samples were used to enzymatic hydrolysis of wheat straw. Wheat straw was treated by a cutting mill and sieved to collect the one with the size below 100 μm. The mass ratio of cellulase to wheat straw was 3 : 100 and the reaction was performed for 24 h (60 °C, pH 4). The glucose productivity during 24 h were determined to evaluate the application performance of the immobilized cellulase. Free cellulase could produce 90.3 mg glucose per g wheat straw in the present experiment, whereas ATP@CS immobilized cellulase and ATP–APTES immobilized cellulase produced 76.6 and 11.9 mg glucose per g wheat straw, respectively. The results indicate that ATP@CS immobilized cellulase has the potential in practical applications (Fig. 7).

Fig. 7 Hydrolysis of wheat straw for 24 h (60 °C, pH 4).

4. Conclusion

Clay@chitosan nanocomposite, consisting of chitosan coated on ATP, was used to covalently immobilize enzyme. The unique flocculent structure of ATP@CS nanocomposite made it a promising support for immobilization. The amount of cellulase on ATP@CS support was found to be as high as 88.3 mg g⁻¹, with an activity of 25.7 IU mg⁻¹ cellulase. Compared to cellulase immobilized on silane coupling agent modified ATP and free cellulase, ATP@CS immobilized cellulase showed higher activity over a broader pH and temperature range as well as better reusability. The immobilized cellulase was explored to enzymatic hydrolyze wheat straw. It could produce 76.6 mg glucose per gram of wheat straw which was ∼85% of production for free cellulase (90.3 mg glucose per g wheat straw). These results indicate that cellulase successfully immobilized on ATP@CS support has good potential for practical application.

Acknowledgements

The work was supported by the National Natural Science Foundation of China (51303035); the Foundation of Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University (2015BCE005).

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