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A reusable multipurpose magnetic nanobiocatalyst for industrial applications
International Journal of Biological Macromolecules 103 (2017) 16–24
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A reusable multipurpose magnetic nanobiocatalyst for industrial applications Mohammad Perwez, Razi Ahmad, Meryam Sardar ∗ Department of Biosciences, Jamia Millia Islamia, 110025 New Delhi, India
a r t i c l e
i n f o
Article history: Received 6 March 2017 Received in revised form 4 May 2017 Accepted 5 May 2017 Available online 8 May 2017
a b s t r a c t A multipurpose magnetic nanobiocatalyst is developed by conjugating Pectinex 3XL (a commercial enzyme containing pectinase, xylanase and cellulase activities) on 3-aminopropyl triethoxysilane activated magnetic nanoparticles. The nanobiocatalyst retained 87% of pectinase, 69% of xylanase and 58% of cellulase activity after conjugation on modified nanoparticles as compared to their soluble counterparts. Thermal stability data at 70 ◦ C showed increase in enzyme stability after conjugation to nanoparticles and the kinetic parameters (Km and Vmax ) remain unaltered after immobilization. The immobilized enzyme system can be successfully used upto 5th cycle after that slight decrease in enzyme activities was observed. The nanobiocatalyst retained high pectinase activities in organic solvents and chemical reagents as compared to free enzymes. DLS data shows that the nanoparticles size increases from 63 nm to 86 nm after immobilization. Atomic Force Microscopy data confirms the deposition of enzymes on the nanoparticles. The nanobiocatalyst was used for the clarification of pine apple and orange juice and was also used for the production of bioethanol. Hydrolysis of pretreated wheat straw produced 1.39 g/l and 1.59 g/l after treatment with free Pectinex 3xL and nanobiocatalyst respectively. The concentration of bioethanol also increases by 1.4 fold as compared to the free enzyme. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hemicellulases hydrolyse the plant cell wall polymer hemicellulose, one of the most abundant polysaccharides in nature. Plant biomass in terms of dry weight comprises of 23% lignin, 40% cellulose and 33% hemicellulose. The potential of plant biomass as a renewable raw material is immense [1]. The judicious use of hemicellulases and cellulases in industries could result in higher yields. These enzymes have many biotechnological applications such as deinking of paper waste, pulp biobleaching, clarification of fruit juices, in baking industry, up gradation of feed, fodder and fibres, and saccharification [2]. Before using these enzymes at industrial scale several criteria have to be fulfilled. In industrial processes the reactions are generally performed at high temperature and with harsh chemical reagents to reduce the crystalline structure of cellulose making it available to cellulases and hemicellulases [3]. Therefore, the properties of these enzymes need to be greatly improved. Immobilization is one such powerful tool for increasing the stability and reusability of the enzymes [4,5]. Different immobilization methods such as covalent, ionic bonding, adsorption,
∗ Corresponding author. E-mail address: [email protected] (M. Sardar). http://dx.doi.org/10.1016/j.ijbiomac.2017.05.029 0141-8130/© 2017 Elsevier B.V. All rights reserved.
entrapment, and encapsulation provide better stability to enzymes. Recently, nanomaterials have emerged as the potential immobilization matrices because of their surface size to volume ratio, large amount of enzyme can be loaded to them [6–10]. Moreover, the diffusion limitations are also minimized when dealing with macromolecular substrates [6,8,11]. Graphene based nanomaterials have also been used as support system for use in immobilizing enzymes [12]. Due to the physiochemical properties like electrostatic and hydrophobic properties, enzymes can easily bind to the graphene based nanomaterial but catalytic activity has been found to be reduced due to which graphene is modified with functional groups and then covalently attached with enzymes which improves its catalytic activity [13]. It can be used for waste water treatment and for industrial applications [13]. Magnetic nanoparticles (MNPs) have been a field of study in recent years due to their applications in pharmacy, biology, diagnostics and biotechnology, etc. [14,15]. The use of MNPs offers many benefits because of their nano-scale size and physical properties [16]. hence, immobilization of bioactive substances such as proteins/enzymes on magnetic iron oxide nanoparticle is preferred [10,11]. Mostly enzyme bound magnetic nanoparticles have been found to be stable towards heat, pH and resistant to denaturation [10]. Moreover, MNPs due to their magnetic properties can be
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separated from the solution and reused in enzyme immobilization systems [9]. In the present work the Pectinex 3XL enzyme that is used in the food processing industry for hydrolysing pectin was covalently conjugated to the surface modified iron oxide nanoparticles. It has been reported earlier by several investigators [17–19] that this enzyme preparation is rich in pectinase, xylanase and cellulase. Thus, a multipurpose magnetic nanobiocatalyst is prepared which can hydrolyse xylan, pectin and cellulose. So, far either cellulase or hemicellulase has been immobilized on iron oxide nanoparticles; in this study we report a co-immobilized enzyme system containing cellulases and hemicellulases activity which enhances its application for biotechnological applications. 2. Materials and methods 2.1. Materials and methods Iron (II, III) Oxide nanopowder (<50 nm by TEM), (3Aminopropyl)triethoxysilane (APTS), Pectinex 3XL and Polygalacturonic acid, all above chemicals were purchased from Sigma Aldrich (USA). Xylan from birchwood was purchased from Sisco Research Laboratories (SRL), Mumbai. Throughout the experiment analytical grade solvents and chemicals were used. 2.2. Determination of pectinase activity Pectinase activity in free and immobilized enzyme was determined using polygalacturonic acid as the substrate according to the method described earlier [20]. One unit of enzyme activity is defined as the amount of enzyme required to produce one mol of galacturonic acid per minute under assay conditions. The amount of galacturonic acid was determined using the dinitrosalicylic acid method [21]. 2.3. Determination of xylanase activity Xylanase activity in free and immobilized enzyme was determined using xylan as the substrate [22]. One unit of enzyme activity is defined as the amount of enzyme required to produce one mol of reducing sugar per minute under assay conditions. The amount of reducing sugar was determined using the dinitrosalicylic acid method [21]. 2.4. Determination of cellulase activity Cellulase activity in free and immobilized enzyme was determined as described earlier [23] using Carboxymethyl cellulose as the substrate. 2.5. Filter paper assay for cellulase activity Cellulase activity in free and immobilized enzyme was determined according to the method described earlier [24]. The reaction mixture was prepared by adding 1 ml of sodium acetate buffer (0.05 M, pH 4.8) in each test tube, 0.5 ml of each enzyme (free and nanobiocatalyst) dilution; 50 mg Whatman No.1 filter paper strip (1.0 × 6.0 cm) was rolled and placed into each test tube. The mixture was incubated at 50 ◦ C for 1 h. 3 ml of DNSA reagent was added to the reaction mixture and the reaction mixture was incubated for 5 min in boiling water. The reaction mixture was cooled till it reaches room temperature and then 20 ml of milli-Q water was added. Finally the absorbance was taken by spectrophotometer at 540 nm.
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2.6. Modification of iron oxide nanoparticles with (3-Aminopropyl)triethoxysilane (APTS) Modification of the Iron oxide nanoparticles with APTS was carried out as described by Campo et al. [25]. APTS (0.306 ml) was added to 0.15 gm of Iron oxide nanoparticles pre-equilibrated with assay buffer and the final volume was made to 15 ml with distilled water. The mixture was kept at 50 ◦ C with constant stirring. After 24 h the nanoparticles were washed extensively with 0.05 M sodium citrate buffer pH 7.4. 2.7. Preparation of nanobiocatalyst Glutaraldehyde (5%) solution in 0.05 M sodium citrate buffer, pH 7.4 was added to the APTS modified nanoparticles and the mixture was stirred for 3 h at 25 ◦ C. The nanoparticles were washed for removing excess glutaraldehyde with assay buffer. Pectinex 3XL enzyme (1 ml containing 55 U of pectinase) was added to the above nanoparticles and left overnight at 4 ◦ C with constant shaking. The enzyme bound nanoparticles were removed by magnet. The nanoparticles were washed with the sodium acetate buffer to remove the loosely bound enzymes. The enzyme activities were calculated in the supernatant and washings. Immobilization efficiency was calculated by varying the enzyme load (25 U–75 U of pectinase) and the immobilization was carried as above. A control was run to check the adsorption of enzymes on the unmodified nanoparticles, for this one ml of enzyme (containing 75 U) was incubated with 0.15 gm of nanoparticles and the final volume was made to 15 ml. The mixture was kept overnight at 4 ◦ C with constant shaking. The unbound enzymes in the supernatant can be easily separated by attracting the bound enzyme with the magnet. The enzyme activities were calculated in the supernatant. A control was run to study the physical adsorption of enzymes on unmodified iron oxide nanoparticles and it was observed that the adsorption of all the three enzymes (pectinase, xylanase and cellulase) on nanoparticles was in the range of 10% only. All the experiments were carried out in the batch mode. Further work was carried out with the nanobiocatalyst showing the best immobilization efficiency of Pectinase i.e 87%. 2.8. Determination of kinetic parameters of enzyme and nanobiocatalyst Km and Vmax values of free enzyme and nanobiocatalyst were determined by measurement of enzyme activities with various concentrations of respective substrates. The kinetic parameters were calculated using Lineweaver-Burk plot [26,27]. 2.9. Thermal stability of enzyme and nanobiocatalyst The stability of enzyme in free and in nanobiocatalyst was studied at 70 ◦ C at different time interval. Free (dissolved in assay buffers) and nanobiocatalyst (suspended in assay buffers) were incubated separately at 70 ◦ C [17]. Proper aliquots of each enzyme sample were taken out at different time interval, the samples were cooled and activities were calculated using their respective substrates. The activity of enzymes at respective optimum temperature was taken as 100%. 2.10. Reusability of the nanobiocatalyst For the first cycle nanobiocatalyst (containing 55U of pectinase) was made to 1.0 ml with the assay buffer and incubated with 0.5 ml of the polygalacturonic acid under shaking condition at 30 ◦ C. After 10 min nanobiocatalyst was removed by magnet and enzyme activity was estimated in the solution. The nanobiocatalyst was
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washed three times with 3 ml of assay buffer. For second cycle the nanobiocatalyst was incubated with 0.5 ml of fresh substrate and the reaction was carried out as before. Similar experiment was performed for xylanase and cellulase activities using xylan and carboxymethyl cellulose as the substrate at 50 ◦ C. 2.11. Effect of organic solvents and chemical reagents on pectinase activity Effect of different organic solvents (DMSO, Toulene, and Chloroform) and chemical reagents [Triton-X 100 (0.1%), Tween 80 (1%), EDTA (0.28%), SDS (1%), and -mercaptoethanol (1%)] on the pectinase activity were examined. Free and nanobiocatalyst was incubated with 50% concentration of organic solvents and different concentration of organic reagents for 1.5 h in 0.05 M sodium acetate buffer at pH 5.0. Residual activity was determined under optimal enzyme assay condition. The activity of the untreated enzyme was considered as control (100%) for calculating percent activity.
Table 1 Optimization of Immobilization of Pectinex 3xL. Enzyme load (U)
25 35 45 55 65 75
Enzyme Efficiency in% (B/A) Pectinase
Xylanase
Cellulase
30 ± 2 48 ± 3 63 ± 4 87 ± 4 69 ± 3 52 ± 3
20 ± 2 38 ± 2 42 ± 2 69 ± 4 56 ± 3 40 ± 2
15 ± 1 23 ± 2 38 ± 2 58 ± 4 48 ± 3 38 ± 2
All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
DLS measurements were performed for determining the average size and size distribution of the iron oxide (Fe3 O4 ) nanoparticles and nanobiocatalyst. It was carried out using the spectroscatter RiNA, GmbH class3B. The dried powder (0.5 mg/ml) was dispersed in milli-Q water and all the analysis were done at 20 ◦ C.
2.16. Fermentation of hydrolysate for bioethanol production. Hydrolysate obtained after saccharification were provided with 10 g/l yeast extract and 20 g/l peptone which was autoclaved at 121 ◦ C for 30 min. Fermentation was carried out after sterilization by adding 1 g/l of baker’s yeast at 30 ◦ C and 150 rpm. Samples of bioethanol produced during fermentation were withdrawn after 0, 12, 24, 48 and 60hr. Bioethanol produced during fermentation was determined by chromic acid method as described by Caputi et al. [29]. Different concentration of ethanol was added to each test tubes and the volume was made up to 5 ml with distilled water. 5 ml chromic acid was added to each test tube and was kept for incubation at 60 ◦ C for 20 min. Absorbance was taken at 660 nm using spectrophotometer (Mechasys Optizen 3220). Concentration of bioethanol was determined using standard curve of ethanol.
2.13. Atomic force microscopy (AFM)
2.17. Statistical analysis
2.12. Dynamic light scattering
®
AFM images were recorded using Brukers Multi-Mode 8 instru® ment in Scan Asyst mode. The samples were first diluted using deionized water. Silica was cleaned with ethanol and air dried. 50 l of the diluted samples was spread on cleaned silica wafer surface and then dried at room temperature. WSxM software (version 12.0) was used to analyse the images [28]. 2.14. Application of nanobiocatalyst 2.14.1. Clarification of juices Pineapple and orange were bought from the local market and fresh juice was extracted in the lab using juicer. To check the clarification, the pineapple and orange juice were incubated with different concentrations of nanobiocatalyst (0.5, 1, 2, 3, 5 and 10 mg/ml) and free enzyme at 45 ◦ C for 10 min. The change in percentage transmittance was measured at 650 nm. Two controls were taken(control 1, and control 2), in control 1, orange and pineapple juice were incubated with the modified MNPs without enzyme and in control 2, orange and pineapple juice were taken, both were incubated at 45 ◦ C for 10 min. 2.15. Bioethanol production 2.15.1. Pretreatment of wheat straw. Wheat straw was milled to 4–5 mm particle size with the help of milling machine. Milled straw was dried at 70 ◦ C to obtain constant weight. 1% w/v of wheat straw was treated with 1N NaOH solution and then autoclaved at 121 ◦ C for 20 min. Pretreated wheat straw was washed five times to remove alkaline traces and after drying it was stored at 4 ◦ C. 2.14. Saccharification of pretreated wheat straw with free enzyme and nanobiocatalyst. 2 g/l of alkaline pretreated wheat straw (in 50 mM sodium acetate buffer at pH 4.8) was hydrolysed with free pectinex 3XL and nanobiocatalyst (each containing 100U of pectinase enzyme); the mixture was kept at 50 ◦ C and 200 rpm. Samples were withdrawn after 12, 24, 36, 48, 60 h for analysing the concentration of reducing sugar using dinitrosalicylic acid method [12].
ANOVA was performed followed by Dunnets test and values were represented as means of three replicate (n = 3) ±SD. The significance level was maintained as p- value <0.05. 3. Results and discussion Commercial enzyme Pectinex 3XL was immobilized by covalent binding onto the surface of modified iron oxide nanoparticles to obtain nanobiocatalyst. The nanoparticles were modified by silane group (APTS) which is most commonly used to modify the hydroxyl group present on the surface. The enzyme was conjugated to the amine activated surface using glutaraldehyde. The immobilization efficiency was determined at various enzyme load as given in Table 1. The immobilization efficiency (B/A) is determined as the ratio of the enzyme activity (calculated by subtracting the unbound activity in the supernatant and wash) in the immobilized enzyme (B) to the total bound activity (A). The immobilization efficiency increases by increasing the load of enzyme unit on nanoparticles. The maximum immobilization efficiency achieved is 0.87 in the case of pectinase, 0.69 in case of xylanase and 0.58 in case of cellulase. Immobilization efficiency decreases by further increase in enzyme load because aggregation occurs at higher protein concentration, this data is in agreement with the earlier report [27,30,31]. This immobilized preparation was used for further studies using standard techniques like DLS and AFM. DLS data shows that the nanoparticles size increases from 63 nm to 86 nm after immobilization (Fig. 1a, b). In order to visualize the deposition of enzymes on iron oxide nanoparticles AFM studies were carried out. Figs. 2 D and 3 D images of the iron oxide nanoparticles and nanobiocatalyst are shown in the Fig. 2(a–d). Fig. 2D images (2a and 2b) clearly show the deposition of enzymes on the surface of the nanoparticles. 3D AFM images in Fig. 2c and d) shows that the height of nanobiocatalyst is more than the height of iron oxide nanoparticles which further confirms the deposition of enzymes on iron oxide nanoparticles. The biochemical parameters of the nanobiocatalyst showing 0.87 pectinase activities were evaluated. The kinetic parameters Km and
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Fig. 1. Dynamic Light Scattering (DLS) a) DLS of iron oxide nanoparticles and b) DLS of nanobiocatalyst.
Vmax of the free enzymes and of nanobiocatalyst remain unchanged (Table 2). However, the nanobiocatalyst was found to be thermally more stable as compared to their soluble enzymes. Fig. 3 shows the remarkable thermo-stabilization of the enzymes present in the preparation. When enzymes were incubated at 70 ◦ C for 90 min
the free enzyme loses all of its activity whereas the immobilized enzymes retained 70% of its activity in the case of pectinase, 56% activity in the case of xylanase and 69% activity of cellulase. These results are in agreement with the earlier reports [6,17] increase in
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Fig. 2. AFM of a) 2D image of iron oxide nanoparticles; b) 2D image of nanobiocatalyst; c) 3D image of iron oxide nanoparticles; d) 3D image of nanobiocatalyst.
120 Pecnase Enzyme
Relative activity (%)
100
Xylanase Enzyme
80
Cellulase enzyme Immobilized Pecnase Enzyme Immobilized Xylanase Enzyme
60 40 20 0 0
20
40
60
80
100
Time in minute Fig. 3. Thermal stability of free Pectinex 3xL and nanobiocatalyst. Free enzyme and nanobiocatalyst were incubated at 70 ◦ C. An appropriate aliquot of free enzyme and nanobiocatalyst were withdrawn at various time intervals of incubation, cooled to 25 ◦ C and their activities were determined using their respective substrate. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05. Table 2 Kinetic parameters of free and immobilized enzymes. Enzymes
Parameters
Free enzyme
Immobilized enzyme
Pectinase
Vmax (mg/ml/min) Km (mg/ml) Vmax/Km (min−1 )
168 14 12
166 12 13.8
Xylanase
Vmax (mg/ml/min) Km (mg/ml) Vmax/Km (min−1 )
296 4.5 66
300 5.0 60
Cellulase
Vmax (mg/ml/min) Km (mg/ml) Vmax/Km (min−1 )
458 30 15.3
462 31 15
All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
covalent bonding reduces the conformational flexibility of enzymes and making it more stable. To check the operational stability of the enzyme the nanobiocatalyst was incubated at 70 ◦ C and the result shows that it can be
successively used for the continuous hydrolysis of pectin and xylan at 70 ◦ C. The amount of reducing sugars formed by nanobiocatalyst after 90 min at 70 ◦ C is 1.5 times more than the reducing sugars formed by free enzymes (Fig. 4a). In addition to the hydrolysis of soluble substrates (xylan, carboxymethyl cellulose and polygalacturonic acid), it can also hydrolyse insoluble substrate (filter paper). Three fold increments are observed in enzymatic hydrolysis of insoluble substrate (filter paper) by nanobiocatalyst as compared to free enzyme which further enhances its potential application in industries (Fig. 4b). Increase in enzyme stability of nanobiocatalyst may be attributed to high temperature and presence of substrate as shown by thermal stability data. The strong binding of the substrate with the enzyme reduces conformational flexibility and enhances the stability against heat denaturation which has been reported earlier by several investigators [32,33]. Operational stability result also indicates that the hydrolysis by nanobiocatalyst is better than free enzyme at higher temperature (Fig. 4a). One of the major issues for industrial applications of enzyme is their reusability and sta-
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Fig. 4. a) Operational stability of free Pectinex 3xL and nanobiocatalyst. Free enzyme and nanobiocatalyst were incubated with substrate at 70 ◦ C. Xylanase and pectinase activity were determined using their respective substrate. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05. b) Filter paper assay of nanobiocatalyst for cellulase activity. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
bility in presence of harsh chemical reagents. The nanobiocatalyst exhibits good reusability for all enzyme activities. It can be successfully used up to 5th cycle after which there is slight decrease in enzyme activities. Fig. 5 shows pectinase loses 3% activity, xylanase loses 9% activity and cellulase loses 13% activity after six cycles. Variation in percent loss of activity may be due to different stabilities of these enzymes towards temperature and repeated use. Other researchers reported decrease in these enzyme activities after third cycle only [17,34]. Dalal et al. [17] prepared cross linked enzyme aggregates of Pectinex 3XL enzyme preparation containing pectinase, xylanase and cellulase enzymes and estimated activity using polygalacturonic acid, xylan and carboxymethyl cellulose respectively as the substrate, they reported decrease in activity for all the three enzymes after third cycle only. Similar decrease in cellulase activity after repeated usage was observed by Li et al. [34]
when they covalently coupled cellulase on liposome and determined its activity using carboxymethyl cellulose as the substrate. The decrease in enzyme activity may be due to end product inhibition or structural distortion of proteins due to repeated usage [6]. The incubation of free enzyme and nanobiocatalyst with different organic solvents and chemical reagents is shown in Fig. 6. The nanobiocatalyst (pectinase) retained high enzymatic activity as compared to free enzyme in 50% of DMSO, Toulene and Chloroform because immobilized enzyme has the potential of retaining native structure of enzyme in presence of organic solvents and different chemical reagent [35]. Organic solvent and reagent tolerant enzymes are highly beneficial in the production of bio-based chemicals. To study the application of nanobiocatalyst in juice industry, it was used for the clarification of pineapple and orange juice. When 1 ml of juice was incubated with different concentrations of
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Fig. 5. Reusability of the nanobiocatalyst. The enzyme activity was determined with their respective substrates as given in the materials and methods section. Immobilized enzyme activities were calculated after each cycle by taking initial activities of the immobilized enzymes as 100%. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
Fig. 6. Effect of organic solvents and chemical reagents on pectinase activity. Different concentration of chemical reagents [Triton-X 100 (0.1%), Tween 80 (1%), EDTA (0.28%), SDS (1%), and -mercaptoethanol (1%)] and 50% concentration of organic solvents were used to check the effect on pectinase activity. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
nanobiocatalyst at 45 ◦ C for 10 min only, the maximum transmittance value for pineapple juice was 53.96% with 2 mg and orange juice was 59.71% with 3 mg nanobiocatalyst (Fig. 7a and b). Initially with increasing concentration of nanobiocatalyst, juice clarification increases due to the enzymatic treatment of nanobiocatalyst on substrate (pectin) leading to the formation of galacturonic acid monomer and oligomer causing depectinization and thus increasing clarity. After maximum transmittance value is achieved, the increase in nanobiocatalyst concentration does not affect clarification of juice because no more pectin is available for degradation by nanobiocatalyst due to which percentage clarification does not change significantly. In the previous study, Chauhan et al. [36]
discussed the clarification of plum juice and reported maximum transmittance of 44.5% only at 45 ◦ C using 15 mg of immobilized enzyme. Similarly Saxena et al. [37] reported maximum transmittance of 55% using 24 U of immobilized enzyme on apple juice kept at 45 ◦ C for 1 h. Nanobiocatalyst was also used for the production of bioethanol from alkali pretreated wheat straw. Pretreatment of 1%w/v of wheat straw was carried out at 121 ◦ C for 20 min in autoclave with 1N NaOH solution. Pretreatment with alkali is shown to be best suited for removal of lignin by breaking down ester bond between lignin and xylan which mostly interferes with the process of hydrolysis, thus making hemicellulose and cellulose available
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Bioethanol concentraon (g/l)
0.6 0.5 0.4 0.3
Free enzyme
0.2
Nanobiocatalyst
0.1 0
-12
0
12
24
36
48
60
72
Time (hr) Fig. 9. Bioethanol concentration produced by hydrolysate obtained from free enzyme and nanobiocatalyst obtained at different time interval (0, 12, 24, 48, 60 h). All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
ciency of the strain, which resulted in greater time duration for converting all reducing sugar to ethanol [39]. Cherion et al. [40] also reported increase in bioethanol production from sugarcane leaves using cellulase immobilized on MnO2 nanoparticle. Nanobiocatalyst shows increase in the concentration of bioethanol by 1.4 fold with respect to free enzyme. As the nanobiocatalyst is magnetic in nature it can be removed and reused for further production of bioethanol from renewable sources. Further research is required to explore its potential in paper, detergent, and bio-based chemical industry. Fig. 7. Effect of free enzyme and nanobiocatalyst concentration on a) pineapple juice and b) orange juice clarification. control 1: orange and pineapple juice were incubated with the modified MNPs without enzyme and control 2: orange and pineapple juice. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
Reducing sugar concentraon (g/l)
1.8 1.6 1.4 1.2 1 0.8
Free Enzyme
0.6
Nanobiocatalyst
0.4 0.2
4. Conclusions Complex carbohydrates are enzymatically degraded into simple sugar by cellulases and hemicellulases which is a potential pathway for biomass conversion. It is difficult to find enzymes with best stability and activity. High cost of enzyme which limits the hydrolysis of biomass needs to be overcome. Hence in the present study we have synthesised nanobiocatalyst which was characterized by different techniques and has the potential for hydrolysing pectin, xylan and insoluble cellulose. It can be effectively used for the clarification of fruit juices. Nanobiocatalyst shows efficient biomass conversion of wheat straw into bioethanol as compared to free enzyme. The nanobiocatalyst shows stability at higher temperature and in organic solvents can be reused successfully which makes it an ideal candidate for biotechnological applications and industries too.
0 0
12
24
36
48
60
72
Time (hr) Fig. 8. Reducing sugar concentration produced by free enzyme and nanobiocatalyst by using pretreated wheat straw as substrate at different time interval (0, 12, 24, 36, 48, 60 h). All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
Acknowledgement The financial support provided by Indian Council of Medical Research (ICMR) and University Grant Commission (UGC) Government of India is greatly acknowledged. References
for hydrolysis [38]. Alkaline pretreated wheat straw (2 g/l) was subjected to hydrolysis by nanobiocatalyst and free pectinase. The maximum concentration of reducing sugar was 1.39 g/l with free pectinase and 1.59 g/l with nanobiocatalyst after 60 h of agitation at 50 ◦ C. (Fig. 8). The data indicates improvement in the formation of reducing sugar by a nanobiocatalyst as compared to free pectinase may be attributed to an increase in stability at high temperature. Maximum concentration of ethanol obtained after fermentation of hydrolysate for free enzyme and nanobiocatalyst with Baker’s yeast for 24 h at 30 ◦ C was 0.37 g/l and 0.5 g/l respectively (Fig. 9). The decrease in ethanol production after 24 h can be attributed to the significant decrease in residual reducing sugars due to the effi-
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A reusable multipurpose magnetic nanobiocatalyst for industrial applications Mohammad Perwez, Razi Ahmad, Meryam Sardar ∗ Department of Biosciences, Jamia Millia Islamia, 110025 New Delhi, India
a r t i c l e
i n f o
Article history: Received 6 March 2017 Received in revised form 4 May 2017 Accepted 5 May 2017 Available online 8 May 2017
a b s t r a c t A multipurpose magnetic nanobiocatalyst is developed by conjugating Pectinex 3XL (a commercial enzyme containing pectinase, xylanase and cellulase activities) on 3-aminopropyl triethoxysilane activated magnetic nanoparticles. The nanobiocatalyst retained 87% of pectinase, 69% of xylanase and 58% of cellulase activity after conjugation on modified nanoparticles as compared to their soluble counterparts. Thermal stability data at 70 ◦ C showed increase in enzyme stability after conjugation to nanoparticles and the kinetic parameters (Km and Vmax ) remain unaltered after immobilization. The immobilized enzyme system can be successfully used upto 5th cycle after that slight decrease in enzyme activities was observed. The nanobiocatalyst retained high pectinase activities in organic solvents and chemical reagents as compared to free enzymes. DLS data shows that the nanoparticles size increases from 63 nm to 86 nm after immobilization. Atomic Force Microscopy data confirms the deposition of enzymes on the nanoparticles. The nanobiocatalyst was used for the clarification of pine apple and orange juice and was also used for the production of bioethanol. Hydrolysis of pretreated wheat straw produced 1.39 g/l and 1.59 g/l after treatment with free Pectinex 3xL and nanobiocatalyst respectively. The concentration of bioethanol also increases by 1.4 fold as compared to the free enzyme. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hemicellulases hydrolyse the plant cell wall polymer hemicellulose, one of the most abundant polysaccharides in nature. Plant biomass in terms of dry weight comprises of 23% lignin, 40% cellulose and 33% hemicellulose. The potential of plant biomass as a renewable raw material is immense [1]. The judicious use of hemicellulases and cellulases in industries could result in higher yields. These enzymes have many biotechnological applications such as deinking of paper waste, pulp biobleaching, clarification of fruit juices, in baking industry, up gradation of feed, fodder and fibres, and saccharification [2]. Before using these enzymes at industrial scale several criteria have to be fulfilled. In industrial processes the reactions are generally performed at high temperature and with harsh chemical reagents to reduce the crystalline structure of cellulose making it available to cellulases and hemicellulases [3]. Therefore, the properties of these enzymes need to be greatly improved. Immobilization is one such powerful tool for increasing the stability and reusability of the enzymes [4,5]. Different immobilization methods such as covalent, ionic bonding, adsorption,
∗ Corresponding author. E-mail address: [email protected] (M. Sardar). http://dx.doi.org/10.1016/j.ijbiomac.2017.05.029 0141-8130/© 2017 Elsevier B.V. All rights reserved.
entrapment, and encapsulation provide better stability to enzymes. Recently, nanomaterials have emerged as the potential immobilization matrices because of their surface size to volume ratio, large amount of enzyme can be loaded to them [6–10]. Moreover, the diffusion limitations are also minimized when dealing with macromolecular substrates [6,8,11]. Graphene based nanomaterials have also been used as support system for use in immobilizing enzymes [12]. Due to the physiochemical properties like electrostatic and hydrophobic properties, enzymes can easily bind to the graphene based nanomaterial but catalytic activity has been found to be reduced due to which graphene is modified with functional groups and then covalently attached with enzymes which improves its catalytic activity [13]. It can be used for waste water treatment and for industrial applications [13]. Magnetic nanoparticles (MNPs) have been a field of study in recent years due to their applications in pharmacy, biology, diagnostics and biotechnology, etc. [14,15]. The use of MNPs offers many benefits because of their nano-scale size and physical properties [16]. hence, immobilization of bioactive substances such as proteins/enzymes on magnetic iron oxide nanoparticle is preferred [10,11]. Mostly enzyme bound magnetic nanoparticles have been found to be stable towards heat, pH and resistant to denaturation [10]. Moreover, MNPs due to their magnetic properties can be
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separated from the solution and reused in enzyme immobilization systems [9]. In the present work the Pectinex 3XL enzyme that is used in the food processing industry for hydrolysing pectin was covalently conjugated to the surface modified iron oxide nanoparticles. It has been reported earlier by several investigators [17–19] that this enzyme preparation is rich in pectinase, xylanase and cellulase. Thus, a multipurpose magnetic nanobiocatalyst is prepared which can hydrolyse xylan, pectin and cellulose. So, far either cellulase or hemicellulase has been immobilized on iron oxide nanoparticles; in this study we report a co-immobilized enzyme system containing cellulases and hemicellulases activity which enhances its application for biotechnological applications. 2. Materials and methods 2.1. Materials and methods Iron (II, III) Oxide nanopowder (<50 nm by TEM), (3Aminopropyl)triethoxysilane (APTS), Pectinex 3XL and Polygalacturonic acid, all above chemicals were purchased from Sigma Aldrich (USA). Xylan from birchwood was purchased from Sisco Research Laboratories (SRL), Mumbai. Throughout the experiment analytical grade solvents and chemicals were used. 2.2. Determination of pectinase activity Pectinase activity in free and immobilized enzyme was determined using polygalacturonic acid as the substrate according to the method described earlier [20]. One unit of enzyme activity is defined as the amount of enzyme required to produce one mol of galacturonic acid per minute under assay conditions. The amount of galacturonic acid was determined using the dinitrosalicylic acid method [21]. 2.3. Determination of xylanase activity Xylanase activity in free and immobilized enzyme was determined using xylan as the substrate [22]. One unit of enzyme activity is defined as the amount of enzyme required to produce one mol of reducing sugar per minute under assay conditions. The amount of reducing sugar was determined using the dinitrosalicylic acid method [21]. 2.4. Determination of cellulase activity Cellulase activity in free and immobilized enzyme was determined as described earlier [23] using Carboxymethyl cellulose as the substrate. 2.5. Filter paper assay for cellulase activity Cellulase activity in free and immobilized enzyme was determined according to the method described earlier [24]. The reaction mixture was prepared by adding 1 ml of sodium acetate buffer (0.05 M, pH 4.8) in each test tube, 0.5 ml of each enzyme (free and nanobiocatalyst) dilution; 50 mg Whatman No.1 filter paper strip (1.0 × 6.0 cm) was rolled and placed into each test tube. The mixture was incubated at 50 ◦ C for 1 h. 3 ml of DNSA reagent was added to the reaction mixture and the reaction mixture was incubated for 5 min in boiling water. The reaction mixture was cooled till it reaches room temperature and then 20 ml of milli-Q water was added. Finally the absorbance was taken by spectrophotometer at 540 nm.
17
2.6. Modification of iron oxide nanoparticles with (3-Aminopropyl)triethoxysilane (APTS) Modification of the Iron oxide nanoparticles with APTS was carried out as described by Campo et al. [25]. APTS (0.306 ml) was added to 0.15 gm of Iron oxide nanoparticles pre-equilibrated with assay buffer and the final volume was made to 15 ml with distilled water. The mixture was kept at 50 ◦ C with constant stirring. After 24 h the nanoparticles were washed extensively with 0.05 M sodium citrate buffer pH 7.4. 2.7. Preparation of nanobiocatalyst Glutaraldehyde (5%) solution in 0.05 M sodium citrate buffer, pH 7.4 was added to the APTS modified nanoparticles and the mixture was stirred for 3 h at 25 ◦ C. The nanoparticles were washed for removing excess glutaraldehyde with assay buffer. Pectinex 3XL enzyme (1 ml containing 55 U of pectinase) was added to the above nanoparticles and left overnight at 4 ◦ C with constant shaking. The enzyme bound nanoparticles were removed by magnet. The nanoparticles were washed with the sodium acetate buffer to remove the loosely bound enzymes. The enzyme activities were calculated in the supernatant and washings. Immobilization efficiency was calculated by varying the enzyme load (25 U–75 U of pectinase) and the immobilization was carried as above. A control was run to check the adsorption of enzymes on the unmodified nanoparticles, for this one ml of enzyme (containing 75 U) was incubated with 0.15 gm of nanoparticles and the final volume was made to 15 ml. The mixture was kept overnight at 4 ◦ C with constant shaking. The unbound enzymes in the supernatant can be easily separated by attracting the bound enzyme with the magnet. The enzyme activities were calculated in the supernatant. A control was run to study the physical adsorption of enzymes on unmodified iron oxide nanoparticles and it was observed that the adsorption of all the three enzymes (pectinase, xylanase and cellulase) on nanoparticles was in the range of 10% only. All the experiments were carried out in the batch mode. Further work was carried out with the nanobiocatalyst showing the best immobilization efficiency of Pectinase i.e 87%. 2.8. Determination of kinetic parameters of enzyme and nanobiocatalyst Km and Vmax values of free enzyme and nanobiocatalyst were determined by measurement of enzyme activities with various concentrations of respective substrates. The kinetic parameters were calculated using Lineweaver-Burk plot [26,27]. 2.9. Thermal stability of enzyme and nanobiocatalyst The stability of enzyme in free and in nanobiocatalyst was studied at 70 ◦ C at different time interval. Free (dissolved in assay buffers) and nanobiocatalyst (suspended in assay buffers) were incubated separately at 70 ◦ C [17]. Proper aliquots of each enzyme sample were taken out at different time interval, the samples were cooled and activities were calculated using their respective substrates. The activity of enzymes at respective optimum temperature was taken as 100%. 2.10. Reusability of the nanobiocatalyst For the first cycle nanobiocatalyst (containing 55U of pectinase) was made to 1.0 ml with the assay buffer and incubated with 0.5 ml of the polygalacturonic acid under shaking condition at 30 ◦ C. After 10 min nanobiocatalyst was removed by magnet and enzyme activity was estimated in the solution. The nanobiocatalyst was
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washed three times with 3 ml of assay buffer. For second cycle the nanobiocatalyst was incubated with 0.5 ml of fresh substrate and the reaction was carried out as before. Similar experiment was performed for xylanase and cellulase activities using xylan and carboxymethyl cellulose as the substrate at 50 ◦ C. 2.11. Effect of organic solvents and chemical reagents on pectinase activity Effect of different organic solvents (DMSO, Toulene, and Chloroform) and chemical reagents [Triton-X 100 (0.1%), Tween 80 (1%), EDTA (0.28%), SDS (1%), and -mercaptoethanol (1%)] on the pectinase activity were examined. Free and nanobiocatalyst was incubated with 50% concentration of organic solvents and different concentration of organic reagents for 1.5 h in 0.05 M sodium acetate buffer at pH 5.0. Residual activity was determined under optimal enzyme assay condition. The activity of the untreated enzyme was considered as control (100%) for calculating percent activity.
Table 1 Optimization of Immobilization of Pectinex 3xL. Enzyme load (U)
25 35 45 55 65 75
Enzyme Efficiency in% (B/A) Pectinase
Xylanase
Cellulase
30 ± 2 48 ± 3 63 ± 4 87 ± 4 69 ± 3 52 ± 3
20 ± 2 38 ± 2 42 ± 2 69 ± 4 56 ± 3 40 ± 2
15 ± 1 23 ± 2 38 ± 2 58 ± 4 48 ± 3 38 ± 2
All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
DLS measurements were performed for determining the average size and size distribution of the iron oxide (Fe3 O4 ) nanoparticles and nanobiocatalyst. It was carried out using the spectroscatter RiNA, GmbH class3B. The dried powder (0.5 mg/ml) was dispersed in milli-Q water and all the analysis were done at 20 ◦ C.
2.16. Fermentation of hydrolysate for bioethanol production. Hydrolysate obtained after saccharification were provided with 10 g/l yeast extract and 20 g/l peptone which was autoclaved at 121 ◦ C for 30 min. Fermentation was carried out after sterilization by adding 1 g/l of baker’s yeast at 30 ◦ C and 150 rpm. Samples of bioethanol produced during fermentation were withdrawn after 0, 12, 24, 48 and 60hr. Bioethanol produced during fermentation was determined by chromic acid method as described by Caputi et al. [29]. Different concentration of ethanol was added to each test tubes and the volume was made up to 5 ml with distilled water. 5 ml chromic acid was added to each test tube and was kept for incubation at 60 ◦ C for 20 min. Absorbance was taken at 660 nm using spectrophotometer (Mechasys Optizen 3220). Concentration of bioethanol was determined using standard curve of ethanol.
2.13. Atomic force microscopy (AFM)
2.17. Statistical analysis
2.12. Dynamic light scattering
®
AFM images were recorded using Brukers Multi-Mode 8 instru® ment in Scan Asyst mode. The samples were first diluted using deionized water. Silica was cleaned with ethanol and air dried. 50 l of the diluted samples was spread on cleaned silica wafer surface and then dried at room temperature. WSxM software (version 12.0) was used to analyse the images [28]. 2.14. Application of nanobiocatalyst 2.14.1. Clarification of juices Pineapple and orange were bought from the local market and fresh juice was extracted in the lab using juicer. To check the clarification, the pineapple and orange juice were incubated with different concentrations of nanobiocatalyst (0.5, 1, 2, 3, 5 and 10 mg/ml) and free enzyme at 45 ◦ C for 10 min. The change in percentage transmittance was measured at 650 nm. Two controls were taken(control 1, and control 2), in control 1, orange and pineapple juice were incubated with the modified MNPs without enzyme and in control 2, orange and pineapple juice were taken, both were incubated at 45 ◦ C for 10 min. 2.15. Bioethanol production 2.15.1. Pretreatment of wheat straw. Wheat straw was milled to 4–5 mm particle size with the help of milling machine. Milled straw was dried at 70 ◦ C to obtain constant weight. 1% w/v of wheat straw was treated with 1N NaOH solution and then autoclaved at 121 ◦ C for 20 min. Pretreated wheat straw was washed five times to remove alkaline traces and after drying it was stored at 4 ◦ C. 2.14. Saccharification of pretreated wheat straw with free enzyme and nanobiocatalyst. 2 g/l of alkaline pretreated wheat straw (in 50 mM sodium acetate buffer at pH 4.8) was hydrolysed with free pectinex 3XL and nanobiocatalyst (each containing 100U of pectinase enzyme); the mixture was kept at 50 ◦ C and 200 rpm. Samples were withdrawn after 12, 24, 36, 48, 60 h for analysing the concentration of reducing sugar using dinitrosalicylic acid method [12].
ANOVA was performed followed by Dunnets test and values were represented as means of three replicate (n = 3) ±SD. The significance level was maintained as p- value <0.05. 3. Results and discussion Commercial enzyme Pectinex 3XL was immobilized by covalent binding onto the surface of modified iron oxide nanoparticles to obtain nanobiocatalyst. The nanoparticles were modified by silane group (APTS) which is most commonly used to modify the hydroxyl group present on the surface. The enzyme was conjugated to the amine activated surface using glutaraldehyde. The immobilization efficiency was determined at various enzyme load as given in Table 1. The immobilization efficiency (B/A) is determined as the ratio of the enzyme activity (calculated by subtracting the unbound activity in the supernatant and wash) in the immobilized enzyme (B) to the total bound activity (A). The immobilization efficiency increases by increasing the load of enzyme unit on nanoparticles. The maximum immobilization efficiency achieved is 0.87 in the case of pectinase, 0.69 in case of xylanase and 0.58 in case of cellulase. Immobilization efficiency decreases by further increase in enzyme load because aggregation occurs at higher protein concentration, this data is in agreement with the earlier report [27,30,31]. This immobilized preparation was used for further studies using standard techniques like DLS and AFM. DLS data shows that the nanoparticles size increases from 63 nm to 86 nm after immobilization (Fig. 1a, b). In order to visualize the deposition of enzymes on iron oxide nanoparticles AFM studies were carried out. Figs. 2 D and 3 D images of the iron oxide nanoparticles and nanobiocatalyst are shown in the Fig. 2(a–d). Fig. 2D images (2a and 2b) clearly show the deposition of enzymes on the surface of the nanoparticles. 3D AFM images in Fig. 2c and d) shows that the height of nanobiocatalyst is more than the height of iron oxide nanoparticles which further confirms the deposition of enzymes on iron oxide nanoparticles. The biochemical parameters of the nanobiocatalyst showing 0.87 pectinase activities were evaluated. The kinetic parameters Km and
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19
Fig. 1. Dynamic Light Scattering (DLS) a) DLS of iron oxide nanoparticles and b) DLS of nanobiocatalyst.
Vmax of the free enzymes and of nanobiocatalyst remain unchanged (Table 2). However, the nanobiocatalyst was found to be thermally more stable as compared to their soluble enzymes. Fig. 3 shows the remarkable thermo-stabilization of the enzymes present in the preparation. When enzymes were incubated at 70 ◦ C for 90 min
the free enzyme loses all of its activity whereas the immobilized enzymes retained 70% of its activity in the case of pectinase, 56% activity in the case of xylanase and 69% activity of cellulase. These results are in agreement with the earlier reports [6,17] increase in
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Fig. 2. AFM of a) 2D image of iron oxide nanoparticles; b) 2D image of nanobiocatalyst; c) 3D image of iron oxide nanoparticles; d) 3D image of nanobiocatalyst.
120 Pecnase Enzyme
Relative activity (%)
100
Xylanase Enzyme
80
Cellulase enzyme Immobilized Pecnase Enzyme Immobilized Xylanase Enzyme
60 40 20 0 0
20
40
60
80
100
Time in minute Fig. 3. Thermal stability of free Pectinex 3xL and nanobiocatalyst. Free enzyme and nanobiocatalyst were incubated at 70 ◦ C. An appropriate aliquot of free enzyme and nanobiocatalyst were withdrawn at various time intervals of incubation, cooled to 25 ◦ C and their activities were determined using their respective substrate. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05. Table 2 Kinetic parameters of free and immobilized enzymes. Enzymes
Parameters
Free enzyme
Immobilized enzyme
Pectinase
Vmax (mg/ml/min) Km (mg/ml) Vmax/Km (min−1 )
168 14 12
166 12 13.8
Xylanase
Vmax (mg/ml/min) Km (mg/ml) Vmax/Km (min−1 )
296 4.5 66
300 5.0 60
Cellulase
Vmax (mg/ml/min) Km (mg/ml) Vmax/Km (min−1 )
458 30 15.3
462 31 15
All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
covalent bonding reduces the conformational flexibility of enzymes and making it more stable. To check the operational stability of the enzyme the nanobiocatalyst was incubated at 70 ◦ C and the result shows that it can be
successively used for the continuous hydrolysis of pectin and xylan at 70 ◦ C. The amount of reducing sugars formed by nanobiocatalyst after 90 min at 70 ◦ C is 1.5 times more than the reducing sugars formed by free enzymes (Fig. 4a). In addition to the hydrolysis of soluble substrates (xylan, carboxymethyl cellulose and polygalacturonic acid), it can also hydrolyse insoluble substrate (filter paper). Three fold increments are observed in enzymatic hydrolysis of insoluble substrate (filter paper) by nanobiocatalyst as compared to free enzyme which further enhances its potential application in industries (Fig. 4b). Increase in enzyme stability of nanobiocatalyst may be attributed to high temperature and presence of substrate as shown by thermal stability data. The strong binding of the substrate with the enzyme reduces conformational flexibility and enhances the stability against heat denaturation which has been reported earlier by several investigators [32,33]. Operational stability result also indicates that the hydrolysis by nanobiocatalyst is better than free enzyme at higher temperature (Fig. 4a). One of the major issues for industrial applications of enzyme is their reusability and sta-
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Fig. 4. a) Operational stability of free Pectinex 3xL and nanobiocatalyst. Free enzyme and nanobiocatalyst were incubated with substrate at 70 ◦ C. Xylanase and pectinase activity were determined using their respective substrate. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05. b) Filter paper assay of nanobiocatalyst for cellulase activity. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
bility in presence of harsh chemical reagents. The nanobiocatalyst exhibits good reusability for all enzyme activities. It can be successfully used up to 5th cycle after which there is slight decrease in enzyme activities. Fig. 5 shows pectinase loses 3% activity, xylanase loses 9% activity and cellulase loses 13% activity after six cycles. Variation in percent loss of activity may be due to different stabilities of these enzymes towards temperature and repeated use. Other researchers reported decrease in these enzyme activities after third cycle only [17,34]. Dalal et al. [17] prepared cross linked enzyme aggregates of Pectinex 3XL enzyme preparation containing pectinase, xylanase and cellulase enzymes and estimated activity using polygalacturonic acid, xylan and carboxymethyl cellulose respectively as the substrate, they reported decrease in activity for all the three enzymes after third cycle only. Similar decrease in cellulase activity after repeated usage was observed by Li et al. [34]
when they covalently coupled cellulase on liposome and determined its activity using carboxymethyl cellulose as the substrate. The decrease in enzyme activity may be due to end product inhibition or structural distortion of proteins due to repeated usage [6]. The incubation of free enzyme and nanobiocatalyst with different organic solvents and chemical reagents is shown in Fig. 6. The nanobiocatalyst (pectinase) retained high enzymatic activity as compared to free enzyme in 50% of DMSO, Toulene and Chloroform because immobilized enzyme has the potential of retaining native structure of enzyme in presence of organic solvents and different chemical reagent [35]. Organic solvent and reagent tolerant enzymes are highly beneficial in the production of bio-based chemicals. To study the application of nanobiocatalyst in juice industry, it was used for the clarification of pineapple and orange juice. When 1 ml of juice was incubated with different concentrations of
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Fig. 5. Reusability of the nanobiocatalyst. The enzyme activity was determined with their respective substrates as given in the materials and methods section. Immobilized enzyme activities were calculated after each cycle by taking initial activities of the immobilized enzymes as 100%. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
Fig. 6. Effect of organic solvents and chemical reagents on pectinase activity. Different concentration of chemical reagents [Triton-X 100 (0.1%), Tween 80 (1%), EDTA (0.28%), SDS (1%), and -mercaptoethanol (1%)] and 50% concentration of organic solvents were used to check the effect on pectinase activity. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
nanobiocatalyst at 45 ◦ C for 10 min only, the maximum transmittance value for pineapple juice was 53.96% with 2 mg and orange juice was 59.71% with 3 mg nanobiocatalyst (Fig. 7a and b). Initially with increasing concentration of nanobiocatalyst, juice clarification increases due to the enzymatic treatment of nanobiocatalyst on substrate (pectin) leading to the formation of galacturonic acid monomer and oligomer causing depectinization and thus increasing clarity. After maximum transmittance value is achieved, the increase in nanobiocatalyst concentration does not affect clarification of juice because no more pectin is available for degradation by nanobiocatalyst due to which percentage clarification does not change significantly. In the previous study, Chauhan et al. [36]
discussed the clarification of plum juice and reported maximum transmittance of 44.5% only at 45 ◦ C using 15 mg of immobilized enzyme. Similarly Saxena et al. [37] reported maximum transmittance of 55% using 24 U of immobilized enzyme on apple juice kept at 45 ◦ C for 1 h. Nanobiocatalyst was also used for the production of bioethanol from alkali pretreated wheat straw. Pretreatment of 1%w/v of wheat straw was carried out at 121 ◦ C for 20 min in autoclave with 1N NaOH solution. Pretreatment with alkali is shown to be best suited for removal of lignin by breaking down ester bond between lignin and xylan which mostly interferes with the process of hydrolysis, thus making hemicellulose and cellulose available
M. Perwez et al. / International Journal of Biological Macromolecules 103 (2017) 16–24
23
Bioethanol concentraon (g/l)
0.6 0.5 0.4 0.3
Free enzyme
0.2
Nanobiocatalyst
0.1 0
-12
0
12
24
36
48
60
72
Time (hr) Fig. 9. Bioethanol concentration produced by hydrolysate obtained from free enzyme and nanobiocatalyst obtained at different time interval (0, 12, 24, 48, 60 h). All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
ciency of the strain, which resulted in greater time duration for converting all reducing sugar to ethanol [39]. Cherion et al. [40] also reported increase in bioethanol production from sugarcane leaves using cellulase immobilized on MnO2 nanoparticle. Nanobiocatalyst shows increase in the concentration of bioethanol by 1.4 fold with respect to free enzyme. As the nanobiocatalyst is magnetic in nature it can be removed and reused for further production of bioethanol from renewable sources. Further research is required to explore its potential in paper, detergent, and bio-based chemical industry. Fig. 7. Effect of free enzyme and nanobiocatalyst concentration on a) pineapple juice and b) orange juice clarification. control 1: orange and pineapple juice were incubated with the modified MNPs without enzyme and control 2: orange and pineapple juice. All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
Reducing sugar concentraon (g/l)
1.8 1.6 1.4 1.2 1 0.8
Free Enzyme
0.6
Nanobiocatalyst
0.4 0.2
4. Conclusions Complex carbohydrates are enzymatically degraded into simple sugar by cellulases and hemicellulases which is a potential pathway for biomass conversion. It is difficult to find enzymes with best stability and activity. High cost of enzyme which limits the hydrolysis of biomass needs to be overcome. Hence in the present study we have synthesised nanobiocatalyst which was characterized by different techniques and has the potential for hydrolysing pectin, xylan and insoluble cellulose. It can be effectively used for the clarification of fruit juices. Nanobiocatalyst shows efficient biomass conversion of wheat straw into bioethanol as compared to free enzyme. The nanobiocatalyst shows stability at higher temperature and in organic solvents can be reused successfully which makes it an ideal candidate for biotechnological applications and industries too.
0 0
12
24
36
48
60
72
Time (hr) Fig. 8. Reducing sugar concentration produced by free enzyme and nanobiocatalyst by using pretreated wheat straw as substrate at different time interval (0, 12, 24, 36, 48, 60 h). All the values are means of triplicate (n = 3) ±SD. ANOVA significant at P ≤ 0.05.
Acknowledgement The financial support provided by Indian Council of Medical Research (ICMR) and University Grant Commission (UGC) Government of India is greatly acknowledged. References
for hydrolysis [38]. Alkaline pretreated wheat straw (2 g/l) was subjected to hydrolysis by nanobiocatalyst and free pectinase. The maximum concentration of reducing sugar was 1.39 g/l with free pectinase and 1.59 g/l with nanobiocatalyst after 60 h of agitation at 50 ◦ C. (Fig. 8). The data indicates improvement in the formation of reducing sugar by a nanobiocatalyst as compared to free pectinase may be attributed to an increase in stability at high temperature. Maximum concentration of ethanol obtained after fermentation of hydrolysate for free enzyme and nanobiocatalyst with Baker’s yeast for 24 h at 30 ◦ C was 0.37 g/l and 0.5 g/l respectively (Fig. 9). The decrease in ethanol production after 24 h can be attributed to the significant decrease in residual reducing sugars due to the effi-
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