Insolubilization of inulinase on magnetite chitosan microparticles, an easily recoverable and reusable support

Journal of Molecular Catalysis B: Enzymatic 113 (2015) 47–55

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Insolubilization of inulinase on magnetite chitosan microparticles, an easily recoverable and reusable support Kalavathy Sairam Paripoorani a , Gurunathan Ashwin a , Prabhakar Vengatapriya a , Venkatesh Ranjitha a , Srikumar Rupasree a , Vaidyanathan Vasanth Kumar b , Vaidyanathan Vinoth Kumar a,∗ a b

Bioprocess Laboratory, Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Chennai 603203, India Department of Electronics and Communication Engineering, SKP Engineering College, Thiruvannamalai 606601, India

a r t i c l e

i n f o

Article history: Received 16 November 2014 Received in revised form 11 January 2015 Accepted 11 January 2015 Available online 17 January 2015 Keywords: Inulinase Insolubilization Fructose Stability Magnetic nanoparticle

a b s t r a c t Inulinase (E.C. 3.2.1.7), a highly productive enzyme could provide a solution to industries looking forward to a robust biocatalyst which is stable and recyclable for the production of high fructose syrup. This objective can be realized by an amalgamation of magnetic nanobiotechnology and enzyme engineering. The present work explores the ramifications of this concept through the insolubilization of inulinase on to magnetite nanoparticles entrapped in chitosan (cMNPs). Statistical methods were used to identify the optimum concentration of EDAC (N-(3-dimethylaminopropyl)-N -ethyl carbodiimide hydrochloride), cMNPs, and cross-linking time which play a pivotal role in deciding the overall efficiency of the biocatalyst. The optimum concentrations for preparation of the magnetic biocatalyst were found to be 15.29 mg and 23.76 mg for EDAC and cMNPs respectively, for which the maximum activity recovery obtained, was 81.40% with an optimum cross linking time of 1.73 h. The optimum pH and temperature for soluble inulinase (SI) was found to be 6.0 and 70 ◦ C whereas that of magnetically insolubilized inulinase (MII) shifted to 4.6 and 50 ◦ C. The insolubilized inulinase could be recycled for up to 15 cycles with substantial activity at least for a year at 4 ◦ C. This feature coupled with ease separation of biocatalyst and a fivefold increase in the t1/2 value at temperature of 80 ◦ C are evidence of enhanced thermal and operational stability inherited by the enzyme after insolubilization, indicating a high stability in practical operation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Inulin, a major carbohydrate reserve (>50%) is mainly found in the roots and tubers of several plants [1,2] and it is a recognized source for the production of high fructose syrup, with d-fructose content over 75% [3]. However the chemical hydrolysis of inulin is faced with hurdles such as the production of undesirable by products and colour forming compounds which tend to reduce the overall product yield. The world is leaning towards a leading technology to meet the burgeoning demand for high quality products for market approval, consumers and industries are on the never ending quest for a process which would be economic, efficient and eco-friendly [4]. Exoinulinase (E.C.3.2.1.80) member of the carbohydrolase family which are involved in the removal of terminal fructose residues from the non-reducing ends of inulin are looked

∗ Corresponding author. Tel.: +91 9943764794. E-mail address: [email protected] (V. Vinoth Kumar). http://dx.doi.org/10.1016/j.molcatb.2015.01.004 1381-1177/© 2015 Elsevier B.V. All rights reserved.

upon as viable alternatives to harmful chemicals for the green synthesis of high fructose syrups in a manner suitable for an industrial setting [3,5]. However their biological origin, does act as a hurdle in their advancement in terms of their solubility in their native state, lower catalytic activity, stability and the fact that they are inhibited by their respective substrate and products [6,7]. These calls for the need to insolubilize inulinase which would help achieve the above goals and could also pave way towards the continuous hydrolysis of inulin for the production of fructose and could improve the reusability of the enzyme [5]. DEAE cellulose [8], amino cellulofine [9], polystyrene [10], porous glass beads [11], streamline DEAE [12], duolite A568 [13], calcium alginate beads [14], polyvinyl alcohol based hydrogel-lentikats [15], Sepabeads [16], sol–gel matrix [17], modified sodium alginate beads [18], chitosan [19], polyurethane foam [20], lentikats [21] and carbon nanotube [22], have been used for inulinase insolubilization as a support material so far for the production of fructose. However the separation process is often tedious involving centrifugation and filtration which could put the enzyme under the risk of leaching [23].

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Combining magnetic nanotechnology with bio catalysis would provide a viable solution to the above challenge as the enzyme after the reaction could be separated from the reaction mixture by application of a simple magnetic field [23]. In the recent years, magnetite nanoparticles (MNP) and its amalgamation with chitosan to yield magnetic bio composites utilized for efficient enzyme insolubilization have caught the eye of several scientist and industries [24]. Some of the perks offered by this robust biocatalyst are the easy separation of the enzyme complex from the reaction mixture making the production approach more cost effective with less fouling, efficient mass transfer and reduced diffusional problems. It is also supplemented with enhanced flexibility [25,26]. Aspergillus niger exo-inulinase was insolubilized onto chitosan functionalized MNP by activating the hydroxyl groups of the enzyme with carbodiimide (EDAC) and N-hydroxysuccinamide (NHS). To the best of our knowledge the insolubilization of inulinase onto magnetic composites is the first of its kind. This paper primarily describes our work, which involves insolubilization of exo-inulinase on magnetic biocomposites followed by comparing the biochemical characteristics such as pH, temperature, stability analysis of the solubilized and magnetic composites, harbouring inulinase. 2. Materials and methods 2.1. Materials Inulin, EDAC, and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (Saint-Louis, MO, USA) while chitosan (medium molecular weight, 75–85% deacetylated, molecular weight ca 400,000) and NHS were purchased from Sisco Research Laboratories, Mumbai, India. All other chemicals used for the present study were of analytical grade and were purchased from local suppliers. The water used throughout this study was deionized and filtered using a U.S. filter purification system. Inulinase preparation from A. niger was produced using culture conditions essentially as described by our group [27] and the culture supernatant centrifuged at 3000 rpm for 20 min. The obtained crude inulinase was purified by two step three phase partitioning technique. 10 mL of crude enzyme solution was saturated with 30% (w/v) ammonium sulphate followed by t-butanol addition in the ratio 1.0:0.5 (v/v). The tubes were centrifuged at 2000 rpm for 10 min. The middle layer obtained was dissolved in pH 6.0 acetate buffer and subjected to a 30% saturation using ammonium sulphate and subsequently added with equal volume of t-butanol addition. All the tubes were once again centrifuged and interfacial layer was separated. The partially purified inulinase (10.2 fold purification and 88% yield) was utilized for insolubilization. 2.2. Synthesis of magnetite nanoparticles Synthesis of magnetic nanoparticles (MNP) by chemical coprecipitation has been previously developed by our group [23]. Magnetite nanoparticles were prepared by slow addition of 1.75 mL of ammonium hydroxide to a mixed solution containing ferric chloride hexahydrate (1.4 g) and ferrous sulphate heptahydrate (0.69 g) in 25 mL of deionized water until the magnetic particles precipitated out. The pH of the solution was adjusted to 7.0 by subjecting it to continuous wash. The precipitate was then centrifuged and dried at 100 ◦ C for a period of 2 h. The dried powder of MNPs was used for the preparation of chitosan nanoparticles entrapped with magnetite. 2.3. Synthesis of chitosan microparticle with entrapped magnetite Functionalization of synthesized MNPs was carried out by dissolving 0.4 g of chitosan in 20 mL of 0.2 M acetic acid. 0.4 g of MNP

was added to the above mixture [28]. After thorough mixing, an excess of 1 M sodium hydroxide was added to convert solubilized chitosan into insoluble magnetite chitosan gel. The chitosan gel containing entrapped magnetite nanoparticles was further dried and ground into smaller pieces and then thoroughly sieved through a 10 ␮m sieve. After sieving, the obtained support was then separated from the mixture by magnetic decantation and washed thrice using deionized water and stored at 4 ◦ C.

2.4. Production of magnetic insolubilized inulinase (MII) A 50 mL centrifuge tube was used to get 10 mL of partially purified inulinase in buffer solution (0.3 mg mL−1 ), (0.1 M acetate buffer, and pH 5.0). Into this inulinase solution, 3 mg mL−1 of EDAC was added slowly and allowed to react for 1 h under agitation. NHS was added to a concentration of 3 mg mL−1 and allowed to react for 60 min under agitation for a stable primary amine group. All these operations were performed in a water-ice bath, with the temperature in the reactor never exceeding 4 ◦ C. MII were produced by cross-linking the cMNP (20 mg) with 10 mL of EDACNHS stabilized soluble inulinase in buffer solution (0.1 M acetate buffer) and shaken for 8 h at room temperature. Cross-linking was done at a controlled pH of 5.0 [29]. After cross-linking, the insolubilized inulinase was then separated from the mixture by magnetic decantation and washed thrice by acetate buffer (pH 5.0) to remove any unbound enzyme and stored in 0.1 M acetate buffer at 4 ◦ C. The results of activity assay and Lowry’s test using BSA as a standard demonstrated the absence of any residual activity or traces of protein in the washings recovered in the above step.

2.5. Design of experiment The designing of an efficient MII is a multivariate process involving many factors which could affect the insolubilization efficiency. Response surface methodology (RSM) is a statistically designed experimental protocol for developing, improving, and optimizing processes. Furthermore, Box Behnken Design (BBD), the most successful factorial design, was used for the optimization of parameters with a limited number of experiments in a cost effective manner [30]. It helps to identify the effect of the interactions of different design variables on the response when they are varied simultaneously. The Design expert 8.0.1 package (SAS Institute Inc., NC, USA) was used for the experimental design, statistical analysis and mathematical modelling. The following factors were taken into account for the optimization of the magnetically separable biocatalyst production (input variables): EDAC concentration (mg mL−1 ), CMNP concentration (mg mL−1 ) and cross-linking time (h). The total number of optimization experimental was based on 17 runs with five centre point replications and the activity recovery of insolubilized inulinase was taken as the dependent variable or response as shown in Table 1. As usual, the experiments were performed in random order to avoid systematic error. In addition, three central replicates were also added to the experimental design to calculate pure experimental error. In this study a three-level BBD full factorial design was employed. Second-order polynomial regression models described estimated response surface contour plots. The explanatory variables and tested ranges were chosen after a series of preliminary experiments. Statistical analysis of the model was performed to evaluate the analysis of variance (ANOVA). This analysis included Fisher’s F-test (overall model significance), its associated probability P (F), and determination coefficient R2 which measures the goodness of fit at regression models.

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Table 1 Experimental runs as generated by BB design and their respective activity recoveries obtained in each run. The difference between predicted and experimental data is tabulated as percentage error. Run

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

A

B

C

Activity recovery (%)

EDAC (mg mL−1 )

cMNP (mg mL−1 )

Cross-linking time (h)

YExp

YPre

% Error

15 32.5 50 50 32.5 32.5 15 32.5 50 32.5 32.5 15 32.5 15 50 32.5 32.5

16 16 16 8 24 16 8 16 16 8 16 16 16 24 24 8 24

1 4 7 4 7 4 4 4 1 7 4 7 4 4 4 1 1

78 52 28 35 39 52.7 58 52.4 48 34 51.8 51 52.7 72 60.3 39.8 66.4

73.98 52 32.01 33.68 38.23 52 61.25 52 48.56 31.31 52 50.43 52.32 73.33 57.05 40.56 69.09

5.15 0 −14.32 3.77 1.97 1.34 5.6 0.76 −1.16 7.91 −0.38 1.11 0.72 −1.84 5.38 −1.9 −4.05

2.6. Leaching test and characterization of magnetic biocatalyst

in the supernatant at the end of the insolubilization procedures after the washing step.

In order to determine any leaching of inulinase activity bonded with cMNPs, MII preparation was incubated in the acetate buffer in a continuous shaking bath for 2 h. After the incubation time, the MII were separated from the buffer with an external magnetic field. The buffer was mixed with 900 ␮L of 1% inulin and the absorption at 540 nm was recorded after 20 min incubation at 60◦ C to characterize the leached amount of inulinase from MII biocatalyst. The MII were recovered later from the mixture with the help of a magnet in not more than 30 s. After washing, the collected products were characterized using a scanning electron microscope (SEM) and an energy dispersive X-ray spectrometer (EDX) element analysis. The shape and surface morphology of the MNPs were characterized by Leo Gemini 1530 SEM analysis at an accelerating voltage of 10 keV. The element analysis of the MNP was carried out using EDX which enables the identification of particular elements and their relative proportions present in a sample. Additionally, the particle size distribution of the MNPs was determined by dynamic light scattering (DLS) (Zetasizer 3000 HSA, UK). 2.7. Determination of enzyme activities SI and MII activities were assayed by measuring the concentration of reducing sugars released from inulin. The reaction mixture containing 100 ␮L of crude enzyme and 900 ␮L of 1% inulin (dissolved in 0.1 M acetate buffer, pH 5.0) was incubated at 60 ◦ C for 20 min. The MII were recovered with a magnet from the mixture in not more than 30 s. The reaction mixture was assayed for reducing sugars (fructose) by the dinitrosalicylic acid method by measuring the absorbance at 540 nm [27]. The calibration curve was drawn with fructose (50–250 ␮g). One unit of inulinase is defined as the amount of enzyme that released 1 ␮mol of fructose per min from inulin at 60 ◦ C. The protein concentration was determined by the Lowry’s method using bovine serum albumin (40–200 ␮g) as the standard [31]. The activity recovery (%) of the MII was calculated as given in Eq. (1): Er =

Ui × 100% UO

(1)

where Er stands for enzyme recovery, Ui corresponds to the total inulinase activity recovered on the support and UO represents the total amount of inulinase activity offered for immobilization. This total enzyme activity recovered on the support is defined as the difference between the total inulinase activity and that remaining

2.8. Physico-chemical properties of soluble and insolubilized inulinase To investigate the effect of pH on the activities of SI and MII, the activities were assayed in different buffer solutions in the pH range of 3.0–8.0. The SI and MII activities were measured in temperature range of 30–90 ◦ C at their optimal pH. Relative activities (calculated as the ratio of the enzyme activity measured at a specific pH and temperature to the maximal activity of the enzyme) were plotted against specific pH values and temperatures. Stability of exo-inulinase was investigated by incubating 20 Unit of SI and MII at distinctive pH range (3.6–7.6) and at diverse temperatures (40–80 ◦ C) for a week. Samples were withdrawn at different time intervals and inulinase activity was assayed according to the method described above. The activities of SI and MII were determined for different inulin concentrations at optimum conditions of each inulinase preparation. Michaelis–Menten constant (Km ), maximum rate of reaction (Vmax ) and catalytic efficiency (Kcat ) values of SI and MII were estimated from their Lineweaver–Burk plots. A comparison of the thermal stability of SI and MII at different temperature (60 ◦ C, 70 ◦ C, and 80 ◦ C) at different time intervals at a pH of 5.0 was carried out. From a semi logarithmic plot of residual activity versus time, the half-life (t1/2 ) of the biocatalysts were calculated. A comparison of the pH stability of SI and MII at different buffers (pH 5.0–8.0) at different time intervals at 60 ◦ C was performed. Samples were taken at specified time intervals and the residual activities were determined using the standard activity assay procedure. Activities were expressed as percentage of the activity of the control. Each experiment was performed in triplicate. The operational stabilities of MII were evaluated in a series of repeated batch experiments, and the retention of the MII activity was tested as described in activity assays. After each batch, the MII were easily recovered using an external magnetic field, and the recovered biocatalyst was washed thrice with acetate buffer (pH 5.0) and subsequently used in the next fresh reaction. The storage stability of the SI and MII was determined after one-year storage in buffer at 4 ◦ C. Taking the initial activity as 100%, the relative activity was defined as the ratio of the activity to the initial activity.

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Fig. 1. Schematic representation of synthesis of magnetic biocatalyst. The first step involves reaction between carboxylic acid residues present on inulinase and the cross linker, EDAC. In step two, the complex formed is stabilized by addition of NHS. The third is covalent immobilization on surface of chitosan magnetic composites via the interaction between the amine group on the chitosan and the carboxyl groups on the surface of the inulinase. After recovery, the insolubilized enzymes are washed with acetate buffer. The picture beside cMNP and MII depict the magnetic property retained before and after enzyme insolubilization.

3. Results and discussion 3.1. Production and characterization of magnetically separable insolubilized inulinase Covalent insolubilization of inulinase on cMNP particles were carried out by EDAC/NHS activation. Fig. 1 illustrates the detail synthetic procedure for cMNP preparation and inulinase activation and insolubilization of inulinase. First step is cross-linking, in which the carboxylic acid residues of A. niger inulinase reacts with cross-linking agent EDAC to form an active amine-reactive O-acylisourea intermediate [32]. However the above complex is highly unstable in aqueous solution which hinders the performance of the enzyme; hence stabilized in the second step using NHS to enhance the cross linking efficiency by stabilizing the intermediate formed. EDAC couples NHS to carboxyls, forming an NHS ester that is considerably more stable than the O-acylisourea intermediate while allowing for efficient conjugation to the amino groups by amide bond formation [33] in the chitosan magnetic composites, forming a stable primary amine group; the third is covalent insolubilization, the amalgamation of the above activated carboxylic group functionalized inulinase with the chitosan magnetic composites via the interaction between the amine group on the chitosan and the carboxyl groups on the surface of the inulinase [28,34]. The final operation is washing, the obtained MII were washed thrice using acetate buffer to remove any soluble enzyme in the reaction. After washing, the activity of the enzyme proved that there was extremely limited due to the covalent insolubilization of the enzyme particles on the surface of cMNP. As all reactions

rendered a black powder at the end of the process, there was no visual difference detected between particles prepared with and without chitosan. Dynamic light scattering (DLS) can be a powerful technique to probe the layer thickness of the inulinase covalently insolubilized onto the cMNP and EDX analysis indicated that these agglomerations predominantly contained ferrous iron (Fig. 2a and b). The standard deviation of the DLS measurement of this particle was 1.3 ␮m and this trend was consistently observed with SEM measurement. Fig. 2c displays the magnetization curves of magnetic biocatalyst by VSM. The saturation magnetization ( s ) which is the value of magnetization to orient the magnetic domains in the prepared magnetic biocatalyst to the applied magnetic field was found to be 42.4 emu g−1 [35]. The data obtained from figure clearly demonstrates that these particles show super paramagnetic behaviour. Fig. 2d shows that the magnetic composite can be easily separated from solution by applying a magnetic field. The magnetic beads can be easily separated from reaction medium within a few seconds by conventional permanent magnet (200 mT). When the applied magnetic force is removed, the magnetic beads can be easily dispersed by simple shaking. Thus the magnetic beads can be removed/recycled in the medium with a simple magnetic device. Although the preparation of carrier is quite important for insolubilization and performance of the insolubilized enzyme, insolubilization conditions are also critical to keep enzyme active. Therefore, three factors affecting the immobilization of inulinase were investigated. RSM was used to evaluate the relationship between the experimental and predicted responses for activity recovery (%) of inulinase insolubilization process. 17 experiments

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Fig. 2. (a) Energy dispersive X-ray analysis of MII shows the presence of ferrous domain in the insolubilized core. (b) Probing of MII with dynamic light scattering (DLS) tool validates the presence of iron domains in the MII. The size of the particle is about 1.3 ␮m which is the same as observed with SEM. (c) Curves of vibrating sample magnetometer (VSM) represent the saturation magnetization which gives a value of 42.4 emu g−1 as the value of magnetization. (d) Depiction of easy separation of magnetic composite from solution by the application of simple magnetic field.

were performed according to the BBD in order to identify optimum concentration of EDAC, CMNP and cross linking time required for highly active MII (Table 1). The empirical relationship between the experimental results obtained on the basis of BBD model and the input variables were expressed by a second-order polynomial equation with interaction terms. The final Eq. (2) obtained in terms of coded factors is given below. Activity recovery (%) = 52.32 − 10.96A + 10.03C − 2.28AB − 1.75AC + 5.4BC + 5.23A2 + 1.22B2 + 6.3C 2

(2)

In Eq. (2) A, B, and C correspond to independent variables of concentration of EDAC, cMNP and cross-linking time, respectively. The obtained activity recovery was close to the activity recovery predicted by the model (Fig. 3a). Fischer F-test with very low probability value (P < 0.05) demonstrates a very high significance for the regression model (Table 2). The goodness of fit of the model was checked by the (R2 ) indicates close agreement between experimental results and theoretical values predicted by the model equation [30]. The value of coefficient of determination (R2 ) 0.9746 indicated 97.46% of the total variation, explained by the model and only 2% of the variations could not be explained by the model. A relatively lower value of the coefficient of variation for biocatalyst production (6.33) indicated improved precision and reliability of the conducted experiments [36]. The model was used to generate response surfaces and contour curves for the analysis of the variable effects on the biocatalyst production (Fig. 3b). The model has high R2 value, significant F-value, an insignificant lack-of-fit value and lower standard deviation, indicating its efficiency in predicting the optimum conditions for synthesis insolubilized inulinase. The effect of the concentration of the EDAC used to activate the carboxyl groups of A. niger inulinase was evaluated (Table 1). The variation of the EDAC concentration modulates the extent of cross-linking by controlling the degree of activation of the carboxyl groups of the inulinase. The contour plots for activity recovery (%) of inulinase insolubilization are presented in Fig. 3b. The result

obtained showed a maximum point on the response surface. From Fig. 3b, it is clearly observed that the activity recovery of the immobilized inulinase increased with increase in cross linking time up to 1.73 h whereas decrease in activity recovery was observed with prolonged incubation time. On the other hand, as time prolonged a large amount of inulinase was insolubilized to the magnetic beads on the basis of protein determination but activity of these preparations was low. The decrease in insolubilized enzyme activity can be due to the multi-point attachment of the enzyme molecules to the activated MNP overcrowding of insolubilized inulinase in cMNP as a result of which substrate diffusion limitations occur. The activity recovery of insolubilized inulinase reached 81.4% under following conditions: EDAC 15.29 mg, cMNP 23.76 mg and cross linking time of 1.73 h. In the subsequent experiments insolubilization was carried out under these optimized conditions. The maximum activity recovery of magnetic biocatalyst reached 81.4% where the amount of protein loading on cMNP support reached about 47.20 ± 1.12 mg g−1 support. In comparison to other supports for insolubilization of inulinase are reported in the literatures, the cMNP presented here provided 81.40% activity recovery (Table 3). This high activity recovery can be attributed to the EDAC form flexible cross-linking leads to the loose super-molecular structures results in high of enzyme activity. 3.2. Effect of temperature on activity and stability of inulinase The temperature dependence of the activity of SI and MII was shown in Fig. 4a. The SI had temperature optima at 70 ◦ C whereas MII exhibited at a lower temperature, 50 ◦ C. The reason for the change in optimum temperature after insolubilization can be attributed to strengthening of protein molecule rigidity that leads to limited conformational alteration at higher temperatures there by depicting maximum activity at lower temperature of 50 ◦ C rather than 70 ◦ C as observed with soluble inulinase [37]. The changes in the optimum temperature after insolubilization of inulinase on various supports were compared in Table 2. The

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Fig. 3. (a) The diagnostic plot between actual and predicated values show the adequacy of model as almost all values on the 45◦ line, (b) the response surface plot shows the effect of process variables on activity recovery.

Table 2 Analysis of variance (ANOVA) showing the interaction between process variables and statistical parameters. Source

Sum of squares

Df

Mean square

F value

p-Value Prob > F

Model A B C BC

2829.736 961.4113 628.3513 804.005 116.64

9 1 1 1 1

314.4151 961.4113 628.3513 804.005 116.64

29.84461 91.25816 59.64376 76.317 11.07159

<0.0001 <0.0001 0.0001 <0.0001 0.012636

Significant

Table 3 Comparison of activity recovery, optimum pH and temperature using different immobilization studies with the present study. Strain

Support

Activity recovery (%)

Aspergillus niger Kluyveromyces marxianus A. ficuum K. marxianus A. niger A. niger K. marxianus A. niger A. niger

Porous cellulose derivatives Macro porous ionic polystyrene beads Porous glass beads Duolite A568 Lentikat, PVA Magnetic sol–gel Sodium alginate beads Chitosan Magnetic chitosan microparticle

96 62 77.2 35.6 48 80 100 83 81.40

Optimum pH and temperature (◦ C) SI

MII

40 4.5 & 50 4.7 & 60 5.5 & 50 4.5 & 60 4.5& 60 5.5 & 55 4.0 & 60 6.0 & 70

5.2 & 50 5.0 & 55 5.0 & 70 5.5 & 55 4.5 & 50 5.0 & 60 5.5 & 55 3.0 & 60 4.6 & 50

References

[9] [10] [11] [13] [15] [17] [18] [19] Present study

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Fig. 4. (a) Temperature activity profile of SI and MII were diagnosed in the temperature range of 60–80 ◦ C. The activity of SI was assayed in pH 6.0 buffer and MII were assayed in pH 4.6 buffer. (b) Half-lives of SI and MII in aqueous solution at different temperature of 60–80 ◦ C. The SI was incubated at pH 4.6 and MII was incubated at pH 6.0 at different temperature of 60–80 ◦ C. All experiments were done in triplicates and the error bars represent the percentage error in each determination.

thermal stability of the enzyme gives us an estimate of the number of times an enzyme can be reused and plays a vital role in designing a bioreactor in a manner that would be cost effective [14]. Thermal stability was determined by incubating at temperatures 60–80 ◦ C at pH 6.0 for SI and 4.6 for MII (Fig. 4b). Identical results were obtained for both forms till 60 ◦ C but at 80 ◦ C, there was vigorous movement of enzyme molecules due to temperature rise and which led to the disruption of bonds in the enzyme structure of soluble enzymes there by a reduced activity at higher temperature [38]. Maximum potential of the two enzyme preparations in terms of their stability is given by the t1/2 value of the two forms of enzymes 593 min for SI while 900 min for MII at 60 ◦ C. With this, it could be inferred that MII were much stable when compared to SI [39]. 3.3. Effect of pH on activity and stability of inulinase The optimum pH for SI was at 6.0 whereas that of MII was 4.6 (Fig. 5a). The reason is that positive charges from the weak basic groups are responsible for the hydrophilic nature of chitosan delivers a higher activity at reduced pH of 4.6. Further, chitosan provides special characteristics when viewed from a technological aspect [24] i.e. operation at lower pH would drastically reduce the risk of microbial contamination thereby ensuring unhindered production of high fructose syrup [19]. The changes in the optimum pH after insolubilization of inulinase on various supports were compared in Table 2. The stability of enzymes to pH ranges 5.0–8.0 was studied and clearly observed that both SI and MII remained active at all pH. The activity of soluble enzyme decreased to 50% in 1200 min when assayed at pH 5.0 whereas MII enzyme depicted an increased resistance and lost 50% activity at 2200 min. At intermediate pH

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Fig. 5. (a) pH activity profiles of SI and MII. The activity of SI (blue solid line) and MII (red solid line) were measured in various buffers of pH ranging from 3.0 to 8.0. (b) pH stability profiles for SI and MII. All experiments were done in triplicates and the error bars represent the percentage error in each determination. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

of 7.0, SI exhibited a t1/2 value of 1200 min whereas insolubilized enzymes had a t1/2 value of 1500 min. pH of 9.0 deactivated the soluble enzymes while insolubilized enzymes sustained 50% of its activity for 600 min (Fig. 5b). This could again be owed to the stabilization of enzyme moiety due to insolubilization and therefore more resistant to pH changes. This increased resistant to changes in pH and temperature is flair to the food industry. 3.4. Enzyme kinetic parameters The effect of change in substrates concentration on reaction rate was studied to measure the affinity of substrates to the enzyme and the rate at which the reaction is catalyzed before and after insolubilization. The Michaelis–Menten constant (Km ) value for the MII was found to be 35.5 mg mL−1 while that of soluble inulinase was 24.2 mg mL−1 (Table 4). Thus, the change in affinity and catalytic efficiency followed by insolubilization was attributed to (a) structural change in enzyme during the covalent binding of the enzyme on cMNPs, and (b) low accessibility of substrate to the active site of the insolubilized enzyme. The Vmax value indicated the intrinsic characters of enzyme which could be hurled by diffusion restraints [40]. The increased Vmax value of the SI (6.25 ␮mol/min) when Table 4 Kinetic constants and catalytic efficiency of SI and MII. Enzyme

KM (mg/ml)

Vmax (␮mol/min)

kcat (s−1 )

SI MII

24.2 ± 0.11 35.5 ± 0.14

6.25 ± 0.72 5.63 ± 0.67

110.8 ± 8.14 99.2 ± 7.38

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4. Conclusion

Fig. 6. Reusability of MII for hydrolysis of inulin to fructose for 15 consecutive cycles. The percentage conversion is represented in terms of relative activity (%). The successive cycles were carried out at a constant temperature and pH. The error bars represent the percentage error.

In summary, A. niger inulinase was successfully insolubilized on to chitosan functionalized MNP by covalent insolubilization using EDAC\NHS chemistry. Statistical optimization technique was successfully used to obtain the concentration of the operating parameters of the insolubilized enzyme towards the hydrolysis of inulin. The ease and efficiency of insolubilization coupled with the simplicity of procuring inulin especially from agricultural waste has rendered the above methodology highly promising for the cost effective production of fructose. Therefore magnetic biocatalyst has greatly bolstered the thermal and operational stability of the enzyme and has the potential to be scaled using magnetically stabilized fluidized bed reactor which would improve its application in an industrial setting. Acknowledgement

compared to the MII (5.63 ␮mol/min) demonstrates the increased rate at which the substrate is converted into product. Gupta et al. [8] and Cattorini et al. [21] also observed a decrease in Vmax , kcat and Km values when producing insolubilized inulinase.

3.5. Operational and storage stability of inulinase Reducing sugar released was studied in batch reactor using both soluble and insolubilized enzyme. There was a similar trend in sugar release by both forms of enzyme during the course of study. It was 23.33 and 20.77 g L−1 by soluble and MII respectively for the first hour. At the end of the reaction, where fructose release from agave inulin was saturated at the 24th hour, it was found to be 34.00 and 30.54 g L−1 respectively. However, MII were fed with fresh substrate to study recyclability of magnetic catalysts. The magnetic biocatalyst highlighted few advantages of promising biotechnological applications because the reutilization, the increase in stability and the use of bioreactors making the separation of the products easier compared to soluble enzyme leading to economical bioprocess. Fig. 6 shows the results of repeated batch hydrolysis of inulin was performed on several consecutive batches for a Jerusalem artichoke inulin containing solution. The percentage of hydrolysis decreased as the number of successive additions of inulin during repeated batch operation. As shown in Fig. 4a, 87% hydrolysis was present in first 5 batches and after the sixth batch run, hydrolysis was decreased dramatically for 10th cycle. This continuous hydrolysis after many cycles could be attributed to the multi-point covalent attachment of enzyme on chitosan because of the presence multiple amine groups [17–22]. The highlighted aspects of the MII further reduce the cost of the HFCS production process which roots them deeply in biotechnology applications. This means, reusability reduces the burden of downstream processing when applied to an industrial operation [23]. In addition, the storage stability of the SI and MII was tested by preparing a stock of enzyme solution and by assaying every day the activity of the same enzyme amount. The storage stability of SI and MII was investigated at 4 ◦ C. It can be seen that the MII was increased to 113% of its original activity after 20 days at 4 ◦ C. After the same time, the soluble inulinase retains only 26% of its initial activity. The results indicate that the MII exhibits good storage stability. This enhanced stability could be attributed to the protective microenvironment and prevention of structural denaturation as a result of the insolubilization of inulinase. Further, magnetic biocatalysts emphasizes that separation of enzymes from products can be done by the application of magnetic field instead of other time consuming methods like filtration or centrifugation which involve physical stress on the biocatalysts.

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