Covalent immobilization of β-amylase onto functionalized molybdenum sulfide nanosheets, its kinetics and stability studies: A gateway to boost enzyme application

Accepted Manuscript Covalent immobilization of β-amylase onto functionalized molybdenum sulfide nanosheets, its kinetics and stability studies: A gateway to boost enzyme application Ranjana Das, Himanshu Mishra, Anchal Srivastava, Arvind M. Kayastha PII: DOI: Reference:

S1385-8947(17)31164-6 http://dx.doi.org/10.1016/j.cej.2017.07.019 CEJ 17285

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

29 April 2017 1 July 2017 5 July 2017

Please cite this article as: R. Das, H. Mishra, A. Srivastava, A.M. Kayastha, Covalent immobilization of β-amylase onto functionalized molybdenum sulfide nanosheets, its kinetics and stability studies: A gateway to boost enzyme application, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.07.019

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Covalent immobilization of β-amylase onto functionalized molybdenum sulfide nanosheets, its kinetics and stability studies: A gateway to boost enzyme application Ranjana Das1¶, Himanshu Mishra2¶, Anchal Srivastava2*, Arvind M. Kayastha1*

1

School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi, India –

221005. 2

Department of Physics, Institute of Science, Banaras Hindu University, Varanasi, India –

221005.

¶

Authors contributed equally

* Corresponding Authors Email id: [email protected] (Prof. Arvind M. Kayastha) [email protected] (Dr. Anchal Srivastava) Ph.: +91-542-2368331 (Prof. Arvind M. Kayastha) Ph.: +91-9453203122(Dr. Anchal Srivastava)

Abstract Present study reports the utilization of molybdenum sulfide nanosheets (MoS 2-NSs) as a novel platform for β-amylase immobilization via glutaraldehyde activation, producing nanobiocatalyst with exotic superiority over the independent enzyme. Confocal microscopy, Fourier transform infrared (FT-IR) spectroscopy, Scanning electron microscopy (SEM) and Atomic force microscopy (AFM) studies demonstrated successful immobilization of β-amylase onto MoS2NSs. Optimizing parameters by Box-Behnken design of Response Surface Methodology, approximately 92% immobilization efficiency was achieved. Thermo-stability, pH stability, reusability and storage stability of immobilized β-amylase were interestingly superior with respect to the soluble enzyme. β-Amylase immobilized onto MoS2-NSs exhibited maximum catalytic activity at the same pH and temperature as the soluble enzyme but with broadening in the range of parameters. In addition, the immobilized enzyme retained almost 80% residual activity, even after 10 reuses. Immobilized enzyme had around 83% residual activity upon storage over a period of 50 days. Changes in Michaelis-Menten constant after immobilization, point that some of the active sites of enzyme were inaccessible to substrate due to strained enzyme structure upon immobilization. The results obtained here suggest that the β-amylaseMoS2-NSs system could be used in industrial processes permitting broader temperature and pH ranges. Besides, the non-toxic nature of matrix, long-term storage and reusability of nanobiocatalyst could be magnificently used for the production of maltose in food and pharmaceutical industries. Keywords β-Amylase, molybdenum sulfide nanosheets, immobilization, nanobiocatalyst, steady state kinetics

Highlights S2-NSs were synthesized by using eco-friendly and facile hydrothermal method. Covalent immobilization of β-amylase onto functionalized MoS2-NSs was investigated. Box-Behnken design optimized parameters, resulting into 92% immobilization efficiency.  XRD, RAMAN, SEM, TEM, FT-IR, AFM and confocal microscopy confirmed immobilization. Improvement of steady state kinetics of β-amylase was observed due to immobilization.

Graphical abstract

1. Introduction Enzymes, being highly effective and selective in catalysis, find widespread use in food, pharmaceuticals and other industrial fields owing to its mild and green reaction. Their exquisite activity, specificity and selectivity assure extravagant application in biocatalysis, biosensors and biomedicines [1-5]. In recent years, glycosidases have drawn immense consideration in biotechnological potential of enzymes like cellulases and amylases in degradation of starch [6, 7]. Amylases act on glycosidic linkages of starch and related polysaccharides. These enzymes are studied extensively from microorganisms, higher plants and animals. Starch is one of the major constituents of food stuff, providing huge amount of calorie, besides, it serves as a raw material for the production of sweeteners, adhesive, thickening and binding agent, etc [8, 9]. An assemblage of amylolytic enzyme of diverse specificity is required for starch degradation to maltodextrins, maltose and glucose so that it could be used further [10]. One of the important enzymes of starch based and food industry is β-amylase (E.C. 3.2.1.2.), skilled to break down polyglucans from the non-reducing end, producing maltose as exclusive end product with traces of β-limit dextrins. This enzyme finds application in baking and brewing. Since it resigns at maltose, β-amylase is used in the structural studies of starch and related polysaccharides [11-13]. The end product maltose, unlike ordinary sugar, is less sweet but works better with flour than sucrose. In the food industry, it extends the shelf-life of certain products [14]. However, in any enzyme technology, the cost consuming material is the enzyme itself, compounded by the fact that it is consequently lost in the reaction mixture. A tedious, downhill process is needed to recover the enzyme. Also, the operational instability and reusability constrains industrial approach of soluble enzyme. These drawbacks could be overcome by immobilizing the enzyme onto some insoluble matrices, which without altering the properties of

enzyme much, are as effective in catalysis as the free counter-part [15-17]. Immobilization is a joint conjuncture of matrix properties and enzyme properties. An ideal matrix for immobilization should possess the properties such as resistance to compression, hydrophilicity, low biodegradability, good mechanical and rheological characteristics, non-toxicity, high diffusion of substrates and product through it, etc [18, 19]. Owing to their large surface to volume ratio, nanomaterials have proven their significance in the field of bio-imaging, nanomedicine, drug delivery, biomolecule functionalization, enzyme loading etc [20, 21]. Enzyme loading using nanoparticles, nanofibers, mesoporous silica etc. as matrix has been reported earlier [22]. Presently, 2D materials are the prominent candidate among research community in different fields of science and technology. The most well-studied 2D material is graphene and its derivatives (graphene oxide, reduced graphene oxide etc.) till date. Graphene, having two accessible sides, provides large surface area and hence it has been intensively investigated for the immobilization of various biomolecules [23]. Pristine graphene, due to absence of functionalities, does not provide suitable platform for the loading of biomolecules while graphene derivatives do so in presence of functionalities over them [24]. Srivastava et al., have used graphene oxide as matrix and achieved a maximum immobilization of enzyme up to 84% [13]. Although graphene oxide provides good enzyme loading yet there are several disadvantages with it, such as synthesis of graphene oxides is time consuming and also requires heavy acid treatment, which is not favorable [25]. Also, graphene derivatives have poor electrical/thermal conductivity which restricts its application as electrode after biomolecule loading for sensing purposes. In this series recently transition metal dichalcogenides (TMDs) have gained attention as an alternative of graphene [26]. TMDs family includes MoS2, MoSe2, WS2, WSe2 etc. Among the various TMDs, MoS2 is the most studied one in the field of next

generation electronics, optoelectronics and biology. Monolayer MoS 2 have three atomic layers, one Mo layer sandwiched between two Sulfur layers. This structure of MoS 2 makes it more robust than pristine graphene to be folded. Thus, it is believed that MoS2 will have small amount of agglomeration in solution in comparison to graphene. These advantages of MoS 2 over graphene and its derivatives make it suitable candidate for the enzyme immobilization. The other motivation behind the present study is that till date to the best of our knowledge there is no report in the literature about the enzyme immobilization and its characterization using MoS2-NSs as matrix. Process optimization is an essential element of experimentation to find out the best operating conditions and elevate the desired response. Response Surface Methodology (RSM) is a statistical tool used to quantify the variable input parameters and the corresponding output parameters. Box-Behnken design of RSM has maximum proficiency for an experiment involving three factors at three levels; moreover, the numbers of experiments administered are less in comparison to the Central Composite design. RSM by Box-Behnken design permits easy assessment of the statistical significance of the variables effect being studied and predicts the interaction between the variables. Optimization of process by RSM involves determination of coefficients by substituting the experimental data to the response function; further, the generated 2D and 3D plots explicitly give an idea of the dominating process variable over the other. Additionally, the plot also exhibits the course of variables interaction in the process [27, 28]. β-Amylase from plants are usually used for producing maltose, but since these enzymes are expensive and fairly unstable, several efforts have been made to prepare other alternatives. We present first report on immobilization of β-amylase from peanut onto functionalized molybdenum sulfide nanosheets (MoS2-NSs), prepared by hydrothermal method. Glutaraldehyde

(pentane 1, 5-dial) modified MoS2-NSs surface, serves as a cross-linker. It binds to the functional moieties on the surface of nanosheets (NSs) through its one arm (-CHO) and binds to enzyme via lysine amino acid through its other arm. Box-Behnken design of RSM has been exploited to optimize immobilization parameters which resulted into a higher immobilization efficiency than the earlier report on graphene oxide [13]. Furthermore, a comparative study of β-amylase on MoS2-NSs and free β-amylase was performed and the results showed that immobilized enzyme has better stability than the free enzyme. 2. Materials and methods 2.1 Materials Sodium molybdate (Na2MoO4.2H2O) and L-cysteine (C3H7NO2S) were purchased from Himedia, India. Peanut (Arachis hypogaea) seeds were purchased from agricultural seed store. The chemicals for preparing buffers were of analytical or electrophoretic grade obtained from Merck Eurolab GmbH Damstadt, Germany. Rest all the chemicals were obtained from Sigma Chem. Co. Milli Q (MQ) water with resistance >18MΩ cm was used throughout the experiment. All the steps were performed at 4 °C and centrifugation was carried out at 8,720 g, unless stated otherwise. 2.2 Enzyme Preparation Soaked peanut seeds (50 g) were coarsely crushed using Waring blender in chilled extraction buffer (50 mM sodium acetate buffer, pH 5.5), and then squeezed through two layers of prewashed muslin cloth followed by centrifugation for 20 min. Obtained crude extract was subjected to 40 % acetone fractionation at -15 °C. Pellet so formed was discarded and the supernatant was subjected to acid precipitation by lowering the pH to 4.0. The precipitate was then centrifuged and pH of the supernatant was brought back to 5.5. This preparation was further

used for affinity precipitation as described by Silvanovich and Hill [29] with minor modifications. The final preparation was used for all immobilization studies. 2.3 Protein assay The amount of protein was determined by the Folin’s Lowry method [30], using crystalline Bovine Serum Albumin as standard protein. Unbound protein was quantified by the difference of protein loaded and that in washing solutions after immobilization. 2.4 Enzyme activity assay The hydrolytic activity of free and immobilized enzyme was estimated by following Bernfeld’s method [31] using UV-VIS double beam spectrophotometer (JASCO ETC-717, Japan), by measuring the absorbance at 540 nm of the reduction of DNS from β-maltose, released by the hydrolysis of starch. Reaction mixture consisted of suitably diluted enzyme (0.5 mL) and 1.0 % starch (0.5 mL) prepared in 50 mM sodium acetate buffer, pH 5.0. Following incubation at 37 °C for 3 min, reaction was stopped by the addition of 3, 5 dinitrosalicylic acid and then the test tubes were placed in boiling water bath for 5 min. After cooling down the test tubes to room temperature, 10 mL of MQ water was added and absorbance was recorded. MoS2-NSs coupled with β-amylase were incubated with 0.5 mL of 1% starch solution prepared in 50 mM sodium acetate buffer, pH 5.0 for 3 min at 37 °C, followed by centrifugation at 6000 rpm for 2 min at 4 °C. Supernatant was pipette out in separate test tubes; 1 mL of 3, 5 dinitrosalicylic acid was added, followed by placing it in boiling water bath for 5 min. Absorbance was recorded at 540 nm after diluting with 10 mL of MQ water. One unit of β-amylase activity is defined as the amount of enzyme that releases 1 µmol of βmaltose from starch in 3 min, under standard test conditions.

2.5 Synthesis of MoS2-NSs A facile and eco-friendly hydrothermal method with slight modification has been used for the synthesis of MoS2-NSs (Fig. 1) [32]. Na2MoO4.2H2O (0.25 g) was dissolved in 25 mL of MQ water and stirred for 10 min at 40 °C. In a separate beaker, 0.50 g of L-cysteine was dissolved in 25 mL MQ and stirred for 10 min at 40 °C. Thereafter, both the solutions were mixed and maintained stirring for 10 min at 40 °C. During the process, HCl (0.1 M) was added to the solution to maintain its pH approximately at 5. Finally, this solution was transferred into a 100 mL capacity stainless steel lined Teflon autoclave. This autoclave was kept into oven, maintained at 220 °C for 30 h. After the completion of the reaction, the autoclave was naturally cooled down. The black color powder was taken out, washed with MQ water and ethanol thrice and dried in open air at 60 °C for 12 h. 2.6 Enzyme immobilization Attachment of enzyme onto the functionalized MoS2-NSs was assisted by a cross-linker glutaraldehyde. MoS2-NSs dispersion of 1 mg/mL was prepared by dissolving functionalized MoS2-NSs in 50 mM sodium phosphate buffer, pH 7.0, sonicated for 15 min and then divided into 17 aliquots according to the design of experiment (Table 1). These aliquots were equilibrated in the same buffer overnight followed by thorough rinsing with the same buffer. MoS2-NSs dispersion was then treated with glutaraldehyde and kept in dark for 4 h at room temperature. Thereafter the aliquots were washed with the same buffer to get rid of unbound glutaraldehyde, followed by incubation with enzyme under dark condition at 4 °C, overnight. The unbound enzyme was washed by thorough rinsing with chilled buffer and successful binding was checked by activity assay under standard conditions as described before. Immobilized enzymes were separated by centrifugation, followed by washing with phosphate buffer (50 mM,

pH 7.0) each time and stored at 4 °C until used. List of the independent variables according to the experimental design and their corresponding response are shown in Table 1. 2.7 Immobilization efficiency The immobilization efficiency was calculated using following formula:

[1]

2.8 Experimental setup and statistical analysis Box-Behnken design of RSM applying multivariate approach leads to a considerable improvement in the method development, by operating fewer experiments, which otherwise leads to high cost, resulting in better optimization of variables. Prior to aiming the multivariate statistical design, the values of factors affecting the process of immobilization were determined by carrying out some preparatory experiments (data not shown). Based on these experiments, a three level study of the significant factors and their interactions were studied by using BoxBehnken design which led to an experiment consisting of 17 trials. The factors and their levels selected for carrying out immobilization of β-amylase onto functionalized MoS2-NSs were: amount of MoS2-NSs (500, 1000, 1500 µg): concentration of glutaraldehyde (2.0, 3.0, 4.0 %) and amount of enzyme (200, 300, 400 µg). ‘Design Expert’ software (version 9.0, Stat-Ease Inc, Minnealpolis, USA) was used for experimental data designing and analysis. The mathematical relationship of the responses to the variables can be calculated by the following quadratic polynomial equation: Yi=βo+ΣβiXi+ΣβiiXi2+ΣβijXiXj

[2]

where, Yi is the predicted response, XiXj are input variables which influence the response variable Yi; βo is the offset term; βi is the ith linear coefficient; βii is the ith quadratic coefficient and βij is the ijth interaction coefficient. The validity of the model was statistically analyzed by ANOVA (Analysis of Variance) based on Fischer’s F-test, associated probability, correlation coefficient R and lack of fit test. 2.9 Optimum pH evaluation The effect of pH on the activity of free and immobilized enzyme was studied at 37 °C in various buffers in the pH range from 3.5 to 8.5 using starch as substrate. The buffers used were 50 mM sodium acetate (pH 3.5-5.5), 50 mM sodium phosphate (pH 6.0-6.5) and 50 mM Tris-HCl (pH 7.0-8.5). 2.10 Optimum temperature and thermal stability assay The optimum temperature was studied by carrying out assay procedures at temperatures between 20 to 90±1 °C, in 50 mM sodium acetate buffer, pH 5.0. Thermal stability was determined by incubating the enzymes at 60 °C for different time interval, followed by residual activity assay under standard conditions. 2.11 Determination of Kinetic parameters To perceive the effect of substrate concentration on immobilized enzyme, starch was varied in the range of 0.5-10.0 mg/mL. The data thus obtained was fitted in Lineweaver-Burk plot and the values of Km and Vmax were obtained. 2.12 Storage stability and reusability

Free enzyme and enzyme immobilized onto MoS2-NSs were stored at 4 °C for a period of 50 days to analyze the stability of enzyme. Residual activity of the enzyme was assayed after regular interval of time as mentioned before. For the assessment of reusability of immobilized enzyme, it was repeatedly used for 10 times and the residual activity was measured with starch as substrate. Following next cycle at the end of each cycle, the aqueous system containing enzyme onto MoS2-NSs was washed with buffer (50 mM phosphate pH 7.0) and centrifuged to get rid of any impurity. This medium was suspended again in a fresh batch of reaction medium. The relative activity of the first assay was defined as 100 %. 2.13 Characterizations Structural and microstructural characterizations of the as synthesized MoS 2-NSs were performed using X-ray diffractometer (XRD, Philips Pan analytical X’Pert powder), transmission electron microscope (TEM), high resolution transmission electron microscope (HRTEM, FEI-Technai G2 F20) operated at accelerating voltage of 200 kV and scanning electron microscope (SEM, Zeiss Germany). Enzyme immobilized samples have also been characterized by using SEM. Raman spectrum of the MoS2-NSs was recorded by Renishaw, In-via spectrometer UK, to find the characteristic Raman modes of MoS2 and to estimate the number of layers. FT-IR spectroscopy (Varian Excalibur 3000, Palo Alto, CA) was performed to determine different functional groups on MoS2-NSs and to show the binding of enzyme over MoS2-NSs via glutaraldehyde crosslinker. Confocal laser scanning microscopy (Carl Zeiss 800) analysis was conducted to determine the fluorescence signal from flourescein isothiocynate (FITC) isomer labeled βamylase after immobilization onto MoS2-NSs. β-Amylase loaded MoS2-NSs and bare MoS2-NSs were stained with a solution of FITC isomer (1 mg/mL in dimethyl formamide (DMF)). Immobilized enzyme was stored in potassium phosphate buffer (0.02 mol/L pH 8.5). 100 µL of

FITC/DMF was added drop-wise to the β-amylase loaded MoS2-NSs and bare MoS2-NSs. Following incubation in dark at 4 °C for 30 min, unlabeled FITC was washed extensively with MQ water. To determine the surface topography of bare as well as enzyme immobilized MoS 2NSs, AFM images were collected in semi contact or tapping mode by nanoscope (NT-MDT Russia, NTEGRA PRIMA model) equipped with silicon tip. Scanning was performed within an area of 6.1 µm x 6.2 µm for bare MoS2-Ns’s and 8.4 µm x 8.8 µm for enzyme immobilized MoS2-Ns’s at a scan rate of 0.5 Hz. Samples were sonicated and deposited onto silicon wafers from aqueous suspension by spin coating method for AFM measurements. 3. Results and discussion β-Amylase was extracted and purified from peanut applying acetone fractionation, acid precipitation and affinity precipitation. Final preparation was found to be homogenous on SDSPAGE and showed a Vmax of 363.63 µmoles/min/mg. 3.1 Characterization Structural and microstructural characterizations of the synthesized MoS 2-NSs were carried out using XRD, Raman, TEM and HRTEM (Fig. 2). XRD pattern for MoS2-NSs is shown in fig. 2(A). The four most intense peaks at ~14.32°, 30.40° and 40.92° corresponding to the planes (002), (100) and (103), have been successfully indexed for the MoS2 (JCPDS Card no. 371492) having hexagonal phase and space group P63/mmc [33]. The XRD peak position corresponding to the (002) plane reveals an interlayer spacing of (d 002 =) 0.618 nm. MoS2-NSs have also been characterized using Raman spectrometer to obtain the characteristic vibrational modes. A typical Raman spectrum of MoS2-NSs is shown in Fig. 2(B). It has two Raman peaks at ~384.02 cm-1 and 409.39 cm-1 ; corresponding to in plane (E12g mode) and out of plane (A1g

mode) vibration of MoS2-NSs. The spacing (Δ) between the E12g and A1g peak positions is found to be ~25.37 cm-1, which confirms that the synthesized MoS2-NSs are multilayer in nature [34]. Fig. 2 (C) shows the TEM image of MoS2-NSs at a scale bar of 20 nm. The image revealed wrinkled as well as flat region of MoS2-NSs. HRTEM image (Fig. 2D) showed the presence of multilayer sheets. Inset in the Fig. 2(D) showed the Fast Fourier transform (FFT) pattern and interlayer spacing of the nanosheets. FFT pattern confirmed the stacking of nanosheets along zdirection. Interlayer spacing was found to be ~0.65 nm, which corresponds to the (002) plane of MoS2-NSs. In the HRTEM image, interlayer spacing of 0.27 nm, corresponding to the plane (100) has been shown [35]. To get an insight of the interaction between functionalized MoS2-NSs, FT-IR spectra of native, glutaraldehyde treated and enzyme immobilized MoS2-NSs were taken. A sharp peak was observed at 467 cm-1(Fig. 3 B), which is characteristic of Mo-S vibration [36]. Peak at 1636.45cm-1 (Fig. 3A) correspond to NH2 scissoring of primary amine (bending). Peak of 3448.23 cm-1 (Fig. 3A) correspond to strong O-H bond (stretching) and amines [37], thereby confirming the presence of amino and oxygen containing functional groups on MoS 2-NSs. Next, the functionalized MoS2-NSs were treated with glutaraldehyde where one arm of glutaraldehyde binds to the –NH2 and –OH functional groups of the functionalized MoS 2 and other arm remains free for attachment with enzyme via amino group. The attachment of –NH2 group with –CHO group of glutaraldehyde occurs through formation of Schiff base, which is a covalent bond in nature. This interaction was confirmed by the observed peak at 1645.23 cm-1(Fig. 3C) which corresponds to –C=N stretching of imines. Finally, the coupling of enzyme to the free arm of glutaraldehyde via amino group was confirmed by bands at 1646.67 cm-1(Fig. 3D) representing carbonyl amide I bond [17]. Other FT-IR peaks such as 2072 cm-1(Fig. 3A), 600 cm-1, 945 cm-1,

1330 cm-1 (Fig. 3C) and 730 cm-1, and 1095 cm-1 (Fig. 3D) correspond to N=C, C-H bend, O-H bend, N-O symmetric stretch, C-H out of plane and C-N stretch vibrational modes, respectively. Immobilization of enzyme over MoS2-NSs was also confirmed by SEM analysis. SEM image of as synthesized MoS2-NSs, before and after enzyme immobilization is shown in Fig. 4. Fig. 4(A) shows the SEM image of the synthesized bare MoS2-NSs before buffer treatment. Fig. 4(B) shows the SEM image of the bare MoS2-NSs after the buffer treatment. It reveals that after the buffer treatment, nanosheets get agglomerated in the form of small sphere and appear like a fluffy structure. White patches appearing in the SEM image of enzyme immobilized onto MoS 2NSs confirms the presence of enzyme over MoS2-NSs (Fig. 4C), being supported by other works [38]. The topography of the bare as well as β-amylase immobilized MoS2-NSs has been examined using AFM technique in tapping or semi-contact mode (Fig. 5(A) and (B), respectively). The 2D and 3D image of MoS2-NSs confirmed that MoS2-NSs were only few nanometers in thickness. Fig. 5(C) and (D) showed the 3D view of the surface morphology of the bare as well as enzyme immobilized MoS2-NSs, respectively. Results clearly reveal the immobilization of enzyme on the surface of the MoS2-NSs. Enzyme was randomly oriented, covering the entire surface of nanosheets. Similar results are reported earlier with other enzymes [39]. Meanwhile, we performed confocal laser scanning analysis for bare MoS 2-NSs and enzyme loaded MoS2-NSs. Flourescein isothiocyanate is one of the most extensively used fluorochrome intermediary agent for protein labeling. A heterogeneous fluorescence distribution was observed in case of MoS2-NSs bio-composite upon treatment with FITC isomer while no fluorescence was observed in bare MoS2-NSs, which was without enzyme (Fig. 6).

3.2 Optimization of immobilization The attachment of enzyme over and onto MoS2-NSs is shown schematically (Fig. 7). Functionalized MoS2-NSs treated with glutaraldehyde binds to the amino, carboxyl or hydroxyl functional group present on the nanosheets through its one arm. Other arm (-CHO) binds to enzyme. Use of glutaraldehyde cross-linker in the MoS2-NSs allows tethering of enzyme molecule, resulting into development of nano-bioconjugate with improved kinetic properties and stability [40-42]. Obtaining optimum conditions for immobilization are essential criteria considering its industrial utility so as not to waste materials, time and labor. Central Composite Design and Box-Behnken design of RSM are frequently used in this respect. Box-Behnken design finds more efficiency and accuracy than Central Composite Design [43]. Therefore, in the present study Box-Behnken design was selected for process optimization. Based on the initial experiments (data not shown), process was designed within operational ranges of variables having maximum effect on response. The details of experiment are described in Table 1 as actual immobilization percentage (±0.5). A model was developed based on these experiments to obtain maximum immobilization response within the given range of variables and following points were determined: MoS 2-NS’s: 1202.47 µg; Glutaraldehyde: 3.4%; Enzyme: 225.68 µg; Immobilization: 90.42%. Affirmation of these predicted values were carried out experimentally and around 92.0% immobilization was achieved (specific activity of soluble and immobilized enzymes being 312 U/mg and 286 U/mg, respectively), which was in accordance to the predicted values. These results also generate 3D and contour plots to show the trend of different parameters on the response i.e. immobilization (Fig. 8) and also the influence of each parameter over the other. The concluding equation in terms of actual factors, which affects the process of immobilization, can be condensed as:

Immobilization (%) = 87.95+0.60×A+6.95×B-16.08×C+2.01×AB+5.50×AC+1.97×BC14.36×A2-16.24×B2-9.03×C2 where, A is MoS2-NSs (µg), B is glutaraldehyde concentration (% v/v) and C is enzyme (µg). Analysis of variance (ANOVA) determines the accuracy and significance of the quadratic model. ANOVA for the response surface is shown in Table 2. The model F-value of 564.69 determines the accuracy of generated model and that there is only 0.01 % chance that this value could have occurred due to noise. The lack of fit F-value of 2.20 states its irrelevancy relative to pure error. Non-significant lack of fit is good; we want the model to fit. The “Adj R-squared” of 0.9968 is in reasonable agreement with “Pre R-squared” of 0.9854. Adequate precision measures signal to noise ratio and a value greater than 4 are desirable. 3.3 Steady state kinetics 3.3.1 Optimum pH The process of immobilization is often accompanied by alteration in kinetics behavior because of its microenvironment. In this study, the optimal pH of free and immobilized enzyme was determined in the range of 3.5 to 8.5. As evident from the Fig 9(A), maximum activity of both the enzymes was recorded at 5.0 pH with similar trend, by varying pH. However, immobilized enzyme had broader pH profile indicating that the rate of catalysis becomes less sensitive to pH. Compared to soluble, immobilized enzyme showed better activity in alkaline range with retention of around 80% relative activity at pH 8.5, while that of its free counterpart was only 30 %. This property is attributed to the enzyme and matrix and to their interaction with each other. In general, β-amylase immobilized onto MoS2-NSs exhibited better adaptability to pH, similar to the results in previous reports [44].

3.3.2 Optimum temperature and thermal stability The immobilized enzyme did not show any change in temperature upon immobilization, when assayed with starch under our test conditions. Immobilized enzyme withstood significant enhancement in tolerance to high temperature, besides broadening. A sharp decline in the activity of soluble enzyme was observed at 80 °C, while the immobilized enzyme retained around 70% activity even at 90 °C (Fig. 9B). Similar changes in temperature optima were also reported by other workers [6, 45]. Thermal stability is benchmark in determining the industrial pursue of enzyme. Whilst studying the thermal stability of β-amylase from peanut immobilized onto MoS2-NS as well as free enzyme at 65 °C, it was found that MoS2-β-amylase retained 85% residual activity after 30 min of storage and around 50% activity after 100 min. The soluble enzyme was estimated to have 68% and 25% values at the same temperature for two incubation periods (data not shown). The decreased distortion of enzyme at high temperature upon immobilization could be the result of conformational rigidity, which allows the enzyme to resist more temperature for longer time. 3.3.3 Kinetic parameters Michaelis-Menten kinetics was studied by varying substrate concentration. A difference in Km value was observed after immobilization as it changed from 1.29 mg/mL to 3.01 mg/mL and Vmax value dropped to 105.26 µmoles/min/mg (Table 3). These changes are attributed to substrate diffusional constrains, steric hindrance of the active site by the support or the loss of enzyme flexibility necessary for substrate binding. Similar results were also observed in case of fenugreek β-amylase immobilized onto chitosan coated PVP and chitosan PVC blend [46]. 3.3.4 Storage stability and Reusability of immobilized β-amylase on MoS2

Enzymes altogether are expensive bio-products, thus unaffordable to be wasted in each cycle of reaction. Knowledge of storage stability of industrial enzymes is one of the major concerns for commercialization because the enzyme must be stabilized for storage and shipping. Hence, stabilization by immobilization is one of the essential topics of research to control the economy of any bioprocess. Storage stability is the intrinsic property of enzyme and it is improvised upon immobilization to any carrier as a consequence of reduction in the rate of denaturation of enzyme. The immobilized β-amylase was stored in wet condition (50 mM phosphate buffer, pH 7.0) at 4 °C and was found to be very stable over a period of 50 days with retention of 83% residual activity, while the soluble enzyme was only 50% active under similar conditions (Fig. 10 A). Other workers from this laboratory have also reported enhanced storage stability upon immobilization to nanomatrices [47]. Reusability is another criterion in determination of industrial applicability of enzymes owing to their expensive and laborious downstream processing. This characteristic of immobilized enzyme is of immense importance in the arena of enzyme biotechnology. MoS2 immobilized βamylase retained around 80% enzymatic activity after 10 uses, at an interval of 1 h each (Fig. 10 B). This feature of immobilized enzyme is a result of high enzyme loading onto MoS2-NSs. At each cycle of reaction, only a fraction of the immobilized enzyme is encountered under normal conditions. Substrate molecule probably penetrates a short distance in the immobilized enzyme system, leaving an ample amount of enzyme in the center that will be taking part in reaction for the next cycle. This condition is known as “Zulu effect”, due to which little loss was observed on reusability of enzyme for 10 times. Frequent administration of substrate to the immobilized enzyme weakens the binding of enzyme and nanomatrix, ensuing leaching of enzyme and

subsequent loss in activity. Also, the distortion of active site reduces the catalytic efficiency of immobilized system [48]. Enzyme catalytic performance of various catalysts immobilized on 2D materials reported so far has been shown in supplementary material Table S1. 4. Conclusion MoS2-NSs were successfully synthesized using facile and eco-friendly hydrothermal method. The synthesized nanosheets provide a novel surface for protein immobilization via functionalization and simple surface treatments. The MoS2-NSs covalently immobilized βamylase using glutaraldehyde as a cross-linker with high protein loading capacity and statistically designing the experiment through Box-Behnken design. Notably, β-amylase exhibited impressive stability, even after 10 reuses with retention of 80% residual activity. The betterment of enzyme upon immobilization in terms of pH, temperature and stability confers wider range of application for maltose production and accordingly, suitable for food and pharmaceuticals industries. We believe the application of functionalized MoS2-NSs as a promising candidate for extension to other enzymes immobilization in industries. Acknowledgement RD is highly thankful to the Indian Council of Medical Research for providing financial support in the form of Junior and Senior Research Fellowship (3/1/3/JRF-2012/HRD-34 80223). HM is thankful to the UGC, New Delhi, India for providing fellowship. AS is thankful to the DST, India (DST Purse Scheme 5050 &DST/TSG/PT/2012/68) for providing financial assistance.

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Figure captions: Figure 1: Schematic representation for hydrothermal synthesis of MoS 2-NSs. Figure 2: (A) XRD, (B) Raman, (C) TEM and (D) HRTEM characterization of MoS 2-NSs. Figure 3: FT-IR spectra of MoS2-NSs (A), magnified view of the yellow encircled region to monitor the peak corresponding to the Mo-S vibration (B), FT-IR spectra of the glutaraldehyde treated MoS2-NSs (C) and FT-IR spectra of Enzyme immobilized MoS2-NSs (D). Figure 4: SEM images of (A) as synthesized bare MoS2 nanosheets (B) buffer treated bare MoS2-NSs and (C) β-amylase from peanut immobilized MoS2-NSs. Figure 5: (A) and (B) show the AFM image of the surface topography of buffer treated bare MoS2-NS and enzyme immobilized MoS2-NS, respectively. (C) and (D) show the 3D view of the bare and enzyme immobilized MoS2-NSs, respectively. Figure 6: Confocal microscopy image (63X) of the FITC treated bare (A) and enzyme immobilized MoS2-NSs (B). Figure 7: Schematic representation of covalent immobilization of β-amylase onto functionalized MoS2-NSs via glutaraldehyde as a cross-linker. Figure 8: Response surface and contour plots showing effects of various parameters on immobilization (%) and the predicted optimal response. 3D and contour plot for (A, B) the effect of glutaraldehyde % and amount of functionalized MoS 2 on immobilization (%), (C, D) the effect of amount of enzyme concentration and glutaraldehyde % and (E, F) the effect of amount of functionalized MoS2 and concentration of enzyme on immobilization (%).

Figure 9: Effect of pH (A) and temperature (B) on the activity of free and immobilized βamylase. For both forms of enzyme, starch prepared in 50 mM of respective buffers was used to determine optimum pH. Effect of temperature on the activity of enzymes were studied by carrying out assay procedure at different temperature using starch as substrate in 50 mM sodium acetate buffer pH 5.0. Figure 10: Storage stability of soluble and immobilized β-amylase on MoS2-NSs (A). Reuse of β-amylase-MoS2-NSs system (B) was performed by assaying activity for 10 times at an interval of 1 h each. After each assay, system was thoroughly rinsed with chilled buffer. Assay was performed at 37 °C, pH 5.0.

Figure 1

Figure 2

Figure 3

A As synthesized bare MoS2 nanosheets

5μm

B

C Agglomerated buffer treated bare MoS2 nanosheets

5μm Figure 4

Enzyme immobilized MoS2 nanosheets

5μm

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Run

Glutaraldehyde Functionalized (%v/v) MoS2 (µg) 3 3 2 3 3 3 4 3 4 4 2 2 3 3 3 2 4

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

1000 1000 1000 1000 1000 500 1500 500 1000 1000 1000 1500 1000 1500 1500 500 500

Enzyme (µg)

Immobilization actual

% predicted

300 300 200 300 300 400 300 200 200 400 400 300 300 400 200 300 300

88.23 89.14 73.89 87.08 87.32 42.13 65.12 85.64 83.28 55.42 38.14 50.63 87.98 54.48 75.98 53.60 60.06

87.95 87.95 74.78 87.95 87.95 42.38 65.91 85.54 82.74 54.53 38.68 49.99 87.95 54.58 75.73 52.81 60.70

Table 1: Box-Behnken experimental design for independent variables and their corresponding responses (% immobilization) for immobilization of β-amylase on MoS2-NSs. Source Model A-MoS2 B-Glutaraldehyde C-Enzyme AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error Cor Total

R2= 0.9986

Sum of Squares 5082.21 2.86 283.46 2067.89 16.12 121.11 15.56 868.40 1109.96 343.43 7.03

df 9 1 1 1 1 1 1 1 1 1 7

Mean Square 564.69 2.86 283.46 2067.89 16.12 121.11 15.56 868.40 1109.96 343.43 1.00

4.38

3

1.46

2.65 5089.24

4 16

0.66

Adj R2 = 0.9968

Pred R2= 0.9854

F Value 562.36 2.84 282.29 2059.36 16.05 120.61 15.50 864.82 1105.38 342.01

p-value

Prob> F significant

< 0.0001 0.1356 < 0.0001 < 0.0001 0.0051 < 0.0001 0.0056 < 0.0001 < 0.0001 < 0.0001 not significant

2.20

0.2301

Df =Degree of freedom

Table 2: Analysis of variance (ANOVA) for response surface model pertaining to % immobilization. Enzyme preparation

Km (mg/mL)

Vmax (µmoles/min/mg)

Free enzyme

1.28

363.63

Enzyme immobilized onto MoS2-NS

3.38

105.26

Table 3: Apparent kinetic parameters of free and immobilized enzyme onto MoS 2-NS.