Chicken feather derived novel support material for immobilization of

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Chicken feather derived novel support material for immobilization of laccase and its application in oxidation of veratryl alcohol Sunil Kumar Suman, Padma Latha Patnam, Sanjoy Ghosh, and Suman Lata Jain ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05679 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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ACS Sustainable Chemistry & Engineering

Chicken feather derived novel support material for immobilization of laccase and its application in oxidation of veratryl alcohol Sunil Kumar Sumana, Padma Lata Patnamb, Sanjoy Ghoshc, Suman Lata Jainb* aBiofuel

Division, CSIR-Indian Institute of Petroleum, Mohkampur, Haridwar Road, Dehradun, Uttrakhand, 248005, India

bChemical

and Material Science Division, CSIR-Indian Institute of Petroleum, Mohkampur,Haridwar Road,Dehradun, Uttrakhand, 248005,India

cBiotechnology

department, Indian Institute of Technology, Roorkee, Haridwar Highway, Uttrakhand , 247667,India *Phone: +91-01352525788 *Email: [email protected]

Abstract Application of biocatalyst at an industrial scale primarily depends on its intrinsic properties, nature of support materials and scalability of the catalyst. Support materials play an important role in the biocatalytic performance with their mechanical and thermal properties, accessibility, non-toxicity, and ease of derivatization for immobilizion of enzyme. Chicken feather, a readily available poultry waste material, was processed and modified for enzyme immobilization. Free Trametes maxima laccase (TML) was immobilized on the amino-functionalized chicken feather particles (TML@ACFP) and an immobilization yield of 74.24 % was achieved. Immobilization improved the pH optimum from 3.0 (TML) to 4.1 (TML@ACFP) and temperature optimum by 5°C. The kinetics and thermodynamics of thermal inactivation of free TML and immobilized TML@ ACFP were studied over the temperature range from 55°C to 65°C. The apparent half-life (t1/2) and decimal reduction time (D-value) for TML was found to be 154.9 min and 514.8 min and 256.8 min and 853.3 min for TML@ACFP respectively at 55°C. The activation energy for deactivation (Ed) was found to be 117.48 kJ/mole and 137.85 kJ/mole for TML and TML@ACFP, respectively. Gibbs free energy (ΔG) and change in enthalpy (ΔH) were increased from 106.58 kJ/mole and 114.75kJ/mole for TML to 107.96 kJ/mole and 135.12 kJ/mole for TML@ ACFP respectively demonstrating its higher stability. The biocatalytic transformation was performed with TML@ ACFP for the oxidation of lignin model compound veratryl alcohol. So far, this is the first strategy that uses chicken feather waste derived novel support material for immobilization of enzyme and its application in the biotransformation.

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KEYWORDS: Chicken feather waste, Biotransformation, Immobilization, Laccase, Lignin oxidization, Veratryl alcohol. Introduction Enzymatic biocatalysis has caused a paradigm shift in the chemical industry paving the way for sustainable processes and green economy with minimal waste generation. Such transformations require an efficient biocatalyst that can carry out reactions in bulk with higher specificity and selectivity. In this context, laccases (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductases) are multi-copper proteins that catalyze the oxidation of a variety of phenolic compounds, such as o- and p-diphenols, polyphenols and aromatic amines.1 Laccases have four copper ions in three centers that are responsible for the transfer of electron from substrate to molecular oxygen thereby reducing it to water.2 Laccase catalyzed oxidation of substrate, generates radicals that further polymerises to form dimmers, oligomers or polymers. In case of methoxy or halogenated phenolic compounds, the oxidation is often associated with partial demethylations and dehalogenations.1 The enzyme has potential to regulate a wide range of reactions with well-established applications in various areas including chemical synthesis3- 4 , dye decolorization,5

- 6

contaminant removal7

- 9

and detoxification of

soils and waste water.10-11 This has led to escalating demand for the use of laccase on an industrial scale12. However, the solubility of laccases in water leads to poor recovery from aqueous medium making the process expensive and less desirable, thereby restricting its use at industrial scale. Immobilization of laccase on insoluble support not only circumvents this problem but also enhances the biocatalytic performance with reusability over substantial periods of time.Enzyme immobilization is a powerful tool to improve enzyme properties13 .It involves confining enzyme molecules to a solid support over which a substrate is passed and converted to products such that it can be used repeatedly while maintaining its catalytic activities. The immobilized enzyme has the advantages of reusability14

-15,

enhanced stability14, enzyme free products and cost efficiency over free enzyme that suffers from difficult recovery and non-recyclability16. It is highly desirable that the support matrix which binds the enzyme can be prepared reproducibly and does not interfere with enzyme activity as these two parameters are of paramount importance for technological efficiency and commercial success. A number of supports have been explored for enzyme immobilization including both inorganic and organic materials17. Various inorganic supports such as silica-based, and other oxide-based materials are used for the immobilization of enzymes

18-20.

In another art of work, enzyme immobilization on carbon nanomaterials 21, graphene 2 ACS Paragon Plus Environment

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oxide22-25 has been used as a carrier for the enzyme because of their large surface area and chemically inertness, it shows high thermal and solvent stability. Synthetic polymers like vinylic and acrylic polymers such as polyacrylamide, polyvinyl alcohol and poly-styrene-divinylbenzene have also been investigated for their enzyme immobilization capabilities26- 27. Among organic materials polysaccharides such as modified celluloses, dextran, chitosan and agarose have displayed the capability of enzyme immobilization28-30 . Besides this, gelatin, sodium alginate, starch, chitosan, magnetic chitosan, polyvinyl alcohol, polyacrylamide have been frequently used for the immobilization of laccase. Another important factor that plays a significant role in the biocatalytic efficiency of enzymes is the method employed for the enzyme immobilization on a solid support. A range of methods have been investigated including adsorption, covalent binding, entrapment and crosslinking

31- 32.

Among the known methods, covalent immobilization is preferred as it gives the

strongest link and provides the most stable support – enzyme conjugates without leaching of the enzymes33. Covalent immobilizations usually a two step process that involves activation of the surface using a linker and covalent coupling of enzyme to the activated support

34.

Linker molecules are

multifunctional reagents mainly used in chemical modifications of proteins and polymers. Different linkers are used for different surfaces (inorganic material, natural or synthetic polymer, membranes) and immobilization protocols. One of the most commonly used linking agent is glutaraldehyde that links covalently to the amine groups of lysine in protein35-37. However, the direction and position of enzyme binding to the support matrix in covalent immobilization is a crucial factor that determines its stability. An active site overlapping or closer to the covalent binding site causes high risk of enzyme denaturization. Moreover covalent bonding requires longer incubation time a compare to other immobilization methods. The above-mentioned immobilization approaches, either use high cost, less accessible supports or require tedious synthetic procedure which further makes the process expensive. Hence, in the recent decades, development of inexpensive, biodegradable and readily available materials that can be easily functionalized, are of particular interest. Chicken feathers (CF), poultry waste, are generated worldwide in billion tones and India alone produces 350 million tons annually creating grave issues of solid waste management. The conventional disposal methods of incineration and landfill burials are not only expensive but lead to air and soil pollution posing a serious threat to the environment38-40. The proximate analysis of chicken feather revealed that the, major component is crude protein (82.36%) followed by crude fibre (2.15%),

and crude lipid (0.83%), it also contains ash (1.49%), and moisture content

(12.33%) whereas the carbon ,nitrogen, oxygen and sulphur content was found to be 64.47 ,10.41, 22.34 3 ACS Paragon Plus Environment


and 2.64 % respectively in ultimate analysis

41.Meanwhile,

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researches focussing on the physical and

chemical properties of these chicken feathers have opened up new opportunities and efforts are being made to utilize these chicken feathers for some valuable applications42-

45.

Chicken feather has been

successfully used as support material for stabilizing the metal-based catalysts for organic transformations43. Chicken Feathers has also been tested for thermoplastic applications46. However, the use of chicken feathers for immobilizing enzymes remains unexplored. The present invention aims to utilize the chicken feather waste as a support for immobilizing the enzyme via covalent attachment (Figure 1). These easily available and inexpensive poultry feathers serve as an excellent support material for enzyme immobilization making them an attractive option for use in biocatalytic transformations in a batch and continuous mode. Additionally, utilization of chicken feather for enzyme immobilization also addresses the emerging problem of solid waste management. The objective of this study was to explore the possibility of the chicken feather as a support material for the immobilization of enzymes by performing different feasibility studies. Further Trametes maxima laccase (TML) was purified using anion exchange chromatography. The chicken feather derived support material was processed and amino functionalised. The TML was covalently attached to the support material in presence of glutaraldehyde which is a bifunctional compound that act as the bridge between support and enzyme via covalent bonding. Studies of variable parameters including the pH, temperature and incubation effect for maximum immobilization yield were performed. For an in-depth analysis of the use of immobilized laccase on the chicken feather, stability, reusability, kinetic and thermodynamic analysis under various conditions were investigated. Finally, the performance of immobilized catalyst was evaluated for oxidation of veratryl alcohol a non-phenolic lignin model compound to veratryl aldehyde.


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Figure 1: Schematic diagram of immobilization course of TML on ACFP Materials and Methods Materials Chicken feathers purchased from a local vendor. Trametes maxima IIPLC-32 laccase (TML) was purified (3mg/ml) in laboratory. Veratryl alcohol was purchased from TCI chemicals, 3-amino propyltrimethoxysilane (APTS), 2,2' azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ABTS, coomassie brilliant blue G 250, glutaraldehyde (GA), all were purchased from Sigma–Aldrich. All other chemicals were of analytical grade, and used as received. Milli-Q water was used to prepare all solutions and reagents.

TML preparation for immobilization Trametes maxima IIPLC-32 strain was grown on potato dextrose agar medium. Mycelial discs (size 10 mm in diameter, 2 in no) were taken from a freshly grown culture and used as an inoculum under optimized parameters for 50 ml of culture volume (pH 6, temperature 320C, rpm 200 in 250ml flasks). The pH of the culture medium was adjusted to 6 for maximum growth, followed by induction after 72 hrs and grown for five days under the same conditions.TML was prepared as described in the previous study47. In brief, the culture supernatant was collected, filtered and the supernatant was concentrated using cut-off membrane followed by ammonium 5 ACS Paragon Plus Environment


sulfate precipitation and dialysis. Dialysed TML was purified using an anion exchange column and used for immobilization.

Preparation of chicken feather particles (CFP) and amino functionalization Chicken feather was obtained from the local poultry farm. Pre-processing of the chicken feather was done by washing with water and subsequently treating with 70% ethanol followed by drying overnight in an oven at 50 0C. To prepare chicken feather particles (CFP), feather barbs were removed from quill and grounded in a ball mill (Retsch) for 6 hr with 12 interval break of 5 min. since the smaller particle size is an important factor for support material that provides additional benefits like lower hindrance of mass transfer and reduced diffusional limitations. Therefore the ball milled CFP was passed through 325 mesh size sieve (44 microns) for separation of fine homogeneous CFP. The amino functionalization of CFP was carried out in the presence of 3aminopropyltrimethoxysilane (APTMS)43. In brief, the CFP (2 gm) was added in 40 ml of toluene followed by the addition of APTMS (5.0 g). After that, the reaction mixture was subjected to overnight reflux at 110 oC in a nitrogen atmosphere with continuous stirring. The amino functionalized chicken feather particles (ACFP) were separated from the reaction mixture by filtration and transferred to a Soxhlet for extraction with dichloromethane for 24 h to remove unreacted APTMS. Lastly, the ACFP was dried under vacuum for 15hr and stored at room temperature (25±2 0C) for further use. Cross-linking and Immobilization of laccase onto ACFP In an immobilization experiment, 100 mg of ACFP were dissolved in distilled water (10ml) and sonicated for 20 min at room temperature (25±2 0C). The mixture was subjected to centrifugation at 5000 rpm and washed thrice with distilled water. Glutaraldehyde (GA) solution was added in a phosphate buffer to reach the final volume 1.5% (v/v) and incubated overnight at 150 rpm at room temperature (25±2 0C). The mixture was again centrifuged for 10 min at 5000 rpm, and the pellet was washed thrice with 10mM of phosphate buffer (pH-7) to remove the excess glutaraldehyde from the solution. Trametes maxima IIPLC32 laccase (TML), 2 ml, 66 IU/ml in phosphate buffer (50mM) pH 6 was added in 100 mg of ACFP and incubated at 4 0C for overnight with shaking at 100 rpm for binding of the enzyme to support material. The incubated mixture was centrifuged at 5000 rpm for 15 minutes to recover the solid support with immobilized TML and washed with the same buffer to remove any non-specific binding. Each wash was collected and measured for activity. TML immobilized on ACFP (TML@ACFP) was stored at 4 0C before use. Effects of varying pH, temperature and incubation time on immobilization were investigated. The pH range 6 ACS Paragon Plus Environment

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3-7 was selected for immobilization. Different temperature conditions (4-37 0C) were studied for the maximum immobilization on ACFP. The optimum incubation period for the binding of TML with ACFP was determined by periodical removal of the TML sample followed by estimation of its residual activity. Furthermore, the enzyme loading capacity on the support material and its associated activity was evaluated by increasing amount of enzyme up to 200unit/100 mg of support material under optimum conditions. The immobilization yield (IY) was calculated by measuring the total residual activity that remained in the solution after immobilization on support. This was calculated by subtracting the residual activity from the total initial activity. The control experiment was carried out with free TML to compensate the enzyme deactivation under the immobilization conditions. The equation for calculating Immobilization yield is IY (%) = 100 × (Immobilization activity/starting activity) Activity of free TML and immobilized TML@ACFP The activity of TML and TML@ACFP was measured spectrophotometrically (Molecular devices Spectra Max M2e) at wavelength 420 nm using ABTS as the substrate. To measure the activity of TML, 125 µl of TML solution (Purified TML diluted with Milli Q water) was added to 375 µl of the 0.1mM of ABTS in Mcilvaine (MI) buffer (0.12M) pH 3 at room temperature (25±2 0C)47. The change in absorbance at 420 nm (ε= 3.6 x104 M-1cm-1) was recorded for two minutes, and the activity was determined by measuring the slope of the initial linear portion of the kinetic curve. All the activity measurements were performed in duplicate. Activity was expressed as an international unit (IU), where one IU represents the amount of enzyme that forms 1µmol/min product under standard assay conditions. The activity of immobilized TML@ACFP was assayed by incubating 100 mg of TML@ACFP in 1ml of Mcilvaine (MI) buffer (0.12M) pH 3 containing 0.1mM ABTS under shaking condition (200 rpm) at room temperature (25±2 0C) for 4 min. A fixed amount of sample was withdrawn at an interval of 1min, centrifuged at 8000 rpm and absorbance was measured spectrophotometrically (Molecular devices Spectra Max M2e) at wavelength 420. The enzyme activities of the immobilized samples were measured as 𝑈 𝑔

=

𝐴𝑏𝑠/min × 𝐹 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 × 𝑉 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 × 106 𝜀 × 𝑀𝑐𝑎𝑟𝑟𝑖𝑒𝑟

Where U/g is the amount of enzyme capable to oxidized 1µmol ABTS per min per mass unit of carrier, Abs/min is absorbance per minute, F dilution is the dilution factor, V reaction is the reaction volume, 10 6 is the conversion factor for M concentration to µM, ε (3.6 x104 M -1cm-1) molar extinction coefficient and M carrier is mass of the support on which enzyme has immobilized. 7 ACS Paragon Plus Environment


Characterization of CFP The surface modified CFP and immobilized TML named TML@CFP were characterized using PerkinElmer spectrometer. KBr method was used to record the FT-IR spectra in the range of 500 and 4000 cm−1. TG-DTA analyzer (Perkin-Elmer) was used for the thermal decomposition analysis of support material. Sample analysis was performed under nitrogen flow within 30 to 500 0C

of the temperature range. A scanning electron microscopy (SEM) was used to observe the

surface morphology of the CFP and ACFP and elemental compositional analysis was performed by EDX.

Temperature and pH optima For the evaluation of optimal pH, several of immobilized TML@ACFP and free TML samples were incubated at varying pH range 2.2-8 in MI buffer and activity of both free TML and immobilized TML@ACFP were measured as the method described above. For the evaluation of optimal temperature, activity were determined at temperature range of 45- 70 0C in the optimized pH condition in the same buffer. pH and Thermal stability of TML and TML@ACFP

To evaluate the pH stability, free TML and immobilized TML@ACFP were incubated for seven days under varying range of pH (3-8), and the activity of free TML and TML@ACFP were measured as described above. The thermal stability of free TML and TML@ACFP was investigated by incubating the sample at different temperatures (50-70 0C) in MI buffer (0.12M) at optimized pH for 120 min. For this purpose, immobilized TML@ACFP samples were suspended in MI buffer (0.12M) and kept in a circulating water chamber at a constant temperature. Samples were taken at regular intervals, and the activity was measured as method described above. Thermal deactivation of free TML and TML@ACFP can kinetically be described by the first-order rate of thermal deactivation a commonly used model 48. In the simplest model of enzyme deactivation, 𝐸𝑎 is the active enzyme; 𝐸𝑖 is the deactivated enzyme and 𝐾𝑑 is the deactivation rate constant.


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𝐾𝑑

𝐸𝑎

𝐸𝑖

(1)

The rate equation for the thermal deactivation can be obtained. 𝑑𝑎 𝑑𝑡

= -𝐾𝑑𝑎

(2)

Where a is the activity at time t during the thermal deactivation process, Eq.(2) can be integrated to obtained Eq. (3)

ln

( )= -𝑘 t a

(3)

𝑑

a0

Where a0 is the initial activity. At different temperature, a semi-log plot of residual activity versus time should give a straight line where the negative slope is the deactivation rate constant 𝑘𝑑 Half- life (𝑡1/2) at different temperature was calculated using the estimated parameter 𝑘𝑑 from Eq. (3) of 𝑡1/2 =

0.693

(4)

𝑘𝑑

The decimal reduction time (D value) was used for the estimation of enzyme stability and is defined as the time required to reduce the initial activity by 90% and mathematically by

𝐷=

ln (10)

(5)

𝑘𝑑

The Arrhenius equation was used to explain the effect of temperature on the rate constant. Therefore 𝐾𝑑 (deactivation rate constant) can be expressed as follows

𝐾𝑑 = 𝐴𝑒 - 𝐸𝑑/𝑅𝑇 Where 𝐸𝑑 (kJ/mol) is the energy for enzyme deactivation, A is the frequency factor or Arrhenius constant, R (8.31 J/mol K) is the universal gas constant, and T (K) is the absolute temperature. Taking the natural logarithm of Arrhenius' equation yields: ln (𝐾𝑑) = ln (𝐴) -

𝐸𝑑 𝑅

1

∙𝑇

Rearranging the equation yields 9 ACS Paragon Plus Environment


ln (𝐾𝑑) =

- 𝐸𝑑 𝑅

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1

∙ 𝑇 + ln⁡ (𝐴)

(6) The value of the activation energy

𝐸𝑑

and 𝑘𝑑 allowed to the determination of various

thermodynamic parameters such as Gibbs free energy of thermal inactivation (ΔG), and deactivation enthalpy (ΔH) The Gibbes free energy of thermal inactivation (ΔG) was determined using the following equation ∆𝐺 = ― 𝑅.𝑇.ln

𝐾𝑑.h

( )

(7)

𝐾𝐵.𝑇

Where h (1.1 × 10

-35

J.min) and 𝐾𝐵 (1.3806 × 10 -23 J K-1) is the Plank’s and Boltzmann’s

constant respectively, 𝐾𝑑 (Min-1) is the rate constant. The value obtained from 𝐸𝑑 was used for the calculation of deactivation enthalpy (ΔH) ∆𝐻 = (𝐸𝑑 ― 𝑅𝑇)

(8)

Reusability and Storage stability of Free TML and TML@ACFP The reusability of immobilized TML@ACFP was assessed by incubating 100mg of TML@ACFP with ABTS solution (1mM) as substrate in MI buffer pH 4. The activity was measured by oxidation of ABTS at room temperature (25±2 0C) under shaking at 200 rpm (Eppendorf Thermo MixerR C 15ml) for 4 min. After each oxidation cycle (4 min cycle) , the sample was centrifuged for 10 min at 5000 rpm to recover TML@ACFP and washed with phosphate buffer solution (50mM) pH 7 for the second and consequent cycles. The fresh substrate (ABTS) was added in each cycle to the TML@ACFP. The activity was determined up to 8 cycles, and the activity was measured in triplicate. The activity of the TML@ACFP was considered 100% in the initial cycle. To examine the storage stability of free TML and immobilized TML@ACFP both were stored at room temperature as well as at 40C and their activity was determined at a certain time interval to evaluate the storage stability. TML@ACFP catalysed biotransformation Biocatalytic oxidation of non-phenolic substrate veratryl alcohol to veratryl aldehyde was performed using covalently bound TML@ACFP in the presence of mediator HOBT. In brief, the reaction medium containing, veratryl alcohol (2mM) 100μl, HOBT (1mM) 200μl, sodium acetate 10 ACS Paragon Plus Environment

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buffer (50 mM, pH 5.5) 9.7ml and TML@ACFP 1000 mg was incubated at 32 0C in an incubator at 120 rpm. The samples were withdrawn at the fixed interval of time and heated in boiling water to stop the reaction. The pH of the samples was lowered to pH 2.0 using HCl and ﬁltered through a 0.4μm- ﬁlter. 50 μl of this sample was mixed with DMSO in NMR tube and used for proton NMR analysis. Reaction mixture without TML@ACFP was performed as a blank control experiment under otherwise identical conditions.

Result and Discussion CFP modification, Cross linking, and Immobilization Chicken feather owing to the presence of various functional groups like -COOH, -NH2, -SH and – OH posses a great potential to be used as a support matrix for further chemical modification and immobilization. The functional groups –OH, NH2 on the chicken feather surface were targeted for the immobilization of the enzyme in the present study. The FTIR spectrum of a bare chicken feather (CFP) exhibits characteristic peaks at 1647, 1533 and 3423 cm-1 correspond to amide I, amide II and (–NH, –OH) stretching, respectively. Subsequently, to achieve and maximize the amino-functionalities on the surface, the CFP was treated with 3-aminopropyl trimethoxy silane (APTMS), and the functionalized material was denoted as ACFP. The presence of two additional peaks in FTIR spectrum of ACFP at 1132 and 1028 cm-1 related to C−NH2 and Si–O stretching respectively confirmed the successful functionalization. In addition, the reduced intensity of the –OH band at 3430 cm−1 suggested the desired amino-functionalization of the support. Further, glutaraldehyde was used as a homo-bifunctional cross-linker to bridge the aminomodified ACFP surface and the free TML. Furthermore, the formation of the Schiff base from the reaction of ACFP with glutaraldehyde was confirmed by the appearance of the characteristic band of imine (C=N) at 1627 cm−1 (Fig.2a) The amino groups on the modified surface were used for Schiff base formation with glutaraldehyde which was confirmed by the color change of the solution as shown in Fig. 2b. The Schiff base containing ACFP was used for the immobilization of TML through covalent bonding (Fig. 2b). Thermal stability of the bare CFP and amino-functionalized CFP (ACFP) was measured by thermogravimetric analysis as shown in Figure 3A. In the TG curve of native CFP, the initial weight loss about 6% in the temperature range between 100-150 0C suggested the removal of adsorbed water molecules. Whereas, the second major weight loss in the region 200 to 300 0C was mainly due to the 11 ACS Paragon Plus Environment


decomposition of organic moieties (Fig.3A). After the amino-functionalization in CFP, the introduction of functional silane groups enhanced the thermal stability of the support matrix. Therefore the thermal degradation for ACFP was found to be in the range 250-330 0C which is higher than CFP. The thermal degradation pattern of the enzyme immobilized ACFP did not show any significant change and was found to be almost identical as ACFP. The SEM images of CFP, ACFP are shown in Fig. 3B (a) and (c) respectively. The morphology of ACFP after the grafting of APTMS has been changed remarkably compared with CFP. In case of CFP, the surface is appearing very rough and layered type structure. Whereas, after grafting of APTMS monomers on the surface, flakes can be clearly seen in ACFP. Furthermore, the elemental composition was determined by EDX analysis (Fig.3B; b and d) indicates the presence of the desired elements C, N, O, S and Si in the CFP; however a strong signal of Si element in ACFP confirmed the successful aminofunctionalization of the chicken feather surface with APTMS. After the immobilization of enzyme the SEM and EDX of TML@ACFP remained nearly similar that is mainly due to the smaller size as well as lower amount of the enzyme on the surface.


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Figure 2. (a) FT-IR spectra of Chicken feather powder (CFP), amino functionalized chicken feather powder (ACFP) and immobilized TML on ACFP TML@ACFP ,(b) Evidence of amino functionalization: (b1) Chicken feather without amino functionalization and reaction with glutaraldehyde (b2) Aminofunctionalized chicken feather after reaction with glutaraldehyde. Change of colour indicates the Schiff base formation.



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(B)

a

b

c

d

Figure 3: (A) TGA curves of (a) CFP, (b) ACFP; (B) SEM image of (a) CFP (b) EDX of CFP (c) ACFP (d) ) EDX of ACFP. Optimization of conditions for immobilization This study was intended to utilize chicken feather waste material for immobilization of the TML enzyme using a simple and affordable process. The immobilization efficiency significantly depends on variables such as pH, temperature and incubation time. In order to attain maximum immobilization yield of TML on ACFP, varying conditions of pH, temperature, and incubation time of enzyme were studied and optimized (Figure 4). Optimum pH for immobilization yield (IY) was tested by varying the pH from 2.2-8. A low immobilization yield (IY) of 12% was recorded at pH 2.2, whereas, 67, 75, 72% and 56 % of IY were observed at pH 5, 6, 7, and 8 respectively, as shown in figure 4a. IY was adversely affected on both lower and higher pH 2 and 8 respectively. To identify the optimum temperature for maximum IY, immobilization was studied under different temperature range (4-37

0C).

Lower temperature favoured the

immobilization while higher temperature displayed a negative effect on IY. Maximum IY, 72% was attended at 4 0C, and it decreased to 53.3% when temperature increased to 37 0C (Figure 14 ACS Paragon Plus Environment

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4b).Immobilization yield also depends on the incubation time and the immobilization strategy. Covalent immobilization of enzyme required longer incubation time when compared to ionic interaction/adsorption. During the incubation period the enzyme interacts with the reactive groups on the carrier leading to the formation of covalent bonds. The TML loading on ACFP was studied until 72 hr of incubation at optimum temperature and pH. The amount of TML loading on ACFP was found to increase through the initial 12 hr of incubation while prolonged incubation (24hr) affected the loading and became stable after 48 hr of incubation (Figure 4c). For incubation period less than 12 hr, the IY is lower probably, because there was not enough time for enzyme to covalently bind to carriers. After optimization of immobilization conditions maximum IY of 74.24% was attained at pH 6, temperature 4 0C and incubation time 24hr. To evaluate the enzyme loading capacity of ACFP, enzyme loading and associated activity on fixed ACFP support (100mg) was studied as a function of the amount of laccase ranging from 80 to 200 units. Maximum activity 14.8 U/g and percent enzyme loading 93% were obtained at laccase concentration of 120 Units. Beyond 120 units, decrease in loading was observed and became almost constant (Fig 4d).At the onset of immobilization, with increase in enzyme amount, increase in enzyme loading is observed that further results in the increase in enzyme activity. However,once the maximum amount has been loaded on the support, no further increase in loading is seen since all the available sites on the supports are occupied by the enzyme. Result also indicates that the activity did not correspond strictly to the enzyme loading on ACFP. The excessive loading of the enzyme on the support causes steric hindrance due to protein- prote in interaction and restricted stretching of protein confirmation49. Similar result was also observed in case of immobilization of pectinase on the macroporous polyacrylamide microspheres support50.



Figure 4. Effect of different operational variables on immobilization of free TML on ACFP; pH (a), temperature (b) and incubation time (c). laccase loading (●) and activity Unit/g (■) of support (d)

pH and temperature effect 16 ACS Paragon Plus Environment

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The influence of pH on immobilized TML@ACFP and free TML was measured at different pH range of MI buffer. Free TML and immobilized TML@ ACFP exhibited activity over a broad pH range (2.2-8 pH) as shown in the figure 5a. Maximum activity of the free TML was recorded at pH 3, the free TML has 10±2 % of lower activity at the pH range 5-7, this is mainly due to the reality that the isoelectric point (pI) of the enzyme lies between 5 to 5.5 that hampered the solubility of enzyme in aqueous solution and may started aggregation. In case of immobilized TML@ACFP, a pH shift was observed with the optimum pH at 4.1, one pH unit higher than the free TML. The enzyme activity of TML@ ACFP was slightly reduced up to pH 6, and a significant reduction in activity was observed at pH 8. Wang et al. reported a similar shift in the optimal pH for immobilized laccase. In contrast, no change in the optimum pH of immobilized laccase on PAN nanofibers was reported by Xu et al. and Catapane et al 51-52. The optimum temperature of free TML and TML@ACFP was investigated at 45-70 0C (Figure 5b). The maximum relative activity (100%) of free TML was observed at 55 0C, the relative activity of free TML at 45 0C and 70 0C was 92.9 and 91.6 respectively. TML@ACFP showed temperature optima 50C higher (60 0C) as compared to free TML. The TML@ACFP showed 88 and 97 % of its activity at 45 0C and 70 0C

respectively with respect to the optimum temperature value at 60 0C. This result clearly indicates that

the heat-resistant capacity of TML increased after immobilization on ACFP. The shift in the optimum temperature towards the higher side in case of TML@ACFP is expected due to the limited movement of the enzyme molecules. The immobilized system is protecting the amino acids at the active sites. However, increase in the temperature enhance the kinetic energy of the substrate molecule that reaches the active site of the immobilized enzyme rapidly and shifting the optimum temperature to the higher side .



Figure 5. Optimal pH and Temperature; (a) pH of TML (●) and TML@ACFP (■) , (b) temperature TML (●) and TML@ACFP (■)

pH and Thermal stability The pH plays an important role in stabilization of enzyme as it can alter the ionization conditions of the constituent amino acids of the enzyme thereby modifying its activity and stability53 . Hence, the pH stability of TML and TML@ACFP was studied in the pH range of 3-8 for an extended period (7days), and the results were evaluated. The obtained result explained that both TML and TML@ACFP were comparatively more active at an acidic range of pH than the alkaline conditions. TML had retained 86% of its initial activity at pH 4 and 57% of the initial activity at pH 8 after seven days of incubation. However, the retained activity of TML@ACFP was 93.26 and 74% at pH 4 and pH 8 respectively. Immobilization increased the stability and pH tolerance capacity of the enzyme as evident from the results (Figure not presented).The thermal stability of the immobilized enzyme is an important factor associated with its commercial applications. Hence, the thermal stability of free TML and immobilized TML@ACFP was studied at the temperature range of 50-70 0C. The free TML and immobilized TML@ACFP were incubated for 2hr at different temperatures. The relative activity of both free TML and TML@ACFP decreased with time at all the temperatures (Figure 6a and 6b respectively). After 2 hrs of incubation, the relative activity of free TML was found to be 49% at 50 0C, whereas the activity of the immobilized counterpart was 75%. When the temperature was increased to 70 0C, drastic loss in activity was observed, free TML losing around 93% of its initial activity, and immobilized TML@ACFP losing around 80% of its initial activity after one hour of incubation. Results indicate that an increase in temperature leads to deactivation of both the free and immobilized enzyme. However, the immobilized TML@ACFP was more stable as compared to free TML. This could be attributed to the confinement 18 ACS Paragon Plus Environment

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in the conformational mobility of the enzyme molecules after getting covalently bound on the support. However, the sharp decrease in the stability could be due to the breakage of bonds in enzymes that leads to loss of protein’s structure and activity

Figure 6. Thermal stability profiles; Free TML (a) and immobilized TML@ACFP (b), temperature T (0C) ; 50 (●), 55 (■), 60 (▲), 65 (▼) and 70 (♦). ABTS (0.1 mM ) was used as the substrate for enzyme assay.

Kinetic and thermodynamic analysis of thermal deactivation The Kinetic and thermodynamic studies of deactivation for the free TML and Immobilized TML@ACFP were carried out at the temperature range of 55-65 0C. Different parameters like deactivation rate constant (Kd), half-life (t1/2), D-value, Gibbes free energy of (ΔG), enthalpy (ΔH), and energy for enzyme deactivation (Ed) were determined. The thermal deactivation rate constant (Kd) of free TML and TML@ACFP was calculated from experimental data Figure 7a and 7b respectively, and values were summarized in Table 1. Both the free TML and TML@ACFP followed the first-order deactivation rate kinetics. The rate constant Kd of both free 19 ACS Paragon Plus Environment


TML and TML@ACFP gradually increased with temperature, while the half-life (t1/2) progressively decreased, indicating that the inactivation gradually increased and the thermo stability declined with a rise in temperature. The Kd value at 55 0C for free TML and immobilized TML@ACFP were found to be 0.0044 and 0.0027 min-1 respectively. Half-life (t1/2) was calculated from the Kd and observed to be 154.9 and 256.8 min, respectively for free TML and immobilized TML@ACFP. With higher t1/2 and lower Kd values, the immobilized TML@ACFP demonstrated higher thermostability as compared to the free TML. Thermal stability was also expressed in terms of decimal reduction time (D value) as shown in table 1. D value of TML@ACFP was significantly higher as compared to the free TML. At temperature 55 0C the Dvalue of free TML and TML@ACFP was 514.8 min and 853.3 min respectively. The D value also decreased with an increase in temperature beyond 50

0C

for both free and immobilized

TML@ACFP. Another critical factor for an enzyme’s stability is the energy required for its denaturation. The complete denaturation of enzyme requires an input of energy denoted as the activation energy of denaturation (Ed). This process of thermal denaturation of enzyme occurs via formation of an unstable intermediate that requires an initial input of energy less than Ed. However, this intermediate form can revert back to its native active state upon removal of thermal effect. An additional increase in energy beyond the Ed barrier causes the complete denaturation of an enzyme such that it cannot achieve it native form even after removal of the thermal effect. In order to estimate the Ed, the graph was plotted between lnKd versus 1/T for both free TML and immobilized TML@ACFP using equation (6). Ed values were calculated from the slope of the straight line obtained (Figure 8). The Ed value of immobilized TML@ACFP (137.8 kJ/mol) was 1.17 times higher than that of the free counterpart (117.48 kJ/mol). This indicates that immobilized TML@ACFP requires more energy for its complete deactivation, and hence is more thermostable as compared to free TML. The activation energy of denaturation (Ed) is related to another important thermodynamic parameter called enthalpy of denaturation (H) that express the total amount of energy required for the denaturation of the enzyme. The larger the  H value, the higher the energy needed to break stabilizing bonds in the thermal deactivation of the enzyme. ΔH were determined for both free and immobilized enzyme at different temperatures (55-65 0C) using equation 8. Within the tested temperature range, no measurable difference in ΔH was obtained for free TML and immobilized TML@ACFP, however significant difference was observed when compared between free TML and immobilized TML@ACFP. Comparing at 55 0C, the ΔH of free TML was 114.75 kJ/mol, and that of immobilized TML@ACFP was 135.12 kJ/mol specifying


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that more energy is required for denaturation of the immobilized TML@ACFP. Thus, the immobilized form of the enzyme was found to be more thermostable than free form. The Gibbs free energy of deactivation (ΔG) evaluates the spontaneity of the inactivation process. The value of ΔG is directly proportional to enzyme’s thermal stability. Higher the ΔG, higher is the stability of the enzyme. The Gibbs free energy (ΔG) changes for free TML and TML@ACFP was determined using equation (7) in the temperature range of 55-65 0C.At 55 0C, ΔG values of the free TML and immobilized TML@ACFP were 106.58 kJ/mol and 107.96 kJ/mol respectively. The positive values of ΔG for both the forms indicate that the inactivation process is thermodynamically non-spontaneous. With an increase in temperature, the ΔG values decline although the drop in values of ΔG is not very significant. The difference in the ΔG value between free TML and immobilized TML@ACFP in the temperature range 55-65 0C was found to be around 1.38 kJ/mol signifying that immobilization not significantly affect the ΔG. Table 1. Comparison of kinetic and thermodynamic parameters for free TML and immobilized TML@ACFP



T 0

( C)

Kd (min

-1)

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t1/2

D-value

ΔG

ΔH

(min)

(min)

(kJ/mol)

(kJ/mol)

154.9 ±8.0

514.8±26.70

106.58±0.14

114.75±1.07

Ed (kJ/mol)

Free TML 55

0.0044

±

0.00023 60

0.00633

117.48±1.07

5 ±

109± 3.0

363.7±8.70

107.23±0.10

114.70±1.08

39.5±4.11

132±13.825

105.49±1.08

114.66±1.07

8

0.0001 65

0.01756± 0.0019

6 Immobilized TML@ACF P 55 60 65

0.00270±0.0000

256.81±8.4

853.31±27.9

107.96±0.08

8

1

7

9

0.00518±0.0002

133.82±6.0

444.65±20.1

107.84±0.12

4

7

7

5

0.01173±0.0008

59.27±4.30

196.95±14.2

107.21±0.20

135.04+4.6

9

6

6

7


135.12±4.67 137.85±4.67 135.07±4.66

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Figure 7. Semi-log plots of thermal denaturation; free TML (a) , TML@ACFP (b) in the temperature range T (0C): 55 (●), 60 ( ■), 65 (▲).The deactivation rate constants Kd were determined from the slopes of ln(A/A0) against time(min)



-4 -4.5 ln (Kd)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

-5 -5.5 -6 -6.5 0.0029

0.003

0.0031

1/T

Figure 8 . Arrhenius plot to calculate the activation energy for deactivation; free TML(▲)and TML@ACFP (■). Each data point is mean value (n=3) Reusability and Storage stability Use of laccases in free form is not feasible at an industrial scale since it is not possible to recover the laccase after the completion of the reaction from the reaction medium leading to an increase in the operational cost. Unlike free laccases, the immobilized counterpart can be recovered from the reaction medium easily and can be reused for multiple cycles. In order to understand the reusability aspect of the immobilized TML@ACFP, most frequently used substrate for laccase (ABTS) was used in this study. Immobilized TML@ACFP was used repeatedly for consecutive cycles with fresh ABTS. The initial activity was considered 100% and the relative activity was calculated for the consecutive cycles. The result obtained from the reusability experiment displays only a mild decrease in the activity of TML@ACFP till eight cycles of oxidation of ABTS as a substrate (Figure 9a). In the first two cycles 100 % of activity was recorded followed by 97.5 and 95.8 % of activity in the third and fourth cycle. TML@ACFP exhibited the residual activity of around 95% after eight consecutive cycles, indicating the strong interaction of the enzyme with the support. This strong interaction of the enzyme with the support is due to covalent bonding and clearly indicating no significant loss of the enzyme activity by leaching during the reaction and repeated washing after every cycle. Since after every substrate – enzyme reaction the product is recovered and the immobilized catalyst is washed with buffer to eliminate any remaining product, hence there is no probability to an accumulation of the immobilized surface. Thus, there is no mass transfer restriction to the proteins molecules even after 8 consecutive cycles. In another research study a slight decrease of activity 8.6 % was observed after twenty two cycles of reaction due to possible interaction with accumulated water molecule in the microenvironment with enzyme molecule 54. 24 ACS Paragon Plus Environment

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The storage stability of enzymes depends upon several factors including buffer, buffering components, temperature, the presence of metal ions, oxidizing and reducing environment of the storage buffer. Out of all, the temperature is an important factor when considering the storage stability. To investigate the storage stability of free TML and immobilized TML@ACFP both are stored at room temperature as well as at 4 0C and their activity were determined at the certain time interval to evaluate the storage stability. As shown in figure 9b, both free TML and immobilized TML@ACFP has excellent storage stability at 4 0C.

The free TML retained 85.5 % of its activity after three weeks of storage; whereas 94.32% of activity

was observed in immobilized TML@ACFP. At the room temperature, the free TML and immobilized TML@ACFP retained their activity by 54.8 and 62.4 % at the end of 3rd week. In a similar study of laccase immobilization on chitosan as a support material, 60 and 70% of the residual activity was reported after 10 and 30 days of storage respectively. Research by Lloret et al., have reported an increase in storage stability after immobilization of laccase on Eupergit supports55. The result shows immobilized TNL@ACFP have higher storage stability in both the conditions.



Figure 9. Reusability and storage stability; reusability study of TML@ACFP (a), Storage stability of free TML and immobilized TML@ACFP (b), TML@ACFP 250C (×), TML 250C (●),TML@ACFP 4 0C (■), TML 4 0C (▲) TML@ACFP catalysed biotransformation

The proton NMR analysis showed a peak at δ 9.8 ppm, which attributed to –CHO moiety of veratrylaldehyde after 48 h. This clearly suggested that the covalently bound TML on ACFP was stable and efficient to convert veratryl alcohol to veratryl aldehyde in the presence to the mediator. The 1H NMR of the product is shown in the supporting information as Fig.S1 Conclusions In the present study, we have demonstrated the first successful application of the chicken feather for the immobilization of the enzyme biocatalyst via covalent attachment. The chicken feather derived support material was prepared and characterized for the immobilization of TML enzyme through covalent bonding using glutaraldehyde as a linker. The independent variables were optimized for the maximum immobilization yield. The optimization experiments indicated the independent variables play an essential role in immobilization yield. The resulting TML@ACFP exhibited an improved pH, temperature, storage stability with a great potential of reusability. Further, the immobilized TML@ACFP was used for the biocatalytic oxidation of non -phenolic substrate (veratryl alcohol). The result of the proton NMR confirmed the conversion of the veratryl alcohol to veratryl aldehyde. This result suggests that chicken feather waste-derived material can be used for the immobilization of enzyme and many industrial applications.


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Conflicts of interest “There are no conflicts to declare”. Acknowledgement Authors are thankful to Director IIP for granting permission to publish these results. Projects CSC-119/6 and OLP-981 are acknowledged for the financial assistance. Analytical division of the Institute is acknowledged for providing support in the analysis of samples.

Supporting Information The 1H NMR of the reaction product veratryl aldehyde is given in the supporting information (Fig. S1).

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TOC/Abstract Graphic

Synopsis First use of sustainable, inexpensive and waste chicken feathers for immobilization of Laccase for chemical transformation with improved stability and reactivity.


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Chicken feather derived novel support material for immobilization of

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