A Novel Approach to Enzymatic Unhairing and Fiber Opening of Skin

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A novel approach to enzymatic unhairing and fibre opening of skin using enzymes immobilized on magnetite nanoparticles Gunavadhi Murugappan, Mohammad Jamal Azhar Zakir, Gladstone Christopher Jayakumar, Yasmin Khambhaty, Kalarical Janardhanan Sreeram, and Jonnalagadda Raghava Rao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.5b00869 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 8, 2016

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ACS Sustainable Chemistry & Engineering

A novel approach to enzymatic unhairing and fibre opening of skin using enzymes immobilized on magnetite nanoparticles Gunavadhi Murugappana, Mohammad Jamal Azhar Zakira, Jayakumar Gladstone Christophera, Yasmin Khambhatyb*, Kalarical Janardhanan Sreerama, Raghava Rao Jonnalagaddaa a

Chemical Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India

b*

Microbiology Division, CSIR- Central Leather Research Institute, Adyar, Chennai 600020,

India KEYWORDS: Dehairing, Fibre Opening, Fibrozyme, Immobilization, Iron Oxide Nanoparticles, Nanozyme

Corresponding author: Dr Yasmin Khambhaty, Microbiology Division, CSIR-Central Leather Research Institute, Adyar, Chennai 600 020, Tamil Nadu, India, Ph No. +91-44-24437135, Email id: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

One of the prerequisites for making leather is to remove the inter fibrillar proteins, noncollagenous materials such as hair, flesh etc. These proteinous and non-proteinous materials are removed in several steps, cumulatively known as the pre-tanning (or beam house) operations. Paradigm shift from chemical to enzyme based processes ensured that these non-collagenous materials were removed using enzymatic digestion rather than brutal osmotic forces employing chemicals like lime and sulphide. In order to make sure that a cocktail of enzymes (protease + amylase) have broad application and stability, their immobilization on to matrices that can enable overcoming such drawbacks is essential. This work, taking clue from the catalytic applications of nanoparticle immobilized enzymes looks at metal oxide nanoparticle immobilized enzymes for unhairing and fibre opening applications in a facile manner. Iron oxide (Fe3O4) nanoparticles have been selected for the present study since; metal oxides are proven as unbeaten matrix for protein tagging. In the present study, a cocktail of protease and amylase (fibrozyme) was used as control and nanoparticle immobilized enzyme cocktail (nanozyme) was used as experimental sample. Enzyme concentration was fixed as 3% of raw skin weight, while drumming in a stainless steel vessel was looked at as the application method. Nanozyme treated leather samples were analyzed for their unhairing and fibre opening efficiency. Histological studies, physical strength and organoleptic studies of control and experimental sample leathers were also carried out. Microstructural analysis based on histo-patholgy studies of fibrozyme and nanozyme treated tissues exhibited no significant change in the tissue morphology, which confirms that nanoparticles, did not give any adverse effect on the skin. Scanning Electron Microscopy (SEM) images of fibrozyme and nanozyme treated leathers shows the degree of fibre opening and Energy Dispersive X-ray spectrum (EDS) shows the elements present on skin matrix. This study


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can provide a newer insight for a cleaner, economical and sustainable method of leather processing.

INTRODUCTION Leather industry has been identified as one of the most environmentally polluting industries.1 The chemicals which are employed in pre-tanning and chrome tanning operations are the source for huge amount of harmful effluent discharged by leather industry.2 Lime and sulphide are the vital chemicals used in beam house operation for the removal of hair from hide/ skin and for fibre opening which are necessary for diffusion of chemicals during subsequent leather processes. The usage of these chemicals leads to numerous adverse effects like consumption of surplus amount of water, degradation of hair and high amount of solid sludge discharged in the effluent. As a remedy for above mentioned problems of conventional chemical unhairing, enzyme based unhairing and fibre opening procedure has been proposed.3 Protease, amylase and lipase are the three enzymes which can perform unhairing, fibre opening and fat removal from skin/hide respectively.4 These enzymes do not degrade hair and hence provides value from hair as a byproduct. In addition to this, there will be huge reduction in the Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), and Total Dissolved Solids (TDS) of effluent water, which are the known dimensions used in the measurement of water pollution.5 However, during industrial applications, some concerns such as poor chemical and thermal stability, high cost involved in production of enzymes and the lack of knowledge in processing and handling of these enzymes by the associated workers have been expressed. For instance, the enzymes tend to lose their stability even due to slight changes in the salinity of the water in


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which they are dissolved, eventually leading to partial or complete loss of the enzyme activity.6 There are reports which ascertain that the tagging of enzymes to a solid support such as thin films, nanoparticles or mesoporous moieties can enhance the performance of enzymes. The reduction in the stability of the enzyme due to fluctuations in temperature and pH can also be controlled through such tagging. The easy separation of these enzymes from reaction mixture which in turn is difficult when used in native state are an added advantage which eventually increases the chance for the reusability of enzymes.7 Nanoparticles have become an intimate part of research and development in almost all the fields in this century. Especially, metal oxide nanoparticles has found place in applications like bioimaging, drug delivery, etc., due to their known high surface to volume ratio, easy modification of surface groups which enhances the scaffolding ability and high scalability at room temperature solution phase synthesis techniques.8 Metal oxide nanoparticles viz., zinc oxide and titanium dioxide possesses photo catalytic activity9 and iron oxide possess ferromagnetic to super paramagnetic behavior10 and hence can be chosen for the immobilization of the enzymes.11 It has also been reported that encapsulation or immobilization of enzymes to metal oxide nanoparticle surface can enhance the activity of the enzymes.12 Among various metal oxides, iron oxide (Fe3O4) nanoparticles were selected for this study due to its super paramagnetic behaviour13 which can aid in the retrieval of enzymes along with nanoparticles with the help of an external magnetic field so that the enzymes can be reused.7The surface –OH groups facilitates the conjugation of protein moieties to the nanoparticle surface with the use of cross linkers like glutaraldehyde.14 The role of enzyme immobilized iron oxide nanoparticles in dehairing and fibre opening steps of leather processing has been thus investigated in this study.


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EXPERIMENTAL General All the chemicals used in the synthesis of iron oxide nanoparticles (FeCl3, FeSO4, and ammonia solution) were of analytical grade and procured from Merck India Pvt. Ltd., poly(oxyethylene) 200 (PEG) used for PEGylation (coating of nanoparticles with PEG 200) was also procured from this firm. A cocktail of protease and amylase enzyme (fibrozyme) were obtained as a kind gift from a company based in Chennai (with a protease activity of 1000 U/g and amylase activity of 100 U/g). Bovine Serum Albumin (BSA), Mucin, Folin-Ciocelatu reagent, Periodic acid was procured from Sigma Aldrich, India and other analytical chemicals were procured from SD Fine Chemicals, India. For the leather trials, wet salted goat skins were used.

Preparation of iron oxide nanoparticles In the present study magnetite nanoparticles were prepared by co-precipitation method as described by Muthukumaran et al. (2012) with a slight modification.15 For this work, the following methodology was optimized. 0.8 M FeCl3 and 0.4 M FeSO4 salts were mixed in 100 mL of distilled water and heated at 70˚C under stirring. The pH of the above solution was rapidly raised to 9.0 by the addition of 30 % ammonia solution. A change in color from brown to black after the addition of ammonia indicates the formation of magnetite nanoparticles. The formed nanoparticles were allowed to stir for 120 mins. The black colored precipitate was either centrifuged at 12,000 rpm or magnetic decanted. The nanoparticles were washed with distilled water until the pH of the discard solution was 7.0. Separated samples were dried in hot air oven


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at 80 °C for 5 hrs. Subsequently, PEGylation was done to enhance the aqueous stability of the synthesized nanoparticles, which eventually leads to the uniform coating on to nanoparticle surface. For achieving PEGylation, 33 mg of nanoparticles was dispersed in 10 mL of double distilled water and sonicated well till attaining uniform dispersion. Five mL of PEG solution was added to the nanoparticle dispersion and few drops of ammonia were added to the solution, keeping at constant stirring conditions for 3 hours. After that the PEG coated magnetite nanoparticles were collected using permanent magnet and washed several times with double distilled water and dried at 40 °C in vacuum oven.16

Characterization of synthesized iron oxide nanoparticles After the synthesis of magnetite nanoparticles, and before PEGylation the nanoparticles were subjected to X-Ray diffraction analysis by Rigaku Mini Flux X-Ray Diffractometer to ensure the elements present and determine the crystallite size. The hydrodynamic radius of the sample was measured by Dynamic Light Scattering (DLS) technique after dispersing the nanoparticles in water under sonication. The solution stability was also measured at consequent stages from the synthesis of nanoparticles to enzyme coating using Malvern Zetasizer Nano ZS. Functional group analysis of synthesized nanozyme was carried out on a JASCO 4200 Fourier Transform Infrared Spectrophotometer (FTIR). The magnetization curve of dried powders was measured using Lakeshore model 7410, Vibrating Sample Magnetometer (VSM).


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Characterization of fibrozyme The cocktail enzyme (fibrozyme) was characterized before proceeding for immobilization step. Enzyme stability and activity with respect to three different pH (4.5, 7.0 and 9.0) and temperature (25° C, 35° C, 45° C) was evaluated. Zeta potential measurement was also undertaken with respect to pH ranging from 3.0-10.0.

Preparation of nanozymes Fibrozyme immobilization on to magnetite nanoparticles was carried out by mixing the suspension of PEGylated nanoparticles to the enzyme solution. An aqueous solution of fibrozyme (1 mg /mL) was prepared by dissolving lyophilized enzyme in deionised water (enzyme was found to be stable at pH 7-8 and hence, deionized water was used to prepare this solution instead of buffer). 1 mg/mL of PEGylated nanoparticles was dispersed using ultrasonicator. Both the solutions were mixed, and stirred at 150 rpm at 25° C. An immediate precipitation was observed as soon as both the solutions were mixed together. The above mixture was incubated at 15 rpm on a shaker, overnight. The precipitates were collected by applying external magnetic field, and washed with deionized water 3 times. The total protein content of the solution was checked before and after nanozyme preparation by Lowry’s method.17 Enzyme activity of solutions before and after immobilization was measured as per standard assay protocol for protease as well as amylase. For protease assay L-Tyrosine was used as standard and casein was used as the substrate for enzyme activity.18, 19 Amylase assay was done using starch as substrate, for which maltose was used as standard.20 Transmission Electron Microscopy (TEM) analysis of enzyme immobilized nanoparticles was performed on Jeol/JEM 2100


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microscope operating at 200 kV, with LaB6 as source. Samples were prepared by drop-casting dilute solution of nanoparticles on carbon-coated copper grids and allowed to dry before the analysis.

Leather trial studies 800 g of wet-salted goat skin was taken for unhairing and fibre opening studies. The skin was cut into two equal halves. They were subjected to soaking in water for removal of salt and rehydration of skin. Three percentage of fibrozyme and nanozyme each were separately added to 30 % of water. One half of the skin was treated with fibrozyme and the other half with nanozyme. Both the trials were performed by drum method (Table1).This was repeated until complete hair removal and fibre opening was achieved. The extent of unhairing and fibre opening depended on enzyme to nanoparticle ratio used. The enzyme treated skins were thoroughly washed and subjected for conventional tanning procedures.21

Proteoglycan estimation After complete dehairing is achieved the liquor was collected and it was subjected to proteoglycan release estimation. Liquor samples were filtered using muslin cloth for the removal of solid contaminants and the analysis was carried out. For this estimation, Mucin was used as standard. The quantification of the proteoglycan was done by matching the absorbance values with the standard curve derived by the Mucin standards. 21


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Histological staining Samples of about 1 cm2 area were cut from leather treated with fibrozyme and nanozyme at similar positions of the unhaired portions of skins. Samples were washed thoroughly and were fixed in formalin solution prepared by adding 0.9 g sodium chloride in 100 mL of 10% formaldehyde solution. Sections of 6 µm were obtained using microtome and were stained using Safranin O to examine the level of proteoglycan released 22 and Haematoxylin & Eosin staining to see the fibre compactness was performed. 23

Physical testing of leather samples The shrinkage temperature, a measure of hydrothermal stability of leather, was determined using SATRA Shrinkage Tester.24 The samples for physical testing were analyzed as per IULTCS methods. 25, 26, 27 Tensile and tear properties of tanned leather were assessed using universal testing machine, and grain crack analysis was carried out on a Lastometer. The samples were conditioned at 26 ˚C and 65% Relative Humidity for 48 hours Physical properties such as tensile strength, % elongation at break, tear strength and grain crack strength was investigated as per standard procedures. Each value reported is an average of four (2 along and 2 across the backbone) measurements.


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Evaluation of organoleptic properties The experimental and control crust leathers were assessed for softness, grain smoothness, fullness and general appearance by tactile evaluation. Three experienced tanners rated the leathers on a scale of 0-10 points for each, where higher points indicate better properties.

Scanning Electron Microscopy and Energy Dispersive X-ray spectrum For SEM analysis, samples from leathers prepared after treatment with both fibrozyme and nanozyme were obtained from official sampling positions.21 SEM analysis was done using Carl Zeiss MA15/EVO18 scanning electron microscope attached with EDS. The crust leathers were mounted on to aluminium stub and coated with thin layer of gold to make the leather conducting. SEM images were obtained in the working voltage of 5 kV at 25 °C.

Results and discussion The simultaneous hydrolysis and dehydration of a mixture of divalent and trivalent iron salt solution lead to the synthesis of magnetite nanoparticles. The overall chemical reaction can be written as follows. To adjust the pH of reaction mixture, under vigorous stirring conditions alkali is added in one spot. This aided in the formation of nanoparticles of ultra-small and similar size. 2Fe3++Fe2++8OH−→FeO· Fe2O3+ 4H2O

Eq (1)


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Characterization of synthesized magnetite nanoparticle The magnetite nanoparticles were subjected to X-Ray diffraction studies. The peak positions at 2 θ values of 29, 35, 42, 53, 56, and 62 indicate the formation of magnetite. Also the XRD pattern was compared with International Centre for Diffraction Data (ICDD)-10716336, which confirmed the formation of magnetite crystals and the average crystal size of the samples was calculated as 25 nm using Scherrer’s equations (Figure1 (a)). The hydrodynamic diameter of the particles was measured using DLS and it was found to be 201 nm (Figure1 (b)). The aqueous stability of the bare nanoparticles is observed to be very low, as the nanoparticles settled during the course of measurement. To increase the stability, the pristine magnetite nanoparticles need to be coated with a synthetic or biological polymer which could stabilize the nanoparticles in the solution. Polyethylene glycol16, poly(vinyl pyridine)28, poly(vinyl alcohol) 29 are some of the examples used for synthetic coating materials. According to reports polyethylene glycol30 can be a better coating and stabilizing material and was hence, used for the present study.

Characterization of enzyme The cocktail enzyme (fibrozyme) was thoroughly characterized before proceeding for immobilization step. Enzyme stability and activity with respect to pH and temperature was evaluated. . Enzyme showed decreasing zeta potential as pH increases, indicative of a reducing stability. The activity of the enzyme was calculated to be minimal in the acidic pH (4.5) and showed maximum activity at pH 7.0. Even at elevated temperature (45 °C) the enzyme was


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stable and showed good activity on its substrate. The ideal condition for enzyme activity was optimized to be pH 7.0 and room temperature (0.997 U/mg of protease and 0.118 U/mg of amylase). After PEG was coated (zeta potential +26 mV), the enzyme loading was carried out. An enzyme to nanoparticle ratio of 1:1 was found to be optimum. The magnetite nanoparticles was separated by magnetic decantation and washed several times with double distilled water to any uncoated enzyme. The supernatant of the solution was subjected to protein quantification by Lowry’s method in order to quantify the amount of free enzyme if any, in the solution. It was observed that 0.003 mg/ mL of enzyme was detected in the supernatant indicating the efficiency of immobilization. It is obvious that a maximal binding efficiency of protein onto magnetite nanoparticles was observed when both interacting allies were oppositely charged.31 Enzyme immobilization on nanoparticle surface could be confirmed from the increase in average size of the nanoparticles. From figure 1b, it is also evident that enzyme immobilization was even and uniform as indicated by the narrow size distribution profile. The zeta potential value of magnetite after enzyme coating was measured as –16.5 mV with a standard deviation value of 4.85. The shift in the zeta potential value could be taken as an indication of binding of nanoparticles to the fibrozyme moieties. Figure 2 shows the TEM images of nanoparticles after immobilization with the enzyme. According to this observation, the particles are spherical in morphology with diameter of 16±5nm. . Figure 3 shows the magnetic nature of the synthesized magnetite nanoparticles. The overlap in hysteresis curve indicates that the prepared nanoparticles have super paramagnetic behavior. The saturation magnetization (Ms) value of bare magnetite nanoparticles was calculated to be 47.41 emu g-1, which is higher than that of fibrozyme coated nanoparticles (40.02 emu g-1). The reduction in the Ms Value can be corroborated to the coating


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of organic moieties on to the nanoparticle surface, which alters the magnetic properties of bare iron.32 The enzyme activity was also checked before and after immobilization. A marginal increase in activities for both the enzymes was observed (given in supporting information).

FTIR spectroscopy is the most efficient characterization tool for the analysis and identification of encapsulated chemicals. The FTIR pattern of lyophilized enzyme powder (fibrozyme) Vs prepared Nanozyme is presented in Figure 5. The fibrozyme sample bears the characteristic bands associated with proteins at 1110, 2920 cm-1 and also the peaks at 1645, 900 cm-1, predominantly arising from the amide groups of the enzyme.33 In the case of Nanozyme, where PEG coated Fe3O4was present, peaks at 560-580 cm-1 region, in addition to that the presence of strong peak 3000-3500 cm-1 was observed. This arises from the -OH stretching of the surface hydroxyl groups of polymer.34 The nanozyme also has additional peaks at 1100, 1640 cm-1 region, clearly indicating the presence of proteins on the surface of metal oxide nanoparticles (given in supporting information).

Unhairing and fibre opening studies The selective removal of proteoglycans from the hair follicles is achieved by the enzyme resulting in the loosening of hair from the skin surface. The degree of hair loosening was recorded hour wise for both the samples. The hair removal started at 3rd h and complete hair removal was observed at 7th h for nanozyme as compared to 9th h for fibrozyme. The reduction in


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the time period could be attributed due to the stable activity of the nanoparticle enzyme conjugate. The removal of intra-fibrillar tissues is an important parameter, which decides diffusion of tanning chemicals and dyes during chrome tanning and post tanning processes. Amylase is reported to remove non-collagenous materials from skin matrix. An hour wise histology sections revealed the reduction in the compactness of fibres, which can be directly correlated to the removal of fibrillar materials (Figure 4). In the early stage of treatment, the hair was seen to be tightly bound to the skin in addition to a very compact fibre orientation. As the enzymatic reaction progresses, there is a disturbance in the proteoglycans present in the hair bulbs, as observed from the histopathological images, which leads to loosening of hair from the skin matrix.35 At final stages of enzymatic process, histological patterns clearly reveals complete removal of proteoglycan from the hair bulbs, reduction in the compactness of the fibres eventually leading to the splitting up of fibre bundle. Histopathological results also exhibited intact microstructural features of pristine enzyme (fibrozyme) and the nanoparticle immobilized enzyme (nanozyme) which proves that the nanoparticle does not cause any adverse effects on the tissues. Proteoglycan release from the raw skin during the enzymatic activity was quantitatively measured using mucin as a standard proteoglycan. From Table 2 it is evident that the release/removal of intra fibrillary material such as chondroitin, dermatan and heparan sulfate proteoglycan from the skin is more in the case of nanozyme treated sample as compared to fibrozyme treated sample. This is a clear proof for the increased activity of enzyme after immobilization. As per the reports the mobility of a catalyst is an important factor in determining their activity, considering high particle mobility of nanoparticle due to ultra-small size, the


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nanoparticle immobilized enzymes possibly show higher activity than native/ free enzyme. As compared to unbound enzymes the nanoparticle immobilized enzyme should be having an easy penetration and enhanced access to the substrate, through skin pores and hair follicles in the skin matrix so they exhibit the higher removal of proteoglycan. 36The inclusion of metal oxide nanoparticles tends to increase the thermal shrinkage of the skin, however, in the present study the shrinkage temperature was in the normal range indicating no adverse effect due to metal oxide nanoparticles.

Physical properties of tanned leather The strength characteristics like tensile, tear and grain crack of both the samples are as mentioned in Table 3.The nanozyme treated leathers passed the grain crack test as per norms. It can be elucidated from the strength characteristics that nanozyme treated crust leathers showed better tensile and shear values as compared to fibrozyme treated leathers as per norms. The shrinkage temperature of tanned nanozyme treated leather samples was 111°C which is comparable to the fibrozyme treated leather.

Organoleptic properties The subjective assessment of leathers by hand evaluation for both control and experimental leathers is presented in a 0-10 point scale. It was observed that the experimental leathers exhibited comparable properties to that of control leathers. Properties like softness, fullness,


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grain tightness are improved in nanoparticle enzyme treated leathers compared to control leathers (Figure 5).

SEM and EDS Analysis Figure 6 (a, b, c, d) shows the surface and cross sectional SEM images of nanozyme and fibrozyme treated leather samples. The cross sectional investigation is important in terms of fibre loosening aspects. The compactness, fibre alignments and orientation of skin fibres have greater influence in the dyeing and diffusion of post tanning chemicals. The nanozyme treated leathers showed comparable fibre splits to that of fibrozyme treated leathers, however in a shorter duration of time when compared to fibrozyme. Energy Dispersive X-ray Spectrum (Figure 6e, 6f) shows the absence of iron peak, which clearly indicates that there is no residual iron present in the skin matrix.

Conclusions The present investigation directs towards a cleaner dehairing and fibre opening process. In order to overcome the issues pertaining to stability while using the fibrozyme in their native forms, the present study addresses the issue by making use of immobilized fibrozyme (nanozyme). The binding efficiency of enzyme to magnetite nanoparticles may be attributed to their opposite charges. It was also concluded that pH plays an important role in the binding efficiency. In terms of leather trial experiments, nanozyme concentration of 3.0 % offers effective unhairing and fibre opening. The nanozyme treated leather revealed faster dehairing and fibre opening as


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evidenced by staining and proteoglycan release studies. Nanozyme treated matrix confirms that there was well ordering and opening of fibre bundles due to enzyme effect. The general parameters like grain smoothness, color and general appearance remains better in nanozyme as compared to fibrozyme treated pelts. The tensile and tear strength of goat upper leather showed 21.49 N/m2 and 28.38 N/mm respectively. The physical characteristics of the crust leathers meet SATRA norms, which is an indicative for effective leather process. The present study paves way for novel bio-processing of leather making eventually meeting the environmental concern faced by the industry during pre-tanning operations.


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FIGURE CAPTIONS Figure 1

XRD pattern of synthesized Fe3O4 nanoparticles (a). Hydrodynamic diameters of nanoparticles before and after immobilization (b).

Figure 2

TEM image of Fe3O4 nanoparticles after enzyme immobilization.

Figure 3

Room temperature hysteresis loop of Fe3O4 nanoparticles (a) and Fe3O4 nanoparticles after enzyme immobilization (b).

Figure 4

Histo-pathology image of skin treated with nanozyme

Figure5

Organoleptic properties of leathers obtained from (a) fibrozyme and (b) nanozyme based unhairing and fibre opening.

Figure 6

SEM images of grain pattern (a,b) and cross section (c, d) of leathers obtained after treatment with fibrozyme and

nanozyme respectively and their

corresponding Energy dispersive X-ray spectrum (e, f).

Table 1

Process flow sheet for the usage of fibrozyme, nanozyme for unhairing and fibre opening.

Table 2

Proteoglycan release from fibrozyme and nanozyme treated skin.

Table 3

Physical strength properties of leathers obtained from fibrozyme and nanozyme based unhairing and fibre opening.


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Figure 1 XRD pattern of synthesized Fe3O4 nanoparticles (a). Hydrodynamic diameters of nanoparticles before and after immobilization (b).


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Figure 2

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TEM image of Fe3O4 nanoparticles after enzyme immobilization.


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Figure 3 Room temperature hysteresis loop of Fe3O4 nanoparticles (a) and Fe3O4 nanoparticles after enzyme immobilization (b).


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Figure 4 Histo-pathology image of skin treated with nanozyme


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Figure 5 Organoleptic properties of leathers obtained from (a) fibrozyme and (b) nanozyme based unhairing and fibre opening.


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Figure 6 SEM images of grain pattern (a,b) and cross section (c, d) of leathers obtained after treatment with fibrozyme and nanozyme respectively and their corresponding Energy dispersive X-ray spectrum (e, f).


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Table 1. Process flow sheet for the usage of fibrozyme, nanozyme for unhairing and fibre opening. Steps

Fibrozyme

Nanozyme

Soaking

Water (300 % of raw Water (300% of raw Wet salted goat skin skin weight)

Details

about 800 g was split

skin weight)

in to 2 halves used for fibrozyme, nanozyme Enzyme unhairing fibreopenning

based Water(30 % of raw Water (30% of raw Drum rpm 7 and skin weight) Fibrozyme

skin weight) (3% of Nanozyme

raw skin weight)

10 min run + 50 min (3%

raw skin weight)

of pause (Till

achieving

complete dehairing)

Collection of liquor and wash liqours for proteoglygan estimation Pickling Tanning


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Page 26 of 33

Table 2. Proteoglycan release from fibrozyme and nanozyme treated skin.

Proteoglycan release in

Proteoglycan release in

fibrozyme liquor (mg/ g of

nanozyme liquor (mg/g of

raw weight)

raw weight)

liquor

5.20±0.2

7.40±0.2

Wash liquor 1

1.20±0.1

1.40±0.1

Wash liquor 2

0.31±0.05

0.36±0.05

Process


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Table 3. Physical strength properties of leathers obtained from fibrozyme and nanozyme based unhairing and fibre opening.

Sample

Tensile

Elongation at

Tear

strength

break (%)

Strength

(N/mm2)

(N/mm)

Grain crack strength

Load(kg)

distension (mm)

Control

20±1

56±4

27±2

38

12

Experimental

21±1

60±4

28±2

39

13.38


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Page 28 of 33

ASSOCIATED CONTENT *S Supporting Information

Figures depicting synthesis of iron oxide nanoparticles,zeta potential of PEG coated nanoparticles, fibrozyme and nanozyme.This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author *Phone No: +91 44 24437135; E mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

Acknowledgements The authors acknowledge CSIR- XIIth Five Year Plan Project- Science & Technology Revolution for Leather with a Green Touch (STRAIT) for financial support (CSIR-CLRI Communication No. A/2015/MIB/CSC0201/1160).


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‘ABBREVIATIONS PEG, polyethylene glycol; DLS, Dynamic Light Scattering; XRD, X-Ray Diffraction; ICDD International Centre for Diffraction Data; VSM, Vibrating Sample Magnetometry; FT-IR, Fourier Transform Infrared Spectroscopy; SEM, Scanning Electron Microscopy; EDS, Energy Dispersive X-ray Spectroscopy

REFERENCES (1) Sivasubramanian, S.; Manohar, B. M.;Rajaram, A.; Puvanakrishnan, R. Ecofriendly lime and sulfide free enzymatic dehairing of skins and hides using a bacterial alkaline protease. Chemosphere. 2008, 70, 1015-1024. (2)Thanikaivelan, P.; Rao, J.R.; Nair, B.U.; Ramasami, T. Progress and recent trendsin biotechnological methods for leather processing. Trends Biotechnol. 2004, 22,181-188. (3) Saran, S.; Mahajan, R. V.; Kaushik, R.; Isar, J.;Saxena, R. K. Enzyme mediated beam house operations of leather industry: a needed step towards greener technology. J. Clean. Prod. 2013. 54, 315-322. (4) Saravanabhavan, S.; Aravindhan, R.; Thanikaivelan, P.; Rao, J. R.; Nair, B. U. Green solution for tannery pollution: effect of enzyme based lime free unhairing and fiber opening in combination with pickle-free chrome tanning. Green. Chem.2003, 5, 707-714. (5) Dettmer, A.; Cavalli, E.; Ayub, M. A. Z.; Gutterres, M. Environmentally friendly hide unhairing: enzymatic hide processing for the replacement of sodium sulfide and deliming. J. Clean. Prod. 2013, 47(1), 11-18. (6) Walti, M.; Roulin, S.; Feller, U. Effects of pH, light and temperature on (1 -> 3,1 -> 4)-betaglucanase stability in wheat leaves. Plant Physiol. Biochem. 2002, 40, 363-371. (7) Yuhong, R.; Rivera J. G.; He, L.; Kulkarni, H.; Lee, D. K.; Messersmith P. B. Facile, high efficiency immobilization of lipase enzyme on magnetic iron oxide nanoparticles via a biomimetic coating. BMC Biotechnol. 2011, 11, 63. (8) Kaushik, A.; Khan, R.; Solanki, P.R.; Pandey, P.; Alam, J.; Ahmad, S.; Malhotra B.D. Iron oxide nanoparticles–chitosan composite based glucose biosensor. Biosens.Bioelectron. 2008, 24, 676-683. (9) Liu, J.; Liu, L.; Bai, H,; Wang, Y.; Sun, D. D. Gram-scale production of graphene oxideTiO2nanorod composites: Towards high-activity photocatalytic materials. Appl. Catal., B. 2011, 106(1-2), 76-82. (10) Wei, C.; Jiaqi, W.; Facile synthesis of superparamagnetic magnetite nanoparticles in liquid polyols. J. Colloid Interface Sci. 2007, 305, 366-370. (11)Venkatanarasimhan, S.; Rani A.E.; Dhamodharan R. Immobilization of amylase on gum acacia stabilized magnetitenanoparticles, an easily recoverable and reusable support. J. Mol. Catal.B: Enzym. 2013, 96, 6-13. (12) Ekta, S.; Paramita, M.; B.Ashutosh V.; Hemant, P. S. Immobilization of Thermomyceslanuginosus lipase on ZnO nanoparticles: mimicking the interfacial environment. RSC Adv. 2015, 5, 26291–26300.


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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

Page 30 of 33

(13) Lun, J.; Wenjing, W.; Huiwen, L.; Yaoliang, Z.; Haibo Z.; Xiaohai,Z.Penicilliumexpansum lipase-coated magnetic Fe3O4–polymer hybrid hollow nanoparticles: a highly recoverable and magnetically separable catalyst for the synthesis of 1,3-dibutylurea. RSC Adv. 2014, 4, 2598325992. (14) Liane, M.R.; Ashley D.Q.; Zeev, R. Glucose oxidase–magnetite nanoparticle bioconjugate for glucose sensing. Anal Bioanal Chem. 2004, 380, 606-613. (15) Muthukumaran, T.; Gnanaprakash G.; John Philip. Synthesis of Stable Magnetic Nanofluids of Different Particle Sizes. J. Nanofluids. 2012, 1, 85-92. (16) Anindita, M.; Nidhi, J.; Krishnananda, C.; Goutam, D. A. A. Facile Synthesis of PEGCoated Magnetite (Fe3O4) Nanoparticles and Their Prevention of the Reduction of Cytochrome C. ACS Appl. Mater. Interfaces. 2012, 4, 142-149. (17) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951,193, 265-275. (18) Anson, M. L. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol.1938, 22(1), 79-89. (19) Folin, O.; Ciocalteu, V. On tyrosine and tryptophane determinations in proteins.J. Biol. Chem 1927, 73, 627–650. (20) Bernfeld, P.; Amylases, α and β.Methods Enzymol. 1955, 1, 149-158. ( (21) Jayakumar, G. C.; Sivaraman, G.; Saravanan, P.; Mohan R.; Rao,J. R. Cohesive system for enzymatic unhairing and fibre opening: an architecture towards eco-benign pretanning operation. J. Clean. Prod. 2014, 83, 428-436. (22) Jayakumar, G. C.; Mehta, A.; Rao J. R.; Fathima,N. N. Ionic liquids: new age materials for eco-friendly leather processing. RSC Adv. 2015, 5, 31998–32005. (23) Kase, S.; Saito, W.; Yokoi, W.; Yoshida, K.,;Furudate, N.; Muramatsu, M.; Saito, A.;Kase, M.; Ohno,S. Expression of glutamine synthetase and cell proliferation in human idiopathic epiretinal Membrane. Br. J. Ophthalmol. 2006. 90(1), 96-98. (24) Kanth, S.V.; Venba, R.;Madhan, B.; Chandrababu, N. K.; Sadulla, S. Cleaner tanning practices for tannery pollution abatement: role of enzymes in ecofriendly vegetable tanning. J. Clean. Prod. 2009,17, 507-515. (25) IUP 2, Sampling. J. Soc. Leather Technol. Chem. 2000, 84, 303. 26) IUP 6, Measurement of tensile strength and percentage elongation. J. Soc. Leather Technol. Chem. 2000, 84, 317. (27) IUP 8, Measurement of tear load-double edge tears. J. Soc. Leather Technol. Chem. 2000, 84,327. (28) Malynych, S.; Luzinov, I.; Chumanov, G. Poly(Vinyl Pyridine) as a Universal Surface Modifier for Immobilization of Nanoparticles. J. Phys. Chem. B. 2002, 106, 1280-1285. (29) Khanna, P. K.; Gokhale, R.; S. Subbarao, V. V. V.; Vishwanatha, A. K.; Dasa B. K.; Satyanarayana, C. V. V. PVA stabilized gold nanoparticles by use of unexplored albeit conventional reducing agent. Mater. Chem. Phys.2005, 92, 229-233. (30) Xie, J.; Xu, C.; Kohler, N.; Hou Y.; Sun,S. Controlled PEGylation of Monodisperse Fe3O4 Nanoparticles For Reduced Non-Specific Uptake by Macrophage Cells. Adv. Mater.2007, 19, 3163-3166.


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(31) Schultz, N; Metreveli, G.; Franzreb, M.; Frimmel F. H.; Syldatk,C. Zeta potential measurement as a diagnostic tool in enzyme immobilisation. Colloids Surf.,B, 2008, 66, 39-44. (32) Ribeiro, V. G. P.; Barreto, A. C. H ; Denardin, J. C.; Mele, G Carbone, L.; Mazzetto, S. E.; Sousa, E. M. B.; Fechine, P. B. A. Magnetic nanoparticles coated with anacardic acid derived from cashew nut shell liquid. J Mater Sci. 2013, 48, 7875-7882. (33) Dandavate, V.; Keharia, H.; Madamwar, D.; Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles. Process Chem. 2009, 44, 349-352. (34) Jimeno, S. G.; Estelrich, J. Ferrofluid based on polyethylene glycol-coated iron oxide nanoparticles: Characterization and properties. Colloid Surface A. 2013, 420, 74-81. (35) Saravanan, P.; Renitha, T. S.; Gowthaman, M. K.; Kamini, N. R. Understanding the chemical free enzyme based cleaner unhairing process in leather manufacturing. J. Clean. Prod. 2014, 79, 258-264. (36) Jia, H.; Zhu, G.; Wang, P. Catalytic Behaviors of Enzymes Attached to Nanoparticles: The Effect of Particle Mobility. Biotechnol.Bioeng. 2003, 84, 406-414.


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“ For table of contents use only”


Chemical Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India

b

Microbiology Division, CSIR- Central Leather Research Institute, Adyar, Chennai 600020, India

Application of enzyme immobilized iron oxide (Fe3O4) nanoparticles in dehairing and fibre opening of skin during leather processing


32

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Chemical Laboratory, CSIR-Central leather Research Institute, Adyar, Chennai 600020, India

b*

Microbiology Division, CSIR- Central Leather Research Institute, Adyar, Chennai 600020, India

Application of enzyme immobilized iron oxide (Fe3O4) nanoparticles in dehairing and fibre opening of skin during leather processing


A Novel Approach to Enzymatic Unhairing and Fiber Opening of Skin

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