Bioconversion of Lignocellulosic Biomass to Fermentable Sugars by

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Bioconversion of Lignocellulosic Biomass to Fermentable Sugars by Immobilized Magnetic Cellulolytic Enzyme Cocktails Karthik Periyasamy, Laishram Santhalembi, Gerard Mortha, Marc Aurousseau, Agnes Boyer, and Sivanesan Subramanian Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00976 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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Bioconversion of Lignocellulosic Biomass to Fermentable Sugars by Immobilized Magnetic Cellulolytic Enzyme Cocktails Karthik Periyasamya,c, Laishram Santhalembib, Gérard Morthac, Marc Aurousseauc, Agnés Boyerc and Sivanesan Subramaniana* a

Department of Applied Science and Technology, Environmental Management Laboratory, A.C. Tech, Anna University, Chennai 600025, India. b

c

Indian Council for Agricultural Research (ICAR), Imphal, Manipur, India.

Univ. Grenoble Alpes, CNRS, Grenoble INP, LGP2, 38000 Grenoble, France.

*Corresponding Author E-mail: [email protected] (Sivanesan); Phone: +91-44-22359168.

ACS Paragon Plus Environment

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ABSTRACT Enzyme cocktails of reusable, highly-stable cellulolytic enzymes play an inevitable role in bioconversion of biomass to biofuels economically. Cellulase, xylanase and β-1,3-glucanase bound silica-amine functionalized iron oxide magnetic nanoparticles (ISN-CLEAs) were prepared and used as the biocatalyst for the depolymerization of cellulosic biomass into monomeric sugar in the present study. The Fe3O4-NPs and Fe3O4@SiO2-NH2-NPs and ISNCLEAs had an average hydrodynamic size of 82.2 nm, 86.4 nm and 976.9 nm respectively, which was confirmed by dynamic light scattering (DLS). About 97% of protein binding was achieved with 135 mM glutaraldehyde at 10 h of crosslinking time and successful binding was confirmed by Fourier transform infrared spectroscopy (FTIR). The ISN-CLEAs exhibited the highest thermal stability of 95% at 50 °C for 2 h and retained extended storage stability of 97% compared to 60% of its free counterpart. Besides, crosslinking allowed ISN-CLEAs reuse for at least eight consecutive cycles retaining over 70% of its initial activity. ISN-CLEAs exhibited approximately 15% increase in carbohydrate digestibility on sugarcane bagasse and eucalyptus pulp than the free enzyme.

KEYWORDS Cellulase, Xylanase, β-1,3-glucanase, Magnetic nanoparticles, Lignocellulose, Fermentable sugars.


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INTRODUCTION Environmental concerns connected with climate change and global warming are of eminent importance for human sustenance and thus has attracted the attention of scientists worldwide. The rising greenhouse gas levels, depletion of oil supply and the increase in energy demands have necessitated the search for an alternative clean and sustainable fuel resource in order to balance the demand supply chain and to improve the quality of life. Currently, fossil fuel resources such as coal and natural gas contribute majorly towards the generation of power in the non-renewable sector. In addition to this, a number of biofuels such as bioethanol, biodiesel, biomethane, biohydrogen and bio-butanol supplement the energy demand by reducing the usage of petroleum-based fuels1–4. Bioethanol has a potential value in the fuel market and several countries like Brazil, United States, Canada, China, Europe and India are interested in ascertaining biofuel based technologies and developing their own biofuel markets5. It was produced from sugars and starch materials, but lignocellulosic biomass can be presumed to be a sustainable feedstock for bioethanol production6. Lignocelluloses are complex heterogeneous natural composites with three main biopolymers such as cellulose, hemicellulose, lignin, ash and other minor organic molecules are also present. Cellulase is a multi-enzyme complex and exhibits the synergetic action of cellobiohydrolase or exo-glucanase, endo-glucanase or carboxymethyl cellulase. Cellobiase or β-glucosidase are required to hydrolyze cellulose into cellooligosaccharides and glucose for fermentation7. Xylanase depolymerizes xylan and release xylooligosaccharides of different size and xylose8. β-1,3-glucanase acts on β-D-glucan and releases glucose units from the non-reducing end of glucan chains. Since commercial enzymes in soluble forms cannot be reused, many strategies have been developed for enzyme immobilization which includes adsorption, covalent bonding, crosslinking, entrapment and encapsulation. The major


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impediment of carrier-free cross-linked enzyme aggregate is not easily recoverable from the reaction medium by conventional separation methods such as filtration and centrifugation9. In order to overcome these problems, covalent linking of enzymes onto the surface of Iron Oxide Magnetic Nanoparticles (Fe3O4 or IOMNPs) have attracted utmost attention because of simple preparation, allowance for high specific enzyme activity and enhanced stability while maintaining maximum catalytic efficiency over several reaction cycles. These particles include three functional parts, typically a magnetic core, an outer coating to shield the magnetic core and functionalized surface coating10,11. IOMNPs are attracted by a magnetic field, but they retained residual magnetism in the absence of magnetic field12. Hence the solution containing suspended IOMNPs can be recovered from a reaction mixture using an external magnetic field, it serves as a highly suitable catalyst support aiding enzyme immobilization and magnetic recovery of the catalyst13. Crosslinking of a large number of different enzymes may be coated on the surface of IOMNPs due to their size and large surface area. This will also help in cut down the additional purification to remove the catalyst from the reaction mixture. It is noteworthy that, the magnetic nanoparticle itself easily aggregates by its magnetic dipolar attraction, which limits their applications. Therefore, it is necessary to do surface modification/functionalization so that the magnetic property helps to separate the magnetic-CLEAs from the reaction mixture. In order to overcome these limitations, we focused in the present study on the preparation of Fe3O4 NPs that were bifunctionalized with silica and amine groups. Then, immobilization of cellulase, xylanase and β-1,3-glucanase onto the bifunctionalized MNPs was achieved. The biochemical characterization of the free enzyme and immobilized form i.e. Iron silica-amine nanoparticles cross linked enzyme aggregates (ISN-CLEAs) was also investigated. Finally,


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saccharification of sugarcane bagasse pulp (SCBP) and eucalyptus pulp (EUCAP) by the free and immobilized enzyme (ISN-CLEAs) was demonstrated.

EXPERIMENTAL Materials and chemicals Beechwood xylan, carboxymethylcellulose sodium salt (CMC), β-D-glucan, Tetraethyl orthosilicate (TEOS), 3-(Aminopropyl)triethoxysilane (APTES), glutaraldehyde solution, 3,5-Dinitrosalicylic acid (DNS), ammonium sulfate, arabinose, galactose, glucose, xylose and mannose were purchased from Sigma-Aldrich. Other reagents were analytical grade and obtained from Carl Roth. Eucalyptus pulp (FIBRIA-EUCA-BRASIL) was procured from FIBRIA paper and pulp industry, Brazil. Sugar cane bagasse (SCB) was locally harvested and was provided by E.I.D-PARRY (I) LTD, Tiruchirappalli, India. The biomass was finely chopped to 2-4 mm size and stored in a container at -20°C until further processing. Cellulase, xylanase and β-1,3glucanase were produced by solid-state fermentation from a strain of Trichoderma citrinoviride AUKAR04. Synthesis of Iron Oxide Magnetic Nanoparticles (Fe3O4 or IOMNPs) There are numerous chemical methods that have been reported for the synthesis of IOMNPs 14– 16

.

In this study, modified co-precipitation method was adopted for the preparation of

IOMNPs17. In this method, 0.4 M of FeCl3.6H2O (6.5 g) and 0.2 M of FeCl2.4H2O (2.4 g) were dissolved in 60 mL of deoxygenated Millipore water and heated up to 80 °C. Then ammonium hydroxide solution (NH4OH-25%) was added dropwise until the appearance of visible precipitates under constant stirring at 500 rpm for 30 min. The solution was left undisturbed for 1


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h for the settling of magnetic nanoparticles at the bottom. Finally, the resulting IOMNPs was separated by magnetic decantation and washed thrice with ultrapure water (MilliQ, Millipore co.,) and freeze-dried at -80 °C with 0.014 mbar pressure for 3 days10. Synthesis of silica anchored-amine Functionalized IOMNPs Two types of functionalization have been carried out to obtain silica anchored-amine functionalized IOMNPs. First, anchoring of silica (SiO2) group onto Fe3O4 NPs was carried out by modified salinization method18. Briefly, 400 mg of Fe3O4 NPs were suspended in 5 mL of ethanol-deionized water (3:1), which was heated to 45 °C and sonicated in a sweeping mode for 20 min. To this solution, 500 µL of tetraethyl orthosilicate (TEOS) and 400 µL of triethanolamine were added and the mixture was kept under sonication for 30 min. Finally, the resulting silica anchored nanoparticles were separated by magnetic decantation and washed thrice with deionized water and ethanol, then freeze-dried in a lyophilizer. Amine (-NH2) functionalization on silica anchored IOMNPs was done by dispersing 200 mg of Fe3O4/SiO2 NPs in a solution mixture of 5 mL deionized water and 5 mL methanol. This solution was sonicated for 30 min and then, 500 µL of 3-(Aminopropyl) triethoxysilane (APTES) and 250 µL of triethanolamine were added and sonicated for 30 min. The resultant silica anchored-amine functionalized nanoparticles were collected by magnetic separation and washed five times with methanol and water and then freeze-dried in the lyophilizer. Production of Enzyme and its assay method Cellulase, xylanase and β-1,3-glucanase were produced in solid-state fermentation (SSF) by a strain of Tricoderma citrinoviride AUKAR04 and the enzymes cocktail were partially purified by three-phase partitioning (TPP) method, then redissolved in 25 mL of 10 mM sodium


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acetate buffer (pH-5.0) and finally, dialyzed against 2 mM sodium acetate buffer (pH-5.0) for 10 h at 4 °C. The obtained enzyme solution was stored in a refrigerator19,20. The activities of cellulase, xylanase and β-1,3-glucanase were assayed and the amounts of reducing sugar liberated was estimated by the method adopted in our previous studies21,22. In this connection, producing enzymes at laboratory scale is a major advantage and minimizes the procurement costs of the enzymes. The total protein concentration was measured by the Bradford’s method with bovine serum albumin as the standard23. Cross linking of cellulase, xylanase and β-1,3-glucanase into silica anchored-amine functionalized IOMNPs The cross-linking of cellulase, xylanase and β-1,3-glucanase onto silica anchored-amine functionalized IOMNPs was carried out as follows: 500 mg of Fe3O4@SiO2-NH2 NPs were dispersed in 10 mL of enzymes solution which contains cellulase (150 U/mg), xylanase (800 U/mg) and β-1,3-glucanase (500 U/mg) in 50 mM sodium acetate buffer (pH-5.0). Subsequently, glutaraldehyde solution with various concentrations (30-150 mM) was added dropwise into the mixture to crosslink the enzymes onto the magnetic nanoparticles. The mixture was incubated at 30 °C under agitation (150 rpm) for different time intervals (2 -14 h). After crosslinking, the resulting immobilized cellulase, xylanase and β-1,3-glucanase onto Fe3O4@SiO2-NH2 NPs were separated by external magnetic field and washed thrice with 50 mM sodium acetate buffer (pH5.0) and stored in the same buffer at 4 °C for subsequent use. The activity recovery (%) of cellulase, xylanase and β-1,3-glucanase in the Fe3O4@SiO2-NH2 NPs-cross linked enzyme aggregates (ISN-CLEAs) were determined by the following equation22 (1). Activity recovery (%) =

× 100

(1)


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The binding efficiency of cellulase, xylanase and β-1,3-glucanase onto ISN-CLEAs was calculated by the equation (2)

Binding efficiency =

!" !#

×v

(2)

Where, Ei as the initial concentration of the free enzymes and Ef as the final concentration of enzymes (mg/mL), m is the mass of Fe3O4@SiO2-NH2 NPs (mg) and v is the volume of solution (mL). The residual activity of cellulase, xylanase and β-1,3-glucanase was estimated by using the equation (3) Residual activity (%) =

%

%

× 100

(3)

Characterization of free ISN-CLEAs Structural characterization The surface morphology of ISN-CLEAs was examined on Environmental Scanning Electron Microscope (ESEM-FEI Quanta 200) and Atomic Force Microscopy (Veeco-icon ScanAsyst, USA). The structural changes were observed in ATR-FTIR (Perkin-Elmer Spectrum 65, USA). The hydrolyzed sugars were quantified by High Performance Liquid Chromatography (HPLC) with Pulse Amperometric Electrochemical Detection on a Dionex ICS5000 HPLC. Dynamic Light Scattering (VASCOTM Particle Size Analyze) used to measure the size distribution of the magnetic nanoparticles.


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Thermal stability and storage study The thermal stability of free enzyme and xylanase, cellulase and β-1,3-glucanase in ISNCLEAs were investigated by incubating them in 50 mM sodium acetate buffer (pH-5.0) in the absence of substrate at 30°C, 40°C, 50°C, 60°C and 70°C. Samples were withdrawn at every 2 h intervals for a total time of 8 h. ISN-CLEAs were separated with an external magnet and assayed to determine xylanase, cellulase and β-1,3-glucanase activities under standard assay method. The residual activity of free enzyme and ISN-CLEAs at each temperature was correlated with the activity at an initial time (0 min) set as 100%. Storage stability of the free and ISNCLEAs was analyzed by incubating in 50 mM sodium acetate buffer (pH-5.0) at 4°C and the activity of each enzyme was measured at intervals of 10, 20, 30, 60, 90 and 120 days. Reusability The reusability of immobilized enzymes is an essential parameter in a large-scale industrial application for an economic reason. The operational stability of xylanase, cellulase and β-1,3glucanase in ISN-CLEAs was studied by the repeated usage in batch operation mode. After each cycle of a hydrolysis reaction, ISN-CLEAs was separated by an external magnet and washed twice with 50 mM sodium acetate buffer (pH-5.0) and then re-suspended in the fresh substrate. In case of free enzymes, it was not able to recover from the reaction mixture. The initial activity of free enzyme and ISN-CLEAs in the first cycle was set as 100%. Enzymatic hydrolysis of Sugarcane Bagasse Pulp (SCBP) and Eucalyptus Pulp (EUCAP) The depolymerization ability of free and ISN-CLEAs was studied based on the hydrolysis of various biomasses that include EUCAP and SCBP. The nature of each pulp can be described as


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follows. Eucalyptus pulp (Kraft cooking treatment followed by Elemental Chlorine-Free bleaching) was procured from FIBRIA-EUCA-BRASIL and it composed approximately 80% of cellulose, about 20 % of hemicelluloses and traces of lignin (less than 0.2%). This pulp was directly used for enzymatic hydrolysis. Besides, SCB raw biomass contains about 55% cellulose, 25% hemicelluloses, 20% lignin and 2% ash and other compounds. The analysis of lignin content and sugar profile of the biomass was performed according to the two-step acid hydrolysis procedure from the NREL protocol24. SCB pulp was prepared by added 50 g of finely chopped (about 2-5 mm) raw SCB (dry basis) in 500 mL of 10% (w/v) liquid ammonia, and it was cooked at 200 °C for 60 min. After this cooking process, the SCB was washed with distilled water until it reaches pH-7.0, and then air dried at room temperature (around 28-30 °C). All the biomasses were comminuted to get the millimetric size (0.5 to 1.0 mm) by using a Forplex hammer mill. In order to check the depolymerization efficiency of free enzyme and ISN-CLEAs, the hydrolysis of EUCAP and SCBP fiber was carried out separately by dispersing 10 g of each biomass in 100 mL of 50 mM sodium acetate buffer (pH-5.0) in a 250 mL conical flask with free and ISN-CLEAs. In each case, cellulase (120 U mg-1), xylanase (800 U mg-1) and β-1,3glucanase (550 U mg-1) were taken for biomass hydrolysis. The depolymerization reaction was carried out at 50 °C in a shaking water bath with 150 rpm for 60 h. At the beginning of the reaction, sodium azide (0.01%) was added to the reaction mixture to inhibit the microbial growth21. Aliquots were withdrawn at 2, 6, 12, 24, 36, 48 and 52 h. Monomeric sugars content was

analyzed

by

High-Performance

Anion-Exchange

Chromatography

with

Pulsed

Amperometric Detection (HPAEC-PAD, Dionex ICS 5000) equipped with a CarboPac PA 10 (250x4 mm, Dionex) column, preceded by a guard column (50 × 4 mm, Dionex). Samples were


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tested against standards consisting of arabinose, galactose, glucose, xylose and mannose. Glucose and xylose yields were calculated as follows. '( )×*.,

Glucose yield (%) = ( -) × 100 Xylose yield (%) =

. )×*.// 0 ( -)

(4)

× 100 (5)

Where, 0.9 is a conversion factor for cellulose to equivalent glucose 0.88 is a conversion factor for hemicelluloses to equivalent xylose

RESULTS AND DISCUSSION Preparation of ISN-CLEAs In order to achieve the maximum cross-linking of xylanase, cellulase and β-1,3-glucanase on functionalized magnetic nanoparticles, it was very important to optimize the basic parameters such as glutaraldehyde concentration and crosslinking time. Glutaraldehyde used as a cross linker, helps to bind the enzymes to nanoparticles via –NH2 terminal groups. As the results show in Figure.1, the maximum activity recovery of xylanase (97.8%), cellulase (97.5%) and β-1,3glucanase (96.3%) in ISN-CLEAs was obtained at 135 mM glutaraldehyde concentration. Further increase of glutaraldehyde concentration up to 150 mM, did not make any significant difference in the enzyme recovery. At the same time, after crosslinking with less than 120 mM glutaraldehyde concentration, unbound enzyme activities were detected in the supernatants indicating insufficient crosslinking with the MNPs. From the results, it was clearly understood that amino functionalization of nanoparticles helps in binding maximum enzyme molecule on its


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surface. Besides, the resultant ISN-CLEAs also retains magnetism and it was easily separated by an external magnet.

Figure 1. Activity recovery of xylanase, cellulase and β-1,3-glucanase in ISN-CLEAs with crosslinking time of 10 h at 30 °C. The 100% corresponds to 150 U mg-1 for cellulase, 800 U mg1

for xylanase and 500 U mg-1 for β-1,3-glucanase. All the experiments were done in triplicate

and the error was lower than 3% in each case of determinations. Crosslinking time was also a major factor for maximum activity recovery of all the enzymes in ISN-CLEAs. Based on the previous results, the amount of glutaraldehyde was fixed at 135 mM. Figure. 2 depicts the effect of crosslinking time on the activity recovery of xylanase, cellulase and β-1,3-glucanase. From the results, it was observed that increased activity recovery of xylanase (98.1%), cellulase (98.3%) and β-1,3-glucanase (97.3%) was attained with a crosslinking time of 10 h. Further increasing the crosslinking time up to 12 h did not make noticeable changes in the activity recovery of all the three enzymes, but extended crosslinking time up to 14 h decreased the enzyme activities resulting in a loss of enzymes’ flexibility due to


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excess crosslinking. Conversely, crosslinking time less than 10 h led to lower activity yield of enzymes due to insufficient crosslinking. The binding of xylanase, cellulase and β-1,3-glucanase on the bifunctionalized nanoparticles were achieved of about 98% with 135 mM glutaraldehyde concentration with 12 h of incubation time. Previous study reported on immobilization of cellulase enzyme on superparamagnetic nanoparticles found 95% of binding efficiency after 7 hours of incubation25.

Figure 2. Effect of cross linking time on ISN-CLEAs preparation with 135 mM glutaraldehyde concentration. The 100% activity recovery corresponds to 800 U mg-1 for xylanase, 120 U mg-1 for cellulase and 500 U mg-1 for β-1,3-glucanase. The percentage activity recovery of each enzyme in ISN-CLEAs was calculated by considering the initial activity as 100%. All the experiments were done in triplicate and the error bar shows the percentage error in each set of readings. Structural analysis by Fourier transform infrared spectroscopy (FTIR)


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FTIR spectra of Fe3O4, Fe3O4@SiO2-NH2 and ISN-CLEAs are illustrated in Figure 3. The peak at 591 cm-1 corresponds to Fe-O bond in Fe3O4 NPs15, which shows the successful synthesis of magnetic nanoparticles. The silica group was attached to the Fe3O4 NPs by Fe-O-Si bonds and this was confirmed by the presence of SiO2 stretching in the range from 691, 1024 and 1120 cm-1 (Figure 3B)15,26. The characteristic peaks at 1317, 1482 and 1557 cm-1 attributed to the bending or vibration of the -NH2 groups thus confirming the functionalization with amino groups onto the silica anchored MNPs27,28. The peaks in the range of 1400 to 1600 cm-1 indicate the symmetric stretching of COO-, C=O and C-O groups29,30. The stretching pattern near 1557 and 1405 cm-1 also represents the binding of the carboxyl groups in the enzymes and amine groups of the MNPs. The observations of FTIR spectra led to the conclusion that the enzymes were successfully cross-linked onto the bi-functionalized magnetic nanoparticles.

Figure 3. FTIR spectra of (A) Fe3O4 (B) Fe3O4@SiO2-NH2 and (C) ISN-CLEAs Surface characterization of ISN-CLEAs by AFM and SEM


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The morphology of the Fe3O4 NPs, Fe3O4@SiO2-NH2 NPs and ISN-CLEAs were examined by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The SEM images (Figure. 4) of Fe3O4@SiO2-NH2 and ISN-CLEAs were the spherical structure of aggregates and the enzymes were bound together into balls onto the functionalized MNPs. The images in Figure. 5 clearly evidence that the morphology of bifunctionalized NPs was significantly modified after immobilization with xylanase, cellulase and β-1,3-glucanase. The nanoparticles are the spherical shape with the average size of 80 nm to 90 nm and the ISN-CLEAs are seen with large accumulated layers due to the binding of enzyme molecules on the bifunctionalized MNPs. To further characterize the variations in the surface topography of Fe3O4 NPs, Fe3O4@SiO2-NH2 and ISN-CLEAs, three-dimensional AFM images (Figure 5.B, 5.D and 5.F) were used to explain the strong visual impact of the structural variations31. It is worth noticing that the surface of the ISN-CLEAs exhibited gaps which showed that there was sufficient crosslinking between the enzymes and nanoparticles. These observations led to the conclusion that the ball structure might pave way for the movement of the substrate to inner enzymes.

Figure 4. SEM of (a) Fe3O4@SiO2-NH2 and (b) ISN-CLEAs


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Figure 5. Topography of (A, B) Fe3O4 (C, D) Fe3O4@SiO2-NH2 and (E, F) ISN-CLEAs Particle size analysis by Dynamic Light Scattering (DLS) The hydrodynamic diameter of the Fe3O4, Fe3O4@SiO2-NH2 and ISN-CLEAs was determined by DLS. The average diameter of the Fe3O4 NPs was 82.2 nm (Figure 6a.) after it functionalized with –NH2 and SiO2 group, the size was increased to 86.4 nm (Figure 6b.). On the other hand, the average diameter of the ISN-CLEAs was about 976.9 nm. This reveals that the immobilization of enzymes was achieved on the surface of functionalized magnetic nanoparticles.


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Figure 6. DLS of Fe3O4(a), Fe3O4@SiO2-NH2 (b) and ISN-CLEAs(c). Thermal stability of free and immobilized enzyme (ISN-CLEAs) The thermal stability of free and immobilized enzyme (ISN-CLEAs) was investigated by incubating them without substrate at a wide range of temperature. The results for thermal stability of free and ISN-CLEAs at 30°C, 40°C, 50°C, 60°C and 70°C with respect to different time are shown in Figure 7. Xylanase, cellulase and β-1,3-glucanase in ISN-CLEAs showed higher thermal stability than that of free enzymes. At 50°C, the enzymes in ISN-CLEAs were retained more than 95% activity for at least 2 hours of incubation time. As observed, ISNCLEAs retained more than 75% of their initial activities at 70°C for 2 hours of incubation. Extended incubation to 4 hours and subsequently 8 hours led to significant reduction in the residual activities of all the three enzymes in ISN-CLEAs (Refer Table S1). These observations led to the conclusion that amine-functionalized magnetic nanoparticles covalently linked with


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xylanase, cellulase and β-1,3-glucanase (ISN-CLEAs) provides more effective conformational stabilization to its secondary structure through a series of hydrogen bonds and hydrophobic interactions leading to increased thermal stability as compared to the free enzymes. Besides, the smaller size of the MNPs may allow enzyme molecules to expand over its surface with the better exposure of the active site channel31–33.

Figure 7. Thermal stability of xylanase, cellulase and β-1,3-glucanase in free enzymes and ISNCLEAs at 30°C, 40°C, 50°C, 60°C and 70°C after 2 h (A), 4 h (B) and 8 h (C). The experiments were performed in triplicates and the error bar shows the percentage error in all set of experiments.


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Storage study The storage stability of free enzymes and ISN-CLEAs was evaluated at 4°C for a period of 120 days. Xylanase, cellulase and β-1,3-glucanase activities in ISN-CLEAs was checked at different intervals of 10, 20, 30, 60, 90 and 120 days. As shown in Figure 8, the free enzymes lost almost 30-40% of their initial activities, but ISN-CLEAs contain enzymes that retained more than 97% of their initial activities even after 120 days of incubation. Therefore, ISN-CLEAs exhibited much extended storage stability compared to that of free enzymes.

Figure 8. Storage stability ISN-CLEAs at 4°C in 50mM sodium acetate buffer (pH 5.0). Reusability of xylanase, cellulase and β-1,3-glucanase in ISN-CLEAs The reusability of a biocatalyst is an important factor in the practical applications for economic reasons. The reusability of xylanase, cellulase and β-1,3-glucanase in ISN-CLEAs were


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examined up to 10 cycles in the reaction mixture containing 10 mL of 1% (w/v) of beechwood xylan, carboxymethyl cellulose (CMC) and β-D-glucan, incubated at pH 5.0 and temperature of 50°C. After each cycle of the hydrolysis reaction (10 min), ISN-CLEAs was separated by an external magnet and washed with 50mM sodium acetate buffer and then re-suspended in a fresh reaction mixture. As shown in Figure.9, the activities of xylanase, cellulase and β-1,3-glucanase in ISN-CLEAs retained at more than 97% up to sixth cycles, but began to decrease after the sixth cycle (80%), and subsequently to about 60% after the ninth cycle. Previous study was conducted with carrier-free cross-linked enzyme aggregates of xylanase, cellulase and β-1,3-glucanase (combi-CLEAs)22. Comparatively, ISN-CLEAs retained better residual activity than combiCLEAs and it was easy to recover from the reaction mixture. The decrease in the enzymes' activity after a certain number of hydrolysis cycles in the ISN-CLEAs could be due to the gradual leakage of enzymes and loss of particles during magnetic separation, and/or enzyme denaturation32.


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Figure 9. Reusability of xylanase, cellulase and β-1,3-glucanase in ISN-CLEAs

Enzymatic hydrolysis of Sugarcane Bagasse Pulp (SCBP) and Eucalyptus Pulp (EUCAP). The enzymatic hydrolysis of the SCBP and EUCAP fiber was performed at 50 °C in a water bath shaker at 150 rpm for 52 h. Xylanase (800 U mg-1), cellulase (120 U mg-1) and β-1,3-glucanase (550 U mg-1) were used either as free enzymes or as an ISN-CLEAs. Free enzymes hydrolyzed the SCBP and released a maximum amount of glucose (30.7 g L-1), xylose (12.3 g L-1) and arabinose (0.7 g L-1) after 24 h of incubation at 50 °C. A prolonged incubation up to 48 hours did not make significant changes in the sugar yield (Figure 10.a). On the other hand, ISN-CLEAs hydrolyzed the SCBP and yielded the maximum amount of about 34.4 g L-1 of glucose, 14.6 g L-1 of xylose and 0.9 g L-1 of arabinose (Figure 10.b) with an incubation time of 48 h. It was found that, extended incubation up to 52 hours, no noticeable sugar yield change


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in the reaction mixture. After the first cycle of hydrolysis, ISN-CLEAs was separated by an external magnet and washed thrice with 50mM sodium acetate buffer (pH-5.0) and then added to the fresh substrate (5%) with the same reaction condition, it produced a maximum of glucose (6.26 g/l), xylose (2.22 g/l) and arabinose (0.11 g/l) after 8 hours of incubation. As shown in Figure 10.c, the hydrolysis of eucalyptus pulp (EUCAP) by free enzymes liberated of about 60.2 g L-1 of glucose, 12.3 g L-1 of xylose and 0.44 g L-1 of arabinose after 36 h of incubation time. Concomitantly, ISN-CLEAs containing xylanase, cellulase and β-1,3glucanase acted on EUCAP and released a maximum amount of glucose (70.4 g L-1), xylose (13.8 g L-1) and arabinose (0.53 g L-1) after 48h of incubation. Similarly, the second cycle of hydrolysis was carried out and yielded of about 11.6 g L-1 of glucose and 4 g L-1 of xylose. The saccharification results evidenced the fact that the immobilized enzyme i.e. ISN-CLEAs would take a longer time to hydrolyze the solid substrate than its free counterpart, which might be due to the limited exposure of enzymes’ active site to the substrate and restriction of the internal mass transport in the accumulated enzyme layers of the NP’s, which was also examined during AFM and SEM analysis. The overall conversion rate of glucose from cellulose and xylose from hemicelluloses was higher than that of free enzymes.


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Figure 10. The yield of glucose, xylose and arabinose from 10 % sugarcane bagasse pulp (SCBP) by free enzyme (a), ISN-CLEAs (b) and the yield of glucose, xylose and arabinose from 10 % Eucalyptus pulp (EUCAP) by free enzyme (c), ISN-CLEAs (d). The error bars indicate the standard errors of three independent experiments. Globally, glucose and xylose are the main components of SCBP and EUCAP. Saccharification of SCBP and EUCAP biomass to glucose and xylose was carried out with free and ISN-CLEAs, and the yield percentage was tabulated in Table.1. From the results, it is clearly evident that the yield of glucose and xylose from both biomasses have been considerably increased (10-15%) by the ISN-CLEAs compared to its free counterpart. Thus immobilization of free enzymes increased its activity as well as preventing loss during hydrolysis reactions.


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Table. 1 Overall hydrolysis yield of sugarcane bagasse pulp (SCBP) and Eucalyptus pulp (EUCAP) by free and ISN-CLEAs

Enzyme form

Hydrolysis yield* (%) of monomeric sugar from SCBP

Hydrolysis yield (%) of monomeric sugar from EUCAP

Glucose

Xylose

Glucose

Xylose

Free enzymes

76.9

61.3

75.3

68.1

ISN-CLEAs

86.0

73.3

88.0

76.7

*The performance of enzymatic hydrolysis was calculated by quantifying glucose and xylose yield from SCBP and EUCAP biomass, and this was expressed as the percentage of glucose and xylose released in relation to the total amount of glucose and xylose present in the pretreated biomass.

CONCLUSION This article evidenced the great interest of ISN-CLEAs, as magnetic NP’s catalysts bearing layers of covalently bound active hydrolytic enzymes that can be reused in several cycles while retaining their activities on different polysaccharides substrates (xylans, glucan). Their best advantages are (1) very easy recovery due to their magnetic properties (a simple magnet placed in the reaction mixture allow their accumulation, recovery, and reuse as suspended NP’s after simple washing with water), and (2) their long time stability compared to free enzymes. Their structure as solid cross-linked enzymes deposited in several layers on a core Fe-O-Si nanoparticle, and the existence of covalent bonds was evidenced by different techniques based on microscopy and spectroscopy. However, it was noticed that such a solid state structure of these catalytic NP’s had the effect of decreasing the rate of substrate hydrolysis, compared to the rate obtained with free suspended enzymes in similar reaction medium, which can be imparted to


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mass transfer limitation in the solid enzyme layers and therefore restriction of the access to the active site of the enzymes.


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ASSOCIATED CONTENT Supporting Information The data of thermal stability of free and Immobilized enzyme are available free of charge on the ACS Publications Website. Author Information Corresponding Author E-mail: [email protected] (Sivanesan); Phone: +91-44-22359168. ORCID Karthik Periyasamy: 0000-0001-6956-5999 Sivanesan Subramanian: 0000-0002-2103-4862

ACKNOWLEDGEMENTS The author Karthik Periyasamy is grateful to the financial support from the European Commission (Erasmus Mundus Action 2 India4EU II–grant number: Indi1200061). LGP2 is part of the LabEx Tec 21 (Investissements d'Avenir – grant agreement no. ANR-11-LABX-0030) and of the Energies du Futur and PolyNat Carnot Institutes. This research was made possible thanks to the facilities of the TekLiCell platform funded by the Region Rhône-Alpes (ERDF: European Regional Development Fund). ABBREVIATIONS ISN-CLEAs, Iron Silica-Amine Nanoparticles- Cross Linked Enzyme Aggregates; SCBP, Sugarcane Bagasse Pulp; EUCAP, Eucalyptus Pulp.


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TABLE OF CONTENT (TOC)

Cellulase, xylanase and β-1,3-glucanase bound silica-amine functionalized iron oxide magnetic nanoparticles (ISN-CLEAs) were prepared and used as the biocatalyst for the depolymerization of cellulosic biomass into monomeric sugars.


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Graphic for manuscript Fe3O4@SiO2

Fe3O4@SiO2-NH2

ISN-CLEAs

Fe3O4 Cellulase

Xylanase β-1, 3-glucanase

Application on lignocellulosic Biomass

Reaction mixture Glucose

Xylose

Glucose

Xylose

Glucose Glucose

Glucose Glucose

Glucose

Sugarcane Bagasse Pulp Glucose

Xylose

Sugarcane Bagasse

Cellulase, xylanase and β-1,3-glucanase bound silica-amine functionalized iron oxide magnetic nanoparticles (ISN-CLEAs) were prepared and used as biocatalyst for the depolymerization of cellulosic biomass into monomeric.


Bioconversion of Lignocellulosic Biomass to Fermentable Sugars by

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