Carrier-free co-immobilization of xylanase, cellulase and β-1,3-glucanase as combined cross-linked enzyme aggregates (combi-CLEAs) for one-pot saccharification of sugarcane bagasse

Abstract

Combined cross-linked enzyme aggregates (combi-CLEAs) are an innovative prospect and a lucrative technology. The present study addresses the preparation, characterization and application of combi-CLEAs with xylanase, cellulase and β-1,3-glucanase to achieve one-pot bioconversion of lignocellulosic biomass to fermentable sugars. A three-phase partitioning (TPP) method was used to aggregate the enzymes. Glutaraldehyde (100 mM) was employed as a cross-linker with the cross-linking time of 7.5 h. Scanning electron microscopy of the tri-enzyme biocatalyst has a coarse-grained appearance. Combi-CLEAs were more thermally stable, retaining about 70% of their initial activity at 70 °C compared to 30% for the free enzyme. The storage stability of combi-CLEAs was more than 97% of their activity after incubation for 11 weeks at 4 °C, whereas the free enzymes retained about 65% of initial activity. The residual activity of combi-CLEAs remained constant at 90% until the sixth cycle. Contrary to free enzymes that remain in the hydrolysate, which prevents their recovery, reuse of combi-CLEAs was possible. Free enzymes hydrolyze the ammonia cooked sugarcane bagasse at about 73%, whereas the combi-CLEAs resulted in maximum hydrolysis of about 83.5% in 48 h.

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1. Introduction

Research on bioethanol production from lignocellulosic biomass has attracted considerable attention, particularly because of depletion of fossil fuels, the high cost of exploration and global warming. Lignocellulosic biomass is considered as a potential and sustainable resource for the production of bioethanol, and can provide sufficient feedstock without threatening food security.¹ Lignocellulosic biomass comprises of two carbohydrate polymers, namely cellulose and hemicelluloses. The specific composition depends on the source of the biomass and the content of lignin linked to polysaccharides into a complex matrix which is highly resistant to biochemical conversion. For the effective production of bioethanol, cellulose and hemicelluloses must be broken down into elemental sugars, mainly glucose and xylose, prior to their conversion into ethanol by fermentation.²

Cellulase is a group of enzymes that catalyze the hydrolysis of cellulose to yield oligosaccharides and glucose. Xylanase randomly hydrolyzes the β-1,4-glycosidic bonds of xylan to produce several xylo-oligomers and xylose. β-1,3-Glucanase can release glucose units from the non-reducing end of the β-1,3-glucan chains. In recent years, a combination of these enzymes has received significant research interest due to their potential industrial applications, especially in biorefineries and bioethanol production. Use of cellulase, xylanase and β-1,3-glucanase to hydrolyze the pretreated lignocellulosic biomass is preferable than chemical hydrolysis processes. However, production of enzyme cocktail is more expensive, and it is often complicated to separate the used enzymes from the hydrolysis mixture. Therefore, it is desirable to recover the proteins after hydrolysis so that the enzymes can be reused several times.³

Three-phase partitioning (TPP) involves the addition of ammonium sulfate to the crude extract followed by the addition of t-butanol, is a promising method employed for separation of proteins. The addition of organic solvents in the presence of salt pushes the protein out of the solution to form an interfacial precipitate layer between lower aqueous and upper organic phase.^4,5 Cross-linked enzyme aggregates (CLEAs) are a versatile immobilization strategy to enhance the stability and re-usability of the biocatalyst.⁶ Insoluble enzyme aggregates engendered by extensive chemical cross-linking of enzyme dissolved in a solution have been recommended as an alternative approach for obtaining stable and reusable enzyme preparations. An interesting characteristic of the CLEAs is that the preparation does not require extensively purified enzymes.⁷ There are several reports on the individual immobilization of laccase,⁸ alpha-amylase,⁹ lipase,¹⁰ penicillin G acylase,¹¹ α-L-rhamnosidase,¹² and only a few reports exist for co-immobilization of two or three different enzymes.^3,13,14

Combi-CLEAs have been mostly used for attaining two sequential steps for a single biotransformation process. Also, utilizing co-immobilized enzymes for multi-step enzymatic reaction is possible into one-pot, as enzymatic cascade processes, without requiring intermediate separation. The enhanced storage stability and reusability can significantly reduce the cost of enzymes and thus makes the industrial application economically feasible.^15,16 Nevertheless, there are no reports on co-immobilization of cellulase, xylanase and β-1,3-glucanase together for the saccharification of pre-delignified lignocellulosic biomass to get simple sugars by a single-step hydrolysis process.

In the present work, we show that this concept is particularly useful in the perspective of preparation of combi-CLEAs with xylanase, cellulase and β-1,3-glucanase to carry out simultaneously three different non-cascade reactions – hydrolysis of xylan, cellulose and β-glucan. The biochemical characterization of the free and immobilized enzyme was investigated. The catalytic efficiency of combi-CLEAs was assessed based on its thermal-stability, re-usability and storage.

2. Materials and methods

2.1 Chemicals

Beechwood xylan, carboxymethylcellulose sodium salt (CMC), β-glucan from barley, glutaraldehyde solution, 3,5-dinitrosalicylic acid (DNS), potassium hydroxide (KOH), ammonium sulfate, arabinose, galactose, glucose, xylose and mannose were procured from Sigma-Aldrich. Other reagents used were of analytical grade and purchased either from Carl Roth or Sigma.

2.2 Enzyme cocktail production

Production of xylanase, cellulase and β-1,3-glucanase by solid-state fermentation from a mutant strain of Trichoderma citrinoviride AUKAR04 [GenBank: KF698728]. The cultivation of Trichoderma sp. in SSF system was carried-out in a shallow aluminum tray of 20 cm × 14 cm × 6 cm. 250 g of wheat bran was moistened with 250 mL of 50 mm sodium acetate buffer (pH 5.0) autoclaved at 121 °C for 45 min. After cooling the tray at room temperature, it was inoculated with 150 mL of seed media with 40–50% of packed mycelium volume. The inoculum and the wheat bran were mixed well by using a sterile spatula in order to ensure a uniform distribution. The trays were incubated at 30 °C for 72 h. The moisture content in this study was 56%. 100 g of fermented solid substrate was mixed with 500 mL of 0.1% Twen-80 in 50 mM sodium acetate buffer (pH-5.0). The sample was then mixed using a rotary shaker (175 rpm) at 25 °C for 2 h. The suspension was centrifuged and the supernatant was passed through Whatman no. 1 filter paper and the clear cell-free filtrate was used for further experimental studies as the enzyme source.⁴ In this context, a major advantage of producing enzyme cocktails at laboratory scale is to minimize the costs for procurement of enzymes.

2.3 Precipitation of enzymes

Free xylanase (800 U mg⁻¹), cellulase (120 U mg⁻¹) and β-1,3-glucanase (550 U mg⁻¹) were precipitated by ammonium sulfate, n-proponol, acetone, ethanol and three-phase partitioning (TPP) method. TPP method was carried out by the following methodology: initially, ammonium sulfate was added to a final concentration of 55% of saturation in 25 mL of crude enzyme solution containing xylanase, cellulase and β-1,3-glucanase. Then, t-butanol was added at a volume ratio of 1 : 0.75 (t-butanol:55% ammonium sulfate saturated crude enzyme solution). The mixture was incubated at 25 °C for 1 h under constant stirring (100 rpm). Then stirring was stopped after 30 min and the solution was left at rest. The mixture was centrifuged (4000g for 10 min) to facilitate phase separation.¹⁷ Then, the interfacial aggregates were collected, redissolved in 10 mL of 10 mM sodium acetate buffer (pH 5.0) and dialyzed against distilled water overnight at 4 °C.

2.4 Preparation of combi-CLEAs

Combi-CLEAs containing xylanase, cellulase and β-1,3-glucanase were prepared by successive aggregation and cross-linking of the enzymes. The latter operation includes the drop-wise addition of various concentrations of glutaraldehyde (20–140 mM) to the mixture and incubated at 30 °C under agitation (220 rpm) for different time intervals (1.5–8.5 h). The suspension was centrifuged at 10 000g for 10 min. The supernatant was discarded, and the pellet was washed until there was no trace of enzyme activity in the supernatant. Finally, the aggregate was stored in 50 mM sodium acetate buffer (pH 5.0) at 4 °C.

The percentage activity recovery of xylanase, cellulase and β-1,3-glucanase in combi-CLEAs was determined by the following eqn (1).

2.5 Enzyme assays

Xylanase activity was routinely measured in a reaction mixture (1.0 mL) containing 0.5 mL of 1% (w/v), beech wood xylan in 50 mM sodium acetate buffer (pH 5.0) and 0.5 mL of each enzyme solution. The substrate and enzyme solution were pre-incubated separately at 50 °C for 5 min, and then the reaction was started by mixing the enzyme with the substrate. After 10 min incubation, the reaction was stopped by the addition of 1 mL DNS reagent.¹⁸ The reaction terminated at zero times was used as control tubes. The absorbance (OD) of the reducing sugar was measured at 540 nm. The standard graph was prepared using 1 to 5 μmol mL⁻¹ xylose in 50 mM sodium acetate buffer. One unit (U) of xylanase activity is defined as the amount of enzymes required to release 1 μmol of xylose per minute in the reaction mixture under the specified assay conditions.

CMCase activity was determined by measuring the amount of glucose released from CMC-Na by DNS method with glucose as the standard. The reaction mixture contained 0.5 mL of 1% CMC-Na in 50 mM citrate buffer (pH 5.0) and 0.5 mL of enzyme solution and was incubated at 50 °C for 10 min.¹⁹ After incubation, the control and the samples were terminated by the addition of 1 mL DNS then kept in the boiling water bath for 10 min and made it to cool down at room temperature. The absorbance of the reaction solutions were measured at 540 nm. One unit (U) of CMCase activity is defined as the amount of enzymes that liberates 1 μmol of glucose equivalents per minute under the assay conditions.

β-1,3-Glucanase activity was measured by mixing 200 μL of enzyme solution with 200 μL of 50 mM sodium acetate buffer (pH 5.0), containing 0.8% β-glucan from barley.²⁰ The enzyme assay was carried out at 50 °C for 10 min. The reducing sugar liberated was quantified by DNS method. One unit (U) of activity is the amount of enzyme required to release 1 μmol of reducing sugar per minute under the above conditions. All assays were performed in triplicates. The amount of total protein was determined by Bradford's method, with bovine serum albumin (BSA) as the standard.

2.6 Characterization of combi-CLEAs

2.6.1 Structural characterization by scanning electron microscopy. The surface morphology of combi-CLEAs was analyzed by scanning electron microscope (SEM). Samples were dried under vacuum and then placed on a carbon tape over a microscope slide to coat with gold particles using a sputter coater.

2.6.2 Thermal stability and storage study. The thermal stability of free enzymes and combi-CLEAs was investigated by incubating in 50 mM sodium acetate buffer (pH 5.0) without substrate at 30 °C, 40 °C, 50 °C, 60 °C and 70 °C. Aliquots were withdrawn at every 2 h interval for a total time of 8 h. Supernatants were collected by centrifugation, and the immobilized biocatalyst was assayed to determine xylanase, cellulase and β-1,3-glucanase activities under standard assay method. The residual activity of each enzyme at each temperature was compared with the activity at initial time (0 min) taken as 100%.²¹ The immobilized enzyme was stored at 4 °C in 50 mM sodium acetate buffer, and the activity of each enzyme was measured at intervals of 1, 3, 5, 7, 9 and 11 weeks.

2.6.3 Reusability. The reusability of immobilized enzymes is one of the crucial factors in industrial applications. The reusability of enzymes in combi-CLEAs was studied under standard assay conditions. After each cycle of a hydrolysis reaction, combi-CLEAs were separated by centrifugation and washed twice with 50 mM sodium acetate buffer (pH 5.0) and then re-suspended in the fresh substrate. The activities acquired in the first cycle for the immobilized enzyme was taken as the control and correspond to 100% activity.

2.7 One-pot hydrolysis of sugarcane bagasse

Sugarcane bagasse (SCB) contained about 55% cellulose, 25% hemicelluloses and 20% lignin. Cooking pretreatment (delignification) consisted in mixing 10 g of SCB (dry basis) in 100 mL of 10% (w/v) liquid ammonia in closed glass bottle. This reaction mixture was autoclaved at 140 °C for 120 min.²² After this cooking step, the SCB was washed with distilled water and air-dried at room temperature, then grinded in a Forplex hammer mill to obtain a biomass powder of mill metric size (0.5 to 1 mm).

The analysis of the lignin and sugar content profile of the SCB pulp was made by classical hydrolysis using sulfuric acid, as follows: about 0.3 g of SCB pulp (as dry matter) was mixed with 3 mL of 72% (w/w) sulfuric acid at room temperature; the mixture was stirred at the beginning with a glass rod, and then occasionally during a total time of one hour; then 84 mL of de-ionized water was added to the mixture and autoclaved at 121 °C for 1 h under saturated water pressure. After cooling, the mixture was filtered on a sintered glass crucible of fine porosity (porosity index of 4). The filtrate was properly diluted (120 times for glucose and 40 times for the other sugars) for elemental sugar content analysis by high performance anion exchange chromatography, and the dry lignin residue on the crucible was weighed after washing and oven-drying at 105 °C during 24 h.

One-pot enzymatic saccharification of SCB was carried out by adding the free enzyme and combi-CLEAs separately in 250 mL shake flasks containing 3 g of pretreated SCB in 100 mL of 50 mM sodium acetate buffer (pH 5.0). The reactions were carried out at 50 °C on a thermostatic rotary shaker at 180 rpm for 57 h. At the beginning of the reaction, 0.01% (w/v) of sodium azide was added to the reaction mixture to inhibit microbial growth or contamination.²³ During the reaction time, aliquots were withdrawn at 2, 9, 24, 33, 48 and 57 h. Monomeric sugar content was analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD, Dionex ICS 5000) equipped with a CarboPac PA 10 (250 × 4 mm, Dionex) column situated after a guard column (50 × 4 mm, Dionex). Columns and detectors were in a compartment regulated at 25 °C. KOH was used as an eluent at a flow rate of 1 mL min⁻¹ and the concentration of KOH was 2 mM during 30 min (analysis time) followed by 100 mM for 15 min (column washing and regeneration). Samples were diluted appropriately, and the pH was adjusted between 8 and 9 and then filtered through 0.45 μm syringe filters prior to injection. 25 μL of sample was injected into the column. Samples were measured against standards consisting of arabinose, galactose, glucose, xylose and mannose.

2.8 Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR)

The structural and chemical changes of SCB raw material, liquid ammonia pretreated SCB and CLEAs-treated SCB were characterized by ATR-FTIR analysis (samples used were solid state dry powders placed on the sample holder of the spectrometer). All samples were dried at 80 °C in an oven prior to analysis. The spectra were recorded by the FTIR spectrometer (Perkin-Elmer Spectrum 65) in the absorbance band mode in the range of 4000 cm⁻¹ to 600 cm⁻¹ with a resolution of 4 cm⁻¹ and 40 scans per sample.

3. Results and discussion

3.1 Effect of the precipitant on the recovery of cellulase, xylanase and β-1,3-glucanase

During combi-CLEAs preparation, the precipitation efficiency and maximum recovery of enzyme activity depend on the nature of the precipitant used, and hence, it is necessary to select the best precipitant.²⁴ In this study, five different types of precipitants were used to determine their effect on enzyme activity recovery prior to the cross-linking step. Fig. 1 shows that the combination of ammonium sulfate and t-butanol (TPP method) was found to be the best precipitant for obtaining maximum enzyme activity recovery (99%) of cellulase, xylanase and β-1,3-glucanase. TPP method exhibits the significant advantage of precipitating all proteins in their native structure.²⁵

Fig. 1 Effect of different precipitants on the residual activity of xylanase, cellulase and β-1,3 glucanase. The 100% activity recovery corresponds to 800 U mg⁻¹ aggregate for xylanase, 120 U mg⁻¹ aggregate for cellulase and 550 U mg⁻¹ aggregate for β-1,3-glucanase. All the experiments were done in triplicate and the error bar shows the error percentage in each set of reading.

3.2 Effect of glutaraldehyde concentration on combi-CLEAs preparation

The concentration of glutaraldehyde is one of the critical parameters in the preparation and recovery of active enzymes in the combi-CLEAs process. A lower concentration of cross-linker leads to insufficient cross-linking and further leaching out of enzymes during the hydrolytic process on the substrate.²⁶ Conversely, a higher concentration of glutaraldehyde results in the loss of enzyme activity due to excessive cross-linking between active sites.²⁷ Fig. 2 shows the influence of glutaraldehyde concentration on the activity and recovery of cellulase, xylanase and β-1,3-glucanase in combi-CLEAs. The results indicate that the highest activity recovery in combi-CLEAs was attained by using 100 mM glutaraldehyde concentration: 99.1 ± 3% for xylanase, 98.3 ± 3% for cellulase and 98.8 ± 3% for β-1,3-glucanase. Correspondingly, there was no significant activity of cellulase, xylanase and β-1,3-glucanase detected in the supernatant. The efficiency of reducing sugar release by the combi-CLEAs was checked against the appropriate substrates as described in the enzyme assay section. In this regard, the maximum amount of reducing sugar (24.5 mg mL⁻¹) was obtained by combi-CLEAs generated with the concentration of 100 mM glutaraldehyde (Fig. 2).

Fig. 2 Effect of glutaraldehyde concentration on the activity recovery of xylanase, cellulase and β-1,3 glucanase with the cross-linking time of 7.5 h at 28 °C. The 100% activity recovery corresponds to 800 U mg⁻¹ aggregate for xylanase, 120 U mg⁻¹ aggregate for cellulase and 550 U mg⁻¹ aggregate for β-1,3-glucanase. All the experiments were done in triplicate and the error bar shows the percentage error in each set of reading.

3.3 Effect of cross-linking time on combi-CLEAs

For the optimization of the cross-linking step, the effect of cross-linking time on the activity recovery of cellulase, xylanase and β-1,3-glucanase in the resulting combi-CLEAs was studied. Based on the previous results, the amount of glutaraldehyde concentration was fixed at 100 mM. Results in Fig. 3 show that an increased activity recovery is observed when the cross-linking time was increased up to 7.5 h at 30 °C. This was checked for each of the three enzymes, and no enzyme activities remained in the supernatant at a time of 7.5 h. Samples collected before that time exhibited some enzyme activity in the supernatant, due to incomplete cross-linking.

Fig. 3 Effect of cross linking time on combi-CLEAs preparation with 100 mM glutaraldehyde concentration. The 100% activity recovery corresponds to 800 U mg⁻¹ aggregate for xylanase, 120 U mg⁻¹ aggregate for cellulase and 550 U mg⁻¹ aggregate for β-1,3-glucanase. The percentage activity recovery of each enzyme in combi-CLEAs was calculated by taking the initial activity as 100%. All the experiments were done in triplicate and the error bar shows the percentage error in each set of reading.

In the samples collected after 7.5 h, we found a decreased activity of each enzyme in combi-CLEAs, but still no enzyme activity in the supernatant. This can be explained by the fact that prolonged cross-linking time hampers enzyme flexibility, due to more intensive cross-linking. Similar results were previously reported.^28–30

3.4 Characterization of combi-CLEAs

3.4.1 SEM of combi-CLEAs. The morphology of combi-CLEAs was characterized by SEM. Schoevaart et al., 2004,³¹ reported that CLEAs have either a spherical aggregate form (type 1) or a less-structured appearance (type 2). SEM of the combi-CLEAs (Fig. 4A) before the first cycle illustrated that it had coarse-grained appearance; more structured than type 2, but does not have the ball-like appearance of type 1 either. Similar structure of CLEAs in the presence of bovine serum albumin was also reported for lipase.³² Fig. 4B has a somewhat ball-type structure with few cavities (marked in redline) which might be due to the leaching out of enzymes.

Fig. 4 (A) SEM of the combi-CLEAs before first cycle; (B) after tenth cycle.

3.4.2 Thermal stability and storage study. The thermal stability of free enzymes (xylanase, cellulase and β-1,3-glucanase) and combi-CLEAs was analyzed by incubating them without substrate as specified in section 2.5. Residual activities are given in Fig. 5. All the three enzymes in combi-CLEAs exhibited higher thermal stability than that of free enzymes: xylanase, cellulase and β-1,3-glucanase in combi-CLEAs retained about 95% of their initial after 2 h incubation at 50 °C, while retention by free enzymes was 20–30% lower. At 70 °C, combi-CLEAs retained more than 70% of their original activities, while about 60% was lost by the free enzymes. The increased thermal stability of enzymes in combi-CLEAs might be due the covalent cross-linking between the enzyme aggregates.³³ Investigations in the literature for different enzymes at different temperatures (30 °C to 75 °C) for various incubation times gave similar results as the herein presented ones.^21,34,35 Prolonged incubation to 4 h and subsequently 8 h led to the significant reduction in the residual activities (Table S1 “ESI”†).

Fig. 5 Thermal stability of xylanase, cellulase and β-1,3-glucanase in combi-CLEAs and free enzymes at 30 °C, 40 °C, 50 °C, 60 °C and 70 °C after 2 h (A),4 h (B) and 8 h (C) of incubation.

For storage study, combi-CLEAs and free enzymes were stored in 50 mM sodium acetate buffer (pH 5.0) at 4 °C for a period of 12 weeks. Xylanase, cellulase and β-1,3-glucanase activities were checked at intervals of 1, 3, 5, 7, 9 and 11 weeks. As shown in Fig. 6, free xylanase, cellulase and β-1,3-glucanase retained only 69%, 65% and 63.4% of its initial activities respectively, while combi-CLEAs retained more than 97% of their initial activities after 11 week of incubation. Clearly, the combi-CLEAs have an extended storage stability compared to that of free enzymes.

Fig. 6 Storage stability of free xylanase, cellulase and β-1,3 glucanase and combi-CLEAs at 4 °C in 50 mM sodium acetate buffer (pH 5.0). The experiments were made in triplicate and the error bar stands for the percentage error in all set of experiments.

3.4.3 Reusability of xylanase, cellulase and β-1,3-glucanase in combi-CLEAs. The reusability of combi-CLEAs was studied up to 10 cycles in batch operation mode at pH 5.0 at 50 °C. After each cycle of reaction, combi-CLEAs was separated by centrifugation at 5000g and washed in 50 mM sodium acetate buffer and then re-suspended in a fresh reaction mixture. As shown in Fig. 7, the activities of xylanase, cellulase and β-1,3-glucanase in combi-CLEAs retained more than 98% up to five cycles, but started to decrease after the sixth cycle (90%), and subsequently to 50% after the ninth cycle. some enzyme contamination during the hydrolysis step would take place and be responsible of the loss of activity after a certain number of hydrolysis cycles. After reaching a certain level of contamination, reusability might decrease,³⁶ possibly due to enzyme leaching from combi-CLEAs during the centrifugation step which exerts a high shear strength on the combi-CLEAs aggregates.³³

Fig. 7 Reusability of xylanase, cellulase and β-1,3-glucanase in combi-CLEAs.

3.5 One-pot hydrolysis of SCB

One-pot enzyme hydrolysis was carried out on ammonia-cooked SCB pulp as a substrate. The release of monomeric sugars was followed by HPLC analysis of the liquid phase hydrolysate. SCB hydrolyzed by free enzymes released a maximum amount of arabinose (150 mg L⁻¹), galactose (20 mg L⁻¹), glucose (1005 mg L⁻¹), xylose (911 mg L⁻¹) and mannose (135 mg L⁻¹) after 24 h of incubation. The reaction of combi-CLEAs on SCB pulp liberated higher amounts of sugars, as illustrated in Fig. 8: arabinose (185 mg L⁻¹), galactose (32 mg L⁻¹), glucose (2023 mg L⁻¹), xylose (1015 mg L⁻¹) and mannose (150 mg L⁻¹). However, this was observed after 48 h of incubation time, which is remarkably higher than the time for free enzymes (Table S2 “ESI”†).

Fig. 8 Sugar concentrations of HPLC analysis of combi-CLEAs hydrolyzed SCB pulp. The concentration of free enzyme xylanase (800 U mg⁻¹), cellulase (120 U mg⁻¹) and β-1,3-glucanase (550 U mg⁻¹) as well as the combi-CLEAs used were same.

It can also be noticed that in the case of combi-CLEAs, the rate of hydrolysis increased significantly over the first period of 24 h, and prolonged incubation time up to 48 h did not make a remarkable change in the sugar yield. Also, it was observed that increasing agitation could increase the maximum sugar yield while decreasing the time to reach it, which is probably linked to the increased accessibility of the substrate to combi-CLEAs (data not shown).

Xylose and glucose recovery yields, which were the main sugars in the SCB pulp after enzyme treatment, are given in Table 1. This results show clearly that the enzyme treatments allow very high recovery yields, about 85% in the case of glucose, and over 70% for xylose in the case of combi-CLEAs treatment. Once again, it appears clearly that combi-CLEAs allow superior hydrolysis yields than free enzymes.

Table 1 Sugar recovery yield by free or CLEAs applied on the SCB pulp

Enzymes	Yield of recovered glucose and xylose (% on initial substrate content)^a
	Glucose	Xylose
a Yields were calculated as % amount of sugar in the enzyme hydrolysate on amount of sugar in the substrate. Substrate analysis was made by sulfuric acid hydrolysis. Only minor or negligible amounts of mannose, galactose and arabinose were found in the substrate by this method, and the results after enzyme hydrolysis were of the same order, even superior in the case of arabinose.
Free enzymes	77	54
Combi-CLEAs	84	71

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3.6 FT-IR analysis of SCB

Fig. 9 allows to compare the FTIR spectra of the untreated SCB, the ammonia-pretreated SCB pulp and combi-CLEAs treated SCB pulp. The band at 3338 cm⁻¹ is ascribed to the stretching of –OH groups. There is no significant variation there, showing the large abundance of polysaccharides in the pulps. Peaks between 1640 cm⁻¹ and 1700 cm⁻¹ are generally ascribed to C=O stretching in carbonyl functions, which is typical of the presence of aldehydes, ketones, carboxylic acids and esters.^37,38 The peak at 1740 cm⁻¹ refers to the acetyl group in hemicelluloses, which appears only in the untreated SCB. Absence of this peak in the ammonia-cooked SCB and in the combi-CLEAs treated SCB pulp implies that ammonia cooking in alkaline medium removes all esters. Variations of the bands in the region between 1500 cm⁻¹ and 1100 cm⁻¹, typical of aromatic C=C stretching³⁹ (1430 cm⁻¹) and various C–C and C–O linkages in lignin tend to indicate that delignification takes place not only during ammonia-cooking, but also during the enzyme treatment of the SCB pulp. The aromatic (C–O) stretching peak in lignin at about 1250 cm⁻¹ decreased significantly after ammonia-cooking of SCB. The peak in the region of 890 cm⁻¹ indicates the presence of C–O–C vibration of β-glycosidic linkage in hemicelluloses and cellulose.³⁹ It became weaker in combi-CLEAs treated SCB but not absent. This allows to conclude that hemicelluloses and cellulose are partially de-polymerized by the synergetic action of xylanase, cellulase and β-1,3-glucanase in combi-CLEAs.

Fig. 9 FTIR spectra of untreated, liquid-ammonia pretreated and combi-CLEAs treated SCB pulp.

4. Conclusion

This research study provides experimental evidence of the efficiency of carrier free combined aggregated enzymes (combi-CLEAs) formulations that include highly active xylanase, cellulase and β-1,3-glucanase. It was shown that combi-CLEAs could surpass the activity of isolated enzymes in terms of thermal stability and preservation during storage.

Here tested on ammonia-cooked sugarcane bagasse as lignocellulosic substrate to be hydrolyzed, their main advantage over single non-aggregated enzymes is the possibility of recovery by centrifugation and their reusability during several hydrolysis cycles.

Therefore, the glutaraldehyde cross-linking technique used here, applied with different enzymes to form combi-CLEAs structures, appears to be a promising tool for the pragmatic commercialization of lignocellulolytic enzymes with improved thermal stability, enhanced operational stability and reusability. Also, it evidently proves the effective synergy of the enzyme cocktail used in this study on lignocellulosic substrates breakdown into fermentable sugars for bioethanol production.

Acknowledgements

This work has been funded with support from the European Commission (Erasmus Mundus Action 2 India4EU II; 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 Région Rhône-Alpes (ERDF: European regional development fund).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00929h

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