{"paper_id":"0200b1e9-2b31-4dde-b3dc-dbbe3f0db705","body_text":"Layered polymeric carbon nitride as a green support for cellulase immobilization: Improved stability, activity, and reusability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Layered polymeric carbon nitride as a green support for cellulase immobilization: Improved stability, activity, and reusability Nuri Gulesci, Orhan Altan, Ali Toprak, M. Serkan Yalçın, Ramazan Bilgin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7657582/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jan, 2026 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted 4 You are reading this latest preprint version Abstract Polymeric carbon nitride (PCN) attracted considerable attention in recent years due to its semiconducting properties. In addition to its semiconducting properties, it is renowned as a support material due to its layered structure and donor groups such as terminal amines and triazine units. In this study, PCN was employed for the first time as a novel support material for the immobilization of cellulase, a key enzyme used in many industrial applications. Cellulase from Aspergillus sp . was immobilized onto PCN in three methods. In the first way, cellulase was adsorbed on PCN (PCN@cellulase). In the second way, PCN@cellulase was crosslinked with glutaraldehyde (PCN@cellulase/Glu). Finally, the primary amino group of PCN was modified with glutaraldehyde and the cellulase was immobilized on this support by covalent attachment (PCN/Glu@cellulase). Each cellulase preparation was individually assessed for its optimal pH, temperature conditions, heat stability, and enzyme kinetics. The optimal pH was 5.5 for all cellulase preparations, while the optimal temperature was 45°C for free cellulase and 55°C for all immobilized cellulase preparations. Thermal stability of PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase increased by 2.1-, 2.7-, and 3.7-fold, respectively, compared to the free cellulase. PCN/Glu@cellulase showed 1.4-fold higher catalytic efficiency than the free cellulase and retained 80% of its initial activity after 10 reuses. These results indicate that the use of metal-free, nitrogen-rich PCN, synthesized from abundant and low-cost melamine, aligns with the principles of green chemistry and offers a sustainable alternative to traditional immobilization supports. Cellulase immobilization polymeric carbon nitride stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cellulases (EC 3.2.1.4) are a group of hydrolytic enzymes that catalyze the breakdown of cellulose, the most abundant renewable biopolymer on Earth, into glucose and other soluble sugars ( 33 ). These enzymes have attracted considerable attention due to their wide range of industrial applications, including biofuel production, textile processing, animal feed formulation, food and beverage production, and waste management ( 30 , 26 ). Cellulase systems are typically composed of three main types of enzymes: endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases, which act synergistically to convert cellulose into fermentable sugars ( 43 , 19 ). Despite their broad utility, the industrial application of free cellulase enzymes is often limited by several factors, such as low stability, high cost, and the inability to reuse the enzymes in continuous processes ( 44 , 31 , 37 ). To overcome these limitations, enzyme immobilization has emerged as a promising strategy. Immobilization refers to the confinement of enzyme molecules to or within solid supports, enabling their repeated use, enhanced operational stability, and improved resistance to changes in environmental conditions such as pH and temperature ( 25 , 28 ). Various techniques, including adsorption, covalent binding, entrapment, and cross-linking, have been employed for the immobilization of cellulase enzymes ( 34 , 11 , 5 , 36 ). Choosing a suitable immobilization technique and support material is essential for preserving the enzyme’s activity and stability ( 39 ). Natural and synthetic polymers, inorganic carriers, and hybrid materials have all been explored for this purpose ( 8 ). Among all other materials, carbon nitrides have gained significant interest in recent years because of their 2-D layered structure, ease of manufacture, excellent thermal and chemical stability, and semiconductor properties ( 16 , 42 , 38 ). When carbon and nitrogen-containing molecular compounds such as melamine, urea, cyanamide, dicyandiamide etc. are pyrolyzed over 525°C, a carbon nitride material can be produced as a yellow-orange solid. This structure is very commonly called \"graphitic carbon nitride\", \"graphitic CN\" or \"g-C3N4\" in the scientific literature ( 22 ). In fact, the structure obtained in this way is a polymeric carbon nitride (PCN) ( 14 , 15 ). In addition to being a semiconductor, PCN has been used to stabilize and support numerous metal nanoparticles due to the nitrogen donors (amine and tri-s-triazine units) in its structure. On the other hand, these donor groups in the structure, especially the amine groups, allow other molecules to chemically bind to PCN. In recent years, PCN has emerged as the perfect metal-free scaffold for enzyme immobilization because of its hydrophobic qualities, surface-active sites, high biocompatibility, and economical manufacturing ( 35 , 6 , 24 ). However, there is no work in the literature about the immobilization of cellulase enzymes onto the PCN support. This study presents a comparative approach for the immobilization of cellulase enzymes onto PCN. While PCN has been widely studied for photocatalytic applications, its potential as a biocompatible, metal-free scaffold for enzyme immobilization remains relatively underexplored, especially in the context of cellulase. In this study, cellulase from Aspergillus sp . was immobilized onto using three different techniques. All immobilized cellulase preparations were investigated using FTIR, SEM, SEM-EDX and TEM analysis. Both free and immobilized cellulase preparations were analyzed for their optimal pH, temperature conditions, resistance to thermal stress, and kinetic behavior. This side-by-side comparison of immobilization methods on the same support material provides valuable insights into how different strategies affect enzyme binding, activity, and stability. Materials and methods Materials Cellulase from Aspergillus sp . (≥ 1000 units/g), melamine, glutaraldehyde, cellulose microcrystalline (CMC), and 3,5-dinitrosalicylic acid were bought from Sigma-Aldrich Chemie GmbH (Germany). All other chemicals were analytical grade and used without further purification. Methods Synthesis of PCN PCN was synthesized using the literature method ( 13 ), with a few modifications. Briefly, 10 g of melamine was ground in a mortar and then taken into a crucible and heated to 550°C in a muffle furnace at a heating rate of 12°C per minute and kept at this temperature for 4 h. The solid cooled to room temperature was coded as PCN and used in the relevant processes. Activation of PCN 1 g of PCN support was treated with 25 mL of glutaraldehyde activation solution (2.5% (w/w) glutaraldehyde in 50 mM sodium phosphate, pH 7.0) at room temperature for 2 hours ( 3 ). The mixture was stirred continuously at 100 rpm during the activation reaction and then, the activated PCN supports were collected by filtration under vacuum and washed with distilled water to remove unreacted glutaraldehyde molecules. The glutaraldehyde-activated PCN supports were stored at 5°C until use. Immobilization of cellulase Two grams of PCN support was treated with 8 mL of cellulase solution (1 mg/mL in 50 mM pH 5.5 citrate buffer). The mixture was gently shaken for 4 h at 5°C. The resulting immobilized cellulase samples were vacuum-filtered and rinsed with the immobilization buffer to eliminate any unbound enzyme. Filtrates were gathered for subsequent protein analysis. The immobilized enzymes were kept at 5°C until further use. One gram of cellulase-adsorbed PCN support was treated with 4 mL of 1.25% (w/w) glutaraldehyde solution prepared in phosphate buffer (50 mM, pH 7.0). After 2 h of the crosslinking reaction, the immobilized cellulases were filtered under vacuum and stored at 5°C until use. Cellulase was immobilized onto glutaraldehyde-activated PCN according to Alagöz et al ( 3 ). One gram of the activated support was suspended in 4.0 mL of 25 mM sodium phosphate solution at pH 7.0 containing 1 mg/mL cellulase. The immobilization suspension was continually shaken at 100 rpm and at 5°C for 2 h. The immobilized cellulases were filtered under vacuum and filtrates were collected for protein analysis. The immobilized cellulases were stored at 5°C until use. Protein concentrations in the collected filtrates were measured using the Lowry method ( 17 ). The immobilization yield (IY%) and recovered activity (RA%) were determined following the approach described by Boudrant et al ( 7 ). Instrumental analysis FT-IR spectra were collected over the 400–4000 cm − 1 range. SEM images were recorded at different magnifications (10000x-100000x). TEM analysis was operated at an acceleration voltage of 200kV. The detailed information about the instrumental analysis of the samples was given in supplementary information file. Cellulase assay The cellulase activity was measured according to Miller with slight modification ( 20 ). The detailed information about the cellulase assay was given in supplementary information file. Effect of pH and temperature on the activity In order to determine the effect of pH, a series of citrate-phosphate buffer were prepared in a pH range of 4.0-6.5 and free and immobilized cellulase activities were separately measured in these pH solutions at 40°C using 0.5% crystalline micro cellulose (CMC) as substrate. To determine the optimal temperature, the free and immobilized cellulase activities was assayed at different temperatures in a range of 35–65°C at pH 5.5. Kinetic parameters of free and immobilized cellulase preparations The free or immobilized cellulase activity was determined for different CMC concentrations ranging from 0.1 to 2.0% under the optimum pH and temperature of each sample. The apparent Michaelis constant (K M ) and maximum velocity (V max ) of each sample were estimated from Lineweaver-Burk plots. To determine the catalytic efficiency ratio (CER), the V max /K M of each immobilized cellulase was divided by the V max /K M of the corresponding free cellulase. Thermal stress The thermal stress of the cellulase samples was tested at 45°C and 55°C in 50 mM sodium acetate at pH 5.5. The residual activity of each sample was determined for the specified time intervals under optimal conditions. The thermal stress parameters of such as inactivation constant (k d ), half-life (t 1/2 ) and stabilization factor (SF) were calculated according to Alagöz et al ( 3 ). Reuse of the different cellulase biocatalysts The reuse experiments were performed in batch assays by incubating 0.1 g immobilized cellulases with 5 mL of 0.5% cellulose solution at 55°C. After 10 min reaction, the suspension was centrifuged (1 min, 5,000 x g) and the residual activity of each immobilized cellulase preparations was measured. After each cycle, the biocatalysts were washed with 50 mM sodium acetate buffer at pH 5.5, filtrered and added to a new hydrolysis cycle with new substrate solution. The activity after each cycle was expressed in relation to the activity after the first cycle. Statistical analysis All experiments were performed in triplicate. Statistical analysis was conducted using analysis of variance (ANOVA) to determine significant differences among experimental groups. Group means were compared using the Least Significant Difference (LSD) method in SigmaPlot software (Version 12), with a significance level set at P < 0.05. Results and discussion In this study, the use of PCN support for the immobilization of A. niger cellulase and comparison of immobilization methods on the activity and stability of immobilized cellulase were aimed. The pH of the immobilization medium plays a crucial role in determining the efficiency of enzyme binding and its subsequent catalytic performance. Table 1 summarizes the immobilization of cellulase on PCN supports via adsorption at different pH values ranging from 4.5 to 7.0. The highest IY% was obtained at an immobilization pH of 5.0. Increasing the pH resulted in a slight decrease in IY% values. However, RA values decreased significantly from 80.8% to 59.0% as the immobilization pH increased from 5.0 to 7.0. At higher pH values, the enzyme's tertiary structure may undergo conformational changes that adversely affect the integrity of the active site, thus reducing catalytic efficiency. As shown in Table 1 , the amount of bound protein increased as the initial protein concentration increased from 0.25 to 2 mg/mL. However, the highest RA value of 98.7% was obtained at an initial protein concentration of 0.5 mg/mL. Further increasing the protein concentration to 2 mg/mL resulted in a decrease in RA value to 48.9%. At higher protein concentrations, excessive enzyme molecules may adsorb onto the surface of the PCN support. This can lead to molecular crowding, where enzymes are packed closely. Such crowding can hinder proper enzyme orientation, restrict substrate accessibility to the active sites, and increase steric hindrance, all of which reduce the overall catalytic activity ( 18 ). Table 1 Effect of immobilization pH and initial added protein on IY (%) and RA (%) values for the adsorption of cellulase on PCN support. Immobilization pH Immobilization temperature Added protein amount Bound protein amount IY (%) RA (%) 4.5 5°C 1 mg 0.89 mg 89.0 76.4 5.0 0.92 mg 91.8 80.8 5.5 0.91 mg 90.8 77.4 6.0 0.90 mg 89.9 74.1 6.5 0.87 mg 86.9 65.4 7.0 0.86 mg 86.0 59.0 5.0 5°C 0.25 mg 0.24 mg 95.1 62.9 0.5 mg 0.48 mg 95.1 98.7 1.0 mg 0.92 mg 91.8 80.8 2.0 mg 1.67 mg 83.5 48.9 The PCN@cellulase was crosslinked using different concentrations of glutaraldehyde to elucidate the effect of the amount of glutaraldehyde on immobilized cellulase activity. As presented in Fig. 1 , the highest activity was observed in the non-crosslinked PCN@cellulase preparation. A slight decrease in relative activity was noted at a glutaraldehyde concentration of 0.625% (w/v). However, the relative activity further declined to 65%, 32.1%, and 10.7% at glutaraldehyde concentrations of 1.25%, 2.5%, and 5%, respectively. These results indicate that increasing the glutaraldehyde concentration increases the rigidity of the immobilized cellulase, which in turn reduces its enzymatic activity ( 32 , 1 ). Table 2 presents the results of covalent immobilization of cellulase on glutaraldehyde-modified PCN supports at different pH values and initial protein concentrations. The IY value was approximately 87% at both pH 7.0 and 8.0. Increasing the immobilization pH to 10.0 resulted in a slight reduction in IY%. The RA value was about 70% at pH 7.0. A slight decrease in RA% was observed at pH 8.0, while a further increase in pH led to a significant drop in RA%. At higher pH values, conformational changes in the enzyme’s tertiary structure may compromise the integrity of the active site, thereby reducing its catalytic efficiency. As shown in Table 2 , the amount of bound protein increased with the rise in initial protein concentration from 0.25 to 2 mg/mL. However, the highest RA value of 80.0% was achieved at an initial protein concentration of 0.5 mg/mL. Increasing the concentration further to 2 mg/mL led to a decrease in RA to 42.4%. At higher protein concentrations, excessive enzyme molecules may be adsorbed onto the surface of the PCN support, leading to molecular crowding. This crowding can result in improper enzyme orientation, restricted substrate access to active sites, and increased steric hindrance—all of which contribute to reduced catalytic activity ( 12 , 9 ). Table 2 Effect of immobilization pH and initial added protein on IY (%) and RA (%) values for the covalent immobilization of cellulase on glutaraldehyde modified PCN support. Immobilization pH Immobilization temperature Added protein amount Bound protein amount IY (%) RA (%) 7.0 5°C 1 mg 0.872 mg 87.2 70.2 8.0 0.870 mg 87.0 68.0 10.0 0.815 mg 81.5 46.4 7.0 5°C 0.25 mg 0.23 mg 93.6 77.5 0.5 mg 0.46 mg 91.3 80.0 1.0 mg 0.87 mg 87.2 70.2 2.0 mg 1.46 mg 73.0 42.4 Figure 2 shows the SEM images of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase. As demonstrated in Fig. 2 , bare PCN had a typical agglomerated surface appearance of graphitic carbon nitride materials (Fig. 2 a-c) ( 41 ). After cellulase immobilization, the surfaces of PCN supports were clearly different from those of bare PCN, and the roughness of the PCN surfaces increased as a result of the cellulase immobilization (Fig. 2 d-f). After glutaraldehyde crosslinking of the adsorbed cellulase, the surface structure is considerably altered (Fig. 2 g-i) and fracture structures have increased. For PCN/Glu@cellulase, the roughness of the surfaces was highly increased after glutaraldehyde modification and covalent immobilization (Fig. 2 j-l). The results of EDX analysis of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase are shown in Fig. 3 . After cellulase immobilization, the presence of oxygen atom in PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase approved the immobilization of cellulase. TEM images of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase are shown in Fig. 4 . The bare PCN support, and PCN@cellulase are spherical in shape and their surfaces are smooth and homogeneous. However, PCN@cellulase/Glu, and PCN/Glu@cellulase have increased surface roughness, and irregularities. The crosslinking of PCN@cellulase and modification of bare PCN with glutaraldehyde followed by covalent bonding may disturb the structure of PCN and this can result in more amorphous appearances or less distinct edges in TEM images compared to the bare PCN support and PCN@cellulase samples. The particle sizes of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase are measured to be 375 ± 0.31, 228 ± 0.75, 226 ± 0.74, and 219 ± 0.65 nm, respectively. FT-IR spectra of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase are shown in Fig. 5 . A sharp peak at 805 cm − 1 corresponds to the triazine/heptazine units of PCN ( 21 ). The peaks observed in the range of 1230–1650 cm − 1 are attributed to the skeletal vibrations of aromatic C–N heterocycles. For bare PCN, the peak at 1630 cm − 1 is associated with the conjugated imine groups in the heptazine rings ( 23 , 10 ). The broad band between 3000 and 3300 cm − 1 is due to N–H and O–H stretching vibrations ( 4 ). There is no obvious change in the spectra for PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, suggesting the intact structure of PCN ( 38 ). In contrast, the peak at 1637 cm − 1 in PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase corresponds to the amide I band. The optimum pH of the free and immobilized cellulase preparations was studied in the pH range of 4.0-6.5 and the results obtained were presented in Fig. 6 A. All cellulase preparations had an optimum pH at 5.5. As shown in Fig. 6 A, free and immobilized cellulase preparations showed nearly similar behaviour against pH change. This result indicates that the three-dimensional structure of the cellulase is protected upon immobilization on PCN. The effect of temperature on the free and immobilized cellulase preparations was determined in the temperature range of 35°C-65°C of pH 5.5. The free cellulase had an optimum at 45°C, whereas the optimum temperature increased for the immobilized cellulases. All immobilized cellulases showed an optimum at 55°C (Fig. 6 B). Moreover, the free cellulase showed 60% of its maximum activity at 65°C, however; the immobilized cellulases displayed at least 80% of their initial activity at the same temperature. Ahmad and Khare reported that the optimum pH of free cellulase from Aspergillus niger and its covalently immobilized form on carbon nanotubes were both 5.0. Moreover, both cellulase forms had an optimum temperature of 50°C ( 2 ). Zdarta et al. ( 40 ) immobilized A. niger cellulase on a TiO 2 –lignin hybrid support and determined the maximum activity of free and immobilized cellulase preparations to be 5.5 and 6.0, respectively. The free enzyme showed its maximum activity at 50°C, while the maximum activity of the immobilized cellulase increased to 55°C. Thermal stability of the free and immobilized cellulase preparation was investigated at 45°C and 55°C over 24 h. The initial activity of the free cellulase was decreased with the incubation time and its remaining activity was determined to be 60% after 24 h. Under the same conditions, the remaining activities were 76, 80, and 86% for PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively (Fig. 7 A). The k d values were calculated to be 27.9x10 − 3 , 18.7x10 − 3 , 14.0x10 − 3 , and 10.0x10 − 3 h − 1 for the free, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively. The corresponding t 1/2 values were 24.8, 37.0, 49.4, and 69.4 h. According to these results, SF values were calculated to be 1.5, 2.0, and 2.8 for PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively (Table 3 ). At 55°C, the remaining activities were determined to be 35, 66, 76, and 82% for the free, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively after 24 h of incubation (Fig. 7 B). The k d values were found to be 50.2x10 − 3 , 24.3x10 − 3 , 18.8x10 − 3 , and 13.4x10 − 3 h − 1 for the free, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively. The corresponding t 1/2 values were 13.8, 28.5, 36.9, and 51.7. According to these results, SF values were calculated to be 2.1, 2.7, and 3.7 for PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively (Table 3 ). Ahmad and Khare reported that the t 1/2 value of A. niger cellulase immobilized on COOH-functionalized MWCNTs was 4-fold higher than free enzyme at 70°C ( 2 ). Li et al. immobilized Candida rugosa lipase on glutaraldehyde modified C 3 N 4 nanosheets (C 3 N 4 -NS@CRL) and reported that C 3 N 4 -NS@CRL preserved 67% of its initial activity after 180 min of incubation time at 55°C ( 16 ). Table 3 Thermal stability parameters of free cellulase, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase at 45°C and 55°C. Biocatalyst 45°C k d (h − 1 ) t 1/2 (h) SF 55°C k d (h − 1 ) t 1/2 (h) SF free 0.0279 24.8 1.0 0.0502 13.8 1.0 PCN@cellulase 0.0187 37.0 1.5 0.0243 28.5 2.1 PCN@cellulase/Glu 0.0140 49.4 2.0 0.0188 36.9 2.7 PCN/Glu@cellulase 0.0100 69.4 2.8 0.0134 51.7 3.7 The apparent K M and V max values of each cellulase sample are given in Table 4 . The apparent K M values of free cellulase, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase were determined to be 1.14 ± 0.14, 1.39 ± 0.18, 1.52 ± 0.21, and 0.84 ± 0.10 mg/mL for CMC ( Fig. S1 ). The alteration in the K M value may be attributed to changes in the enzyme–substrate affinity resulting from conformational modifications of cellulase induced by immobilization, the onset of substrate diffusional constraints, or a synergistic effect of both phenomena. The corresponding V max values were found to be 1.25 ± 0.09, 1.77 ± 0.22, 1.17 ± 0.09, and 1.30 ± 0.11 U/mg protein. The elevated Vmax values observed for PCN@cellulase and PCN/Glu@cellulase compared to free cellulase may be attributed to the favorable orientation of enzyme molecules on the support surface, which facilitates improved substrate access to the active site. According to these results CER ratios were calculated to be 1.2, 0.7, and 1.4. These results indicate that PCN@cellulase and PCN/Glu@cellulase exhibit 1.2- and 1.4-fold higher activity, respectively, compared to free cellulase. The CER value of cellulase immobilized on COOH-functionalized MWCNTs is approximately 1.66-fold higher than that of free cellulase ( 2 ). The K M and V max values of free cellulase from A. niger were determined to be 2.06 ± 0.85 mM and 159 ± 11 U/mg protein for cellulose, respectively. The corresponding values for its immobilized counterpart on TiO 2 –lignin hybrid support were 2.63 ± 0.96 mM and 125 ± 19 U/mg protein. In addition, CER value was calculated to be 0.62 ( 40 ). Table 4 The apparent K M and V max values of free cellulase, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase for CMC under the optimum conditions of each preparation. Biocatalyst K M (mg/mL) V max (U/mg protein) CER free 1.14 ± 0.14 1.25 ± 0.09 1.0 PCN@cellulase 1.39 ± 0.18 1.77 ± 0.22 1.2 PCN@cellulase/Glu 1.52 ± 0.21 1.17 ± 0.09 0.7 PCN/Glu@cellulase 0.84 ± 0.10 1.30 ± 0.11 1.4 The reuse stability of immobilized cellulase preparations was investigated in a batch type reactor for 10 cycles. Among the tested immobilized cellulases, the covalently immobilized PCN/Glu@cellulase retained 80% of its initial activity, while PCN@cellulase/Glu and PCN@cellulase retained 72% and 50% of their initial activities, respectively (Fig. 8 ). These results highlight that PCN, particularly when modified through covalent attachment, provides a durable and reusable platform for enzyme immobilization, offering great potential for cost-effective and sustainable industrial bioprocesses. Celulase immobilized onto carbon coated Fe 3 O 4 nanoparticles protected 80% of its initial activity after 9 reuses ( 45 ). Rashid et al. modified nickel nanoparticle with 3-APTES to generate free -NH 2 group onto the support surface and immobilized cellulase from Aspergillus niger by adsorption followed by glutaraldehyde crosslinking. After 10 reuses, the immobilized cellulase retained 84% of its initial activity after ten reuses ( 27 ). C 3 N 4 -NS@CRL retained 72% of the initial activity after 10 reuses ( 16 ). Shangguan et al. reported that CALB covalently bound to g-C 3 N 4 maintained 65% of its initial activity after 9 cycles ( 29 ). Conclusion In this study, PCN synthesized from melamine was successfully employed as a sustainable and metal-free support for cellulase immobilization. Three different immobilization strategies - simple adsorption, adsorption followed by glutaraldehyde crosslinking, and covalent binding via glutaraldehyde activation - were comparatively evaluated. All immobilized cellulase preparations retained their catalytic activity and exhibited improved thermal stability compared to the free enzyme. In particular, the covalently bound PCN/Glu@cellulase demonstrated the highest stabilization factor, extended half-life and better reusability, highlighting the effectiveness of covalent immobilization in enhancing enzyme performance. These findings confirm that PCN provides a promising platform for enzyme immobilization due to its biocompatibility, high surface area, and abundance. The results open up new perspectives for the application of PCN-based biocatalysts in sustainable bioprocesses, especially for biomass conversion and other industrial applications requiring robust and reusable cellulase systems. Declarations Credit author statement Nuri Gulesci: Conceptualization , Methodology, Writing – original draft. Orhan Altan : Conceptualization , Methodology, Data curation, Formal analysis. Ali Toprak: Conceptualization , Methodology, Data curation, Formal analysis. Mustafa Serkan Yalcin: Methodology, Data curation, Formal analysis . Ramazan Bilgin: Writing – review & editing. Deniz Yildirim: Methodology, Writing – original draft, Writing – review & editing. Conflict of interest The author declares that they have no conflict of interest. Funding This research received no external funding. Data availability Data will be made available on reasonable request. References Assis Modenez, I., Sastre, D. E., Moraes, C., F. and, & Marques Netto, C. G. C. (2018). 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13:09:31\",\"extension\":\"png\",\"order_by\":21,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":27019,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Onlinefloatimage8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/5a5012e69001d34fc2753b56.png\"},{\"id\":93496705,\"identity\":\"53e54125-f5c4-4c4b-b19e-9db9686dd25e\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:09:32\",\"extension\":\"xml\",\"order_by\":22,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":130430,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"ABABD25030420structuring.xml\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/04ee61c9925cda671ca9a121.xml\"},{\"id\":93496703,\"identity\":\"daded85e-130b-4944-afa9-a6672e95135a\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:09:32\",\"extension\":\"html\",\"order_by\":23,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":141127,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"earlyproof.html\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/dd2271132309a74a09dac753.html\"},{\"id\":93496677,\"identity\":\"c6502e64-967a-4e9f-b936-16f7e14c430d\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:09:31\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":69340,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of glutaraldehyde concentration on the activity of PCN@cellulase.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/77e2d306ec63a94b8c1f65a4.png\"},{\"id\":93497096,\"identity\":\"6dac24ee-dafd-4d45-b68a-c1ca809dae25\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:17:31\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":731340,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSEM images of bare PCN support (a-c), PCN@cellulase (d-f), PCN@cellulase/Glu (g-i), and PCN/Glu@cellulase (j-l) with different magnefications.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/6de17a50c94f3886bf92d277.png\"},{\"id\":93497099,\"identity\":\"75dba022-51cb-4f26-a992-c68b24bf7070\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:17:31\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":57716,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSEM-EDX analysis bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/6545c0f6a8335e2270cf6721.png\"},{\"id\":93496679,\"identity\":\"ab3ae6ed-713a-4619-a477-e49aa72d1923\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:09:31\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":302052,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTEM images of bare PCN support (a-c), PCN@cellulase (d-f), PCN@cellulase/Glu (g-I), and PCN/Glu@cellulase (j-l).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/c0eaf5479379b1cd03582c84.png\"},{\"id\":93497097,\"identity\":\"fc81cfa7-7a08-46f3-aa3a-b7cbe3624029\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:17:31\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":129723,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFT-IR spectra of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/79db000fb166b9d46c845add.png\"},{\"id\":93496684,\"identity\":\"7bc68b37-2127-42b8-ab7f-c9568c5db936\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:09:31\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":92648,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of pH (A) and temperature (B) on the activity of free cellulase, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase. The maximum activity was taken as 100% with the relative activities (%) expressed as the ratios of the activity at the measured pH or temperature to the maximum activity.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/07ac954f42f1730f33a5bf7b.png\"},{\"id\":93497101,\"identity\":\"3289a115-49fb-4146-a9e7-95e2ca9b64ac\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:17:31\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":109660,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThermal stability of free cellulase, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase at 45°C (A) and 55°C (B). The residual activity was calculated as the ratio between the activity at a given time and the activity at the beginning of incubation.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/ac6eb9335051cbd480c20681.png\"},{\"id\":93496690,\"identity\":\"786316ee-c4a0-417a-8fe1-af2c6af6710e\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:09:31\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":112562,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eReuse stability of PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/75921b6e94af2091c1c7ea9a.png\"},{\"id\":99545459,\"identity\":\"b39df6ac-36d7-4b7e-a1da-282673e3ce5f\",\"added_by\":\"auto\",\"created_at\":\"2026-01-05 16:07:52\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2371246,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/dc0c652c-c4a1-411a-a76b-b000acd27408.pdf\"},{\"id\":93496686,\"identity\":\"6de040ce-c6a3-4522-af55-8fb893c05d19\",\"added_by\":\"auto\",\"created_at\":\"2025-10-14 13:09:31\",\"extension\":\"docx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":90842,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryInformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7657582/v1/3439351ad2d4cc493c8d8b9a.docx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Layered polymeric carbon nitride as a green support for cellulase immobilization: Improved stability, activity, and reusability\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eCellulases (EC 3.2.1.4) are a group of hydrolytic enzymes that catalyze the breakdown of cellulose, the most abundant renewable biopolymer on Earth, into glucose and other soluble sugars (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e). These enzymes have attracted considerable attention due to their wide range of industrial applications, including biofuel production, textile processing, animal feed formulation, food and beverage production, and waste management (\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e). Cellulase systems are typically composed of three main types of enzymes: endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases, which act synergistically to convert cellulose into fermentable sugars (\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e). Despite their broad utility, the industrial application of free cellulase enzymes is often limited by several factors, such as low stability, high cost, and the inability to reuse the enzymes in continuous processes (\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). To overcome these limitations, enzyme immobilization has emerged as a promising strategy. Immobilization refers to the confinement of enzyme molecules to or within solid supports, enabling their repeated use, enhanced operational stability, and improved resistance to changes in environmental conditions such as pH and temperature (\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e). Various techniques, including adsorption, covalent binding, entrapment, and cross-linking, have been employed for the immobilization of cellulase enzymes (\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eChoosing a suitable immobilization technique and support material is essential for preserving the enzyme\\u0026rsquo;s activity and stability (\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e). Natural and synthetic polymers, inorganic carriers, and hybrid materials have all been explored for this purpose (\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e). Among all other materials, carbon nitrides have gained significant interest in recent years because of their 2-D layered structure, ease of manufacture, excellent thermal and chemical stability, and semiconductor properties (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e). When carbon and nitrogen-containing molecular compounds such as melamine, urea, cyanamide, dicyandiamide etc. are pyrolyzed over 525\\u0026deg;C, a carbon nitride material can be produced as a yellow-orange solid. This structure is very commonly called \\\"graphitic carbon nitride\\\", \\\"graphitic CN\\\" or \\\"g-C3N4\\\" in the scientific literature (\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). In fact, the structure obtained in this way is a polymeric carbon nitride (PCN) (\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e). In addition to being a semiconductor, PCN has been used to stabilize and support numerous metal nanoparticles due to the nitrogen donors (amine and tri-s-triazine units) in its structure. On the other hand, these donor groups in the structure, especially the amine groups, allow other molecules to chemically bind to PCN. In recent years, PCN has emerged as the perfect metal-free scaffold for enzyme immobilization because of its hydrophobic qualities, surface-active sites, high biocompatibility, and economical manufacturing (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e). However, there is no work in the literature about the immobilization of cellulase enzymes onto the PCN support.\\u003c/p\\u003e\\u003cp\\u003eThis study presents a comparative approach for the immobilization of cellulase enzymes onto PCN. While PCN has been widely studied for photocatalytic applications, its potential as a biocompatible, metal-free scaffold for enzyme immobilization remains relatively underexplored, especially in the context of cellulase. In this study, cellulase from \\u003cem\\u003eAspergillus sp\\u003c/em\\u003e. was immobilized onto using three different techniques. All immobilized cellulase preparations were investigated using FTIR, SEM, SEM-EDX and TEM analysis. Both free and immobilized cellulase preparations were analyzed for their optimal pH, temperature conditions, resistance to thermal stress, and kinetic behavior. This side-by-side comparison of immobilization methods on the same support material provides valuable insights into how different strategies affect enzyme binding, activity, and stability.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eMaterials\\u003c/h2\\u003e\\u003cp\\u003eCellulase from \\u003cem\\u003eAspergillus sp\\u003c/em\\u003e. (\\u0026ge;\\u0026thinsp;1000 units/g), melamine, glutaraldehyde, cellulose microcrystalline (CMC), and 3,5-dinitrosalicylic acid were bought from Sigma-Aldrich Chemie GmbH (Germany). All other chemicals were analytical grade and used without further purification.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eMethods\\u003c/h3\\u003e\\n\\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eSynthesis of PCN\\u003c/h2\\u003e\\u003cp\\u003ePCN was synthesized using the literature method (\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e), with a few modifications. Briefly, 10 g of melamine was ground in a mortar and then taken into a crucible and heated to 550\\u0026deg;C in a muffle furnace at a heating rate of 12\\u0026deg;C per minute and kept at this temperature for 4 h. The solid cooled to room temperature was coded as PCN and used in the relevant processes.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eActivation of PCN\\u003c/h3\\u003e\\n\\u003cp\\u003e1 g of PCN support was treated with 25 mL of glutaraldehyde activation solution (2.5% (w/w) glutaraldehyde in 50 mM sodium phosphate, pH 7.0) at room temperature for 2 hours (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). The mixture was stirred continuously at 100 rpm during the activation reaction and then, the activated PCN supports were collected by filtration under vacuum and washed with distilled water to remove unreacted glutaraldehyde molecules. The glutaraldehyde-activated PCN supports were stored at 5\\u0026deg;C until use.\\u003c/p\\u003e\\n\\u003ch3\\u003eImmobilization of cellulase\\u003c/h3\\u003e\\n\\u003cp\\u003eTwo grams of PCN support was treated with 8 mL of cellulase solution (1 mg/mL in 50 mM pH 5.5 citrate buffer). The mixture was gently shaken for 4 h at 5\\u0026deg;C. The resulting immobilized cellulase samples were vacuum-filtered and rinsed with the immobilization buffer to eliminate any unbound enzyme. Filtrates were gathered for subsequent protein analysis. The immobilized enzymes were kept at 5\\u0026deg;C until further use.\\u003c/p\\u003e\\u003cp\\u003eOne gram of cellulase-adsorbed PCN support was treated with 4 mL of 1.25% (w/w) glutaraldehyde solution prepared in phosphate buffer (50 mM, pH 7.0). After 2 h of the crosslinking reaction, the immobilized cellulases were filtered under vacuum and stored at 5\\u0026deg;C until use.\\u003c/p\\u003e\\u003cp\\u003eCellulase was immobilized onto glutaraldehyde-activated PCN according to Alag\\u0026ouml;z et al (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). One gram of the activated support was suspended in 4.0 mL of 25 mM sodium phosphate solution at pH 7.0 containing 1 mg/mL cellulase. The immobilization suspension was continually shaken at 100 rpm and at 5\\u0026deg;C for 2 h. The immobilized cellulases were filtered under vacuum and filtrates were collected for protein analysis. The immobilized cellulases were stored at 5\\u0026deg;C until use.\\u003c/p\\u003e\\u003cp\\u003eProtein concentrations in the collected filtrates were measured using the Lowry method (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). The immobilization yield (IY%) and recovered activity (RA%) were determined following the approach described by Boudrant et al (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eInstrumental analysis\\u003c/h2\\u003e\\u003cp\\u003eFT-IR spectra were collected over the 400\\u0026ndash;4000 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e range. SEM images were recorded at different magnifications (10000x-100000x). TEM analysis was operated at an acceleration voltage of 200kV. The detailed information about the instrumental analysis of the samples was given in supplementary information file.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eCellulase assay\\u003c/h3\\u003e\\n\\u003cp\\u003eThe cellulase activity was measured according to Miller with slight modification (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e). The detailed information about the cellulase assay was given in supplementary information file.\\u003c/p\\u003e\\n\\u003ch3\\u003eEffect of pH and temperature on the activity\\u003c/h3\\u003e\\n\\u003cp\\u003eIn order to determine the effect of pH, a series of citrate-phosphate buffer were prepared in a pH range of 4.0-6.5 and free and immobilized cellulase activities were separately measured in these pH solutions at 40\\u0026deg;C using 0.5% crystalline micro cellulose (CMC) as substrate. To determine the optimal temperature, the free and immobilized cellulase activities was assayed at different temperatures in a range of 35\\u0026ndash;65\\u0026deg;C at pH 5.5.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eKinetic parameters of free and immobilized cellulase preparations\\u003c/h2\\u003e\\u003cp\\u003eThe free or immobilized cellulase activity was determined for different CMC concentrations ranging from 0.1 to 2.0% under the optimum pH and temperature of each sample. The apparent Michaelis constant (K\\u003csub\\u003eM\\u003c/sub\\u003e) and maximum velocity (V\\u003csub\\u003emax\\u003c/sub\\u003e) of each sample were estimated from Lineweaver-Burk plots. To determine the catalytic efficiency ratio (CER), the V\\u003csub\\u003emax\\u003c/sub\\u003e/K\\u003csub\\u003eM\\u003c/sub\\u003e of each immobilized cellulase was divided by the V\\u003csub\\u003emax\\u003c/sub\\u003e/K\\u003csub\\u003eM\\u003c/sub\\u003e of the corresponding free cellulase.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eThermal stress\\u003c/h2\\u003e\\u003cp\\u003eThe thermal stress of the cellulase samples was tested at 45\\u0026deg;C and 55\\u0026deg;C in 50 mM sodium acetate at pH 5.5. The residual activity of each sample was determined for the specified time intervals under optimal conditions. The thermal stress parameters of such as inactivation constant (k\\u003csub\\u003ed\\u003c/sub\\u003e), half-life (t\\u003csub\\u003e1/2\\u003c/sub\\u003e) and stabilization factor (SF) were calculated according to Alag\\u0026ouml;z et al (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eReuse of the different cellulase biocatalysts\\u003c/h2\\u003e\\u003cp\\u003eThe reuse experiments were performed in batch assays by incubating 0.1 g immobilized cellulases with 5 mL of 0.5% cellulose solution at 55\\u0026deg;C. After 10 min reaction, the suspension was centrifuged (1 min, 5,000 x g) and the residual activity of each immobilized cellulase preparations was measured. After each cycle, the biocatalysts were washed with 50 mM sodium acetate buffer at pH 5.5, filtrered and added to a new hydrolysis cycle with new substrate solution. The activity after each cycle was expressed in relation to the activity after the first cycle.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e\\u003cp\\u003eAll experiments were performed in triplicate. Statistical analysis was conducted using analysis of variance (ANOVA) to determine significant differences among experimental groups. Group means were compared using the Least Significant Difference (LSD) method in SigmaPlot software (Version 12), with a significance level set at P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Results and discussion\",\"content\":\"\\u003cp\\u003eIn this study, the use of PCN support for the immobilization of \\u003cem\\u003eA. niger\\u003c/em\\u003e cellulase and comparison of immobilization methods on the activity and stability of immobilized cellulase were aimed. The pH of the immobilization medium plays a crucial role in determining the efficiency of enzyme binding and its subsequent catalytic performance. Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e summarizes the immobilization of cellulase on PCN supports via adsorption at different pH values ranging from 4.5 to 7.0. The highest IY% was obtained at an immobilization pH of 5.0. Increasing the pH resulted in a slight decrease in IY% values. However, RA values decreased significantly from 80.8% to 59.0% as the immobilization pH increased from 5.0 to 7.0. At higher pH values, the enzyme's tertiary structure may undergo conformational changes that adversely affect the integrity of the active site, thus reducing catalytic efficiency. As shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, the amount of bound protein increased as the initial protein concentration increased from 0.25 to 2 mg/mL. However, the highest RA value of 98.7% was obtained at an initial protein concentration of 0.5 mg/mL. Further increasing the protein concentration to 2 mg/mL resulted in a decrease in RA value to 48.9%. At higher protein concentrations, excessive enzyme molecules may adsorb onto the surface of the PCN support. This can lead to molecular crowding, where enzymes are packed closely. Such crowding can hinder proper enzyme orientation, restrict substrate accessibility to the active sites, and increase steric hindrance, all of which reduce the overall catalytic activity (\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eEffect of immobilization pH and initial added protein on IY (%) and RA (%) values for the adsorption of cellulase on PCN support.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"6\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eImmobilization\\u003c/p\\u003e\\u003cp\\u003epH\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eImmobilization\\u003c/p\\u003e\\u003cp\\u003etemperature\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eAdded protein\\u003c/p\\u003e\\u003cp\\u003eamount\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eBound\\u003c/p\\u003e\\u003cp\\u003eprotein amount\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eIY (%)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eRA (%)\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e4.5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"5\\\" rowspan=\\\"6\\\"\\u003e\\u003cp\\u003e5\\u0026deg;C\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"5\\\" rowspan=\\\"6\\\"\\u003e\\u003cp\\u003e1 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.89 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e89.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e76.4\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e5.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.92 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e91.8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e80.8\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e5.5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.91 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e90.8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e77.4\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e6.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.90 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e89.9\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e74.1\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e6.5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.87 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e86.9\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e65.4\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e7.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.86 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e86.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e59.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e\\u003cp\\u003e5.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e\\u003cp\\u003e5\\u0026deg;C\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.25 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.24 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e95.1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e62.9\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.5 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.48 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e95.1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e98.7\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.0 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.92 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e91.8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e80.8\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2.0 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e1.67 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e83.5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e48.9\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe PCN@cellulase was crosslinked using different concentrations of glutaraldehyde to elucidate the effect of the amount of glutaraldehyde on immobilized cellulase activity. As presented in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, the highest activity was observed in the non-crosslinked PCN@cellulase preparation. A slight decrease in relative activity was noted at a glutaraldehyde concentration of 0.625% (w/v). However, the relative activity further declined to 65%, 32.1%, and 10.7% at glutaraldehyde concentrations of 1.25%, 2.5%, and 5%, respectively. These results indicate that increasing the glutaraldehyde concentration increases the rigidity of the immobilized cellulase, which in turn reduces its enzymatic activity (\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eTable\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e presents the results of covalent immobilization of cellulase on glutaraldehyde-modified PCN supports at different pH values and initial protein concentrations. The IY value was approximately 87% at both pH 7.0 and 8.0. Increasing the immobilization pH to 10.0 resulted in a slight reduction in IY%. The RA value was about 70% at pH 7.0. A slight decrease in RA% was observed at pH 8.0, while a further increase in pH led to a significant drop in RA%. At higher pH values, conformational changes in the enzyme\\u0026rsquo;s tertiary structure may compromise the integrity of the active site, thereby reducing its catalytic efficiency. As shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, the amount of bound protein increased with the rise in initial protein concentration from 0.25 to 2 mg/mL. However, the highest RA value of 80.0% was achieved at an initial protein concentration of 0.5 mg/mL. Increasing the concentration further to 2 mg/mL led to a decrease in RA to 42.4%. At higher protein concentrations, excessive enzyme molecules may be adsorbed onto the surface of the PCN support, leading to molecular crowding. This crowding can result in improper enzyme orientation, restricted substrate access to active sites, and increased steric hindrance\\u0026mdash;all of which contribute to reduced catalytic activity (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eEffect of immobilization pH and initial added protein on IY (%) and RA (%) values for the covalent immobilization of cellulase on glutaraldehyde modified PCN support.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"6\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eImmobilization\\u003c/p\\u003e\\u003cp\\u003epH\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eImmobilization\\u003c/p\\u003e\\u003cp\\u003etemperature\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eAdded protein\\u003c/p\\u003e\\u003cp\\u003eamount\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eBound\\u003c/p\\u003e\\u003cp\\u003eprotein amount\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eIY (%)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eRA (%)\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e7.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e\\u003cp\\u003e5\\u0026deg;C\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e\\u003cp\\u003e1 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.872 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e87.2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e70.2\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e8.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.870 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e87.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e68.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e10.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.815 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e81.5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e46.4\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e\\u003cp\\u003e7.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e\\u003cp\\u003e5\\u0026deg;C\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.25 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.23 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e93.6\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e77.5\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.5 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.46 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e91.3\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e80.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.0 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.87 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e87.2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e70.2\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2.0 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e1.46 mg\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e73.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e42.4\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eFigure\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e shows the SEM images of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase. As demonstrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, bare PCN had a typical agglomerated surface appearance of graphitic carbon nitride materials (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea-c) (\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e). After cellulase immobilization, the surfaces of PCN supports were clearly different from those of bare PCN, and the roughness of the PCN surfaces increased as a result of the cellulase immobilization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed-f). After glutaraldehyde crosslinking of the adsorbed cellulase, the surface structure is considerably altered (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eg-i) and fracture structures have increased. For PCN/Glu@cellulase, the roughness of the surfaces was highly increased after glutaraldehyde modification and covalent immobilization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ej-l).\\u003c/p\\u003e\\u003cp\\u003eThe results of EDX analysis of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e. After cellulase immobilization, the presence of oxygen atom in PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase approved the immobilization of cellulase.\\u003c/p\\u003e\\u003cp\\u003eTEM images of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e. The bare PCN support, and PCN@cellulase are spherical in shape and their surfaces are smooth and homogeneous. However, PCN@cellulase/Glu, and PCN/Glu@cellulase have increased surface roughness, and irregularities. The crosslinking of PCN@cellulase and modification of bare PCN with glutaraldehyde followed by covalent bonding may disturb the structure of PCN and this can result in more amorphous appearances or less distinct edges in TEM images compared to the bare PCN support and PCN@cellulase samples. The particle sizes of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase are measured to be 375\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.31, 228\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.75, 226\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.74, and 219\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.65 nm, respectively.\\u003c/p\\u003e\\u003cp\\u003eFT-IR spectra of bare PCN support, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e. A sharp peak at 805 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e corresponds to the triazine/heptazine units of PCN (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). The peaks observed in the range of 1230\\u0026ndash;1650 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e are attributed to the skeletal vibrations of aromatic C\\u0026ndash;N heterocycles. For bare PCN, the peak at 1630 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e is associated with the conjugated imine groups in the heptazine rings (\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). The broad band between 3000 and 3300 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e is due to N\\u0026ndash;H and O\\u0026ndash;H stretching vibrations (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e). There is no obvious change in the spectra for PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, suggesting the intact structure of PCN (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e). In contrast, the peak at 1637 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e in PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase corresponds to the amide I band.\\u003c/p\\u003e\\u003cp\\u003eThe optimum pH of the free and immobilized cellulase preparations was studied in the pH range of 4.0-6.5 and the results obtained were presented in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA. All cellulase preparations had an optimum pH at 5.5. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA, free and immobilized cellulase preparations showed nearly similar behaviour against pH change. This result indicates that the three-dimensional structure of the cellulase is protected upon immobilization on PCN. The effect of temperature on the free and immobilized cellulase preparations was determined in the temperature range of 35\\u0026deg;C-65\\u0026deg;C of pH 5.5. The free cellulase had an optimum at 45\\u0026deg;C, whereas the optimum temperature increased for the immobilized cellulases. All immobilized cellulases showed an optimum at 55\\u0026deg;C (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB). Moreover, the free cellulase showed 60% of its maximum activity at 65\\u0026deg;C, however; the immobilized cellulases displayed at least 80% of their initial activity at the same temperature. Ahmad and Khare reported that the optimum pH of free cellulase from \\u003cem\\u003eAspergillus niger\\u003c/em\\u003e and its covalently immobilized form on carbon nanotubes were both 5.0. Moreover, both cellulase forms had an optimum temperature of 50\\u0026deg;C (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). Zdarta et al. (\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e) immobilized \\u003cem\\u003eA. niger\\u003c/em\\u003e cellulase on a TiO\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026ndash;lignin hybrid support and determined the maximum activity of free and immobilized cellulase preparations to be 5.5 and 6.0, respectively. The free enzyme showed its maximum activity at 50\\u0026deg;C, while the maximum activity of the immobilized cellulase increased to 55\\u0026deg;C.\\u003c/p\\u003e\\u003cp\\u003eThermal stability of the free and immobilized cellulase preparation was investigated at 45\\u0026deg;C and 55\\u0026deg;C over 24 h. The initial activity of the free cellulase was decreased with the incubation time and its remaining activity was determined to be 60% after 24 h. Under the same conditions, the remaining activities were 76, 80, and 86% for PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA). The k\\u003csub\\u003ed\\u003c/sub\\u003e values were calculated to be 27.9x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e, 18.7x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e, 14.0x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e, and 10.0x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e h\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e for the free, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively. The corresponding t\\u003csub\\u003e1/2\\u003c/sub\\u003e values were 24.8, 37.0, 49.4, and 69.4 h. According to these results, SF values were calculated to be 1.5, 2.0, and 2.8 for PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). At 55\\u0026deg;C, the remaining activities were determined to be 35, 66, 76, and 82% for the free, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively after 24 h of incubation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB). The k\\u003csub\\u003ed\\u003c/sub\\u003e values were found to be 50.2x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e, 24.3x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e, 18.8x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e, and 13.4x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e h\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e for the free, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively. The corresponding t\\u003csub\\u003e1/2\\u003c/sub\\u003e values were 13.8, 28.5, 36.9, and 51.7. According to these results, SF values were calculated to be 2.1, 2.7, and 3.7 for PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase, respectively (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Ahmad and Khare reported that the t\\u003csub\\u003e1/2\\u003c/sub\\u003e value of \\u003cem\\u003eA. niger\\u003c/em\\u003e cellulase immobilized on COOH-functionalized MWCNTs was 4-fold higher than free enzyme at 70\\u0026deg;C (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). Li et al. immobilized \\u003cem\\u003eCandida rugosa\\u003c/em\\u003e lipase on glutaraldehyde modified C\\u003csub\\u003e3\\u003c/sub\\u003eN\\u003csub\\u003e4\\u003c/sub\\u003e nanosheets (C\\u003csub\\u003e3\\u003c/sub\\u003eN\\u003csub\\u003e4\\u003c/sub\\u003e-NS@CRL) and reported that C\\u003csub\\u003e3\\u003c/sub\\u003eN\\u003csub\\u003e4\\u003c/sub\\u003e-NS@CRL preserved 67% of its initial activity after 180 min of incubation time at 55\\u0026deg;C (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eThermal stability parameters of free cellulase, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase at 45\\u0026deg;C and 55\\u0026deg;C.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"9\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eBiocatalyst\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"4\\\" rowspan=\\\"5\\\"\\u003e\\u003cp\\u003e45\\u0026deg;C\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003ek\\u003csub\\u003ed\\u003c/sub\\u003e\\u003c/p\\u003e\\u003cp\\u003e(h\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003et\\u003csub\\u003e1/2\\u003c/sub\\u003e\\u003c/p\\u003e\\u003cp\\u003e(h)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eSF\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\" morerows=\\\"4\\\" rowspan=\\\"5\\\"\\u003e\\u003cp\\u003e55\\u0026deg;C\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003ek\\u003csub\\u003ed\\u003c/sub\\u003e\\u003c/p\\u003e\\u003cp\\u003e(h\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003et\\u003csub\\u003e1/2\\u003c/sub\\u003e\\u003c/p\\u003e\\u003cp\\u003e(h)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003eSF\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003efree\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.0279\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e24.8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e1.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0.0502\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e13.8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e1.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePCN@cellulase\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.0187\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e37.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e1.5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0.0243\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e28.5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e2.1\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePCN@cellulase/Glu\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.0140\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e49.4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e2.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0.0188\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e36.9\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e2.7\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePCN/Glu@cellulase\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e0.0100\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e69.4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e2.8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003e0.0134\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e\\u003cp\\u003e51.7\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e\\u003cp\\u003e3.7\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe apparent K\\u003csub\\u003eM\\u003c/sub\\u003e and V\\u003csub\\u003emax\\u003c/sub\\u003e values of each cellulase sample are given in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e. The apparent K\\u003csub\\u003eM\\u003c/sub\\u003e values of free cellulase, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase were determined to be 1.14\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.14, 1.39\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.18, 1.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21, and 0.84\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.10 mg/mL for CMC (\\u003cb\\u003eFig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e\\u003c/b\\u003e). The alteration in the K\\u003csub\\u003eM\\u003c/sub\\u003e value may be attributed to changes in the enzyme\\u0026ndash;substrate affinity resulting from conformational modifications of cellulase induced by immobilization, the onset of substrate diffusional constraints, or a synergistic effect of both phenomena. The corresponding V\\u003csub\\u003emax\\u003c/sub\\u003e values were found to be 1.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09, 1.77\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.22, 1.17\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09, and 1.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.11 U/mg protein. The elevated Vmax values observed for PCN@cellulase and PCN/Glu@cellulase compared to free cellulase may be attributed to the favorable orientation of enzyme molecules on the support surface, which facilitates improved substrate access to the active site. According to these results CER ratios were calculated to be 1.2, 0.7, and 1.4. These results indicate that PCN@cellulase and PCN/Glu@cellulase exhibit 1.2- and 1.4-fold higher activity, respectively, compared to free cellulase. The CER value of cellulase immobilized on COOH-functionalized MWCNTs is approximately 1.66-fold higher than that of free cellulase (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). The K\\u003csub\\u003eM\\u003c/sub\\u003e and V\\u003csub\\u003emax\\u003c/sub\\u003e values of free cellulase from \\u003cem\\u003eA. niger\\u003c/em\\u003e were determined to be 2.06\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.85 mM and 159\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;11 U/mg protein for cellulose, respectively. The corresponding values for its immobilized counterpart on TiO\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026ndash;lignin hybrid support were 2.63\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.96 mM and 125\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;19 U/mg protein. In addition, CER value was calculated to be 0.62 (\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eThe apparent K\\u003csub\\u003eM\\u003c/sub\\u003e and V\\u003csub\\u003emax\\u003c/sub\\u003e values of free cellulase, PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase for CMC under the optimum conditions of each preparation.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"4\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eBiocatalyst\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eK\\u003csub\\u003eM\\u003c/sub\\u003e\\u003c/p\\u003e\\u003cp\\u003e(mg/mL)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eV\\u003csub\\u003emax\\u003c/sub\\u003e\\u003c/p\\u003e\\u003cp\\u003e(U/mg protein)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eCER\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003efree\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1.14\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.14\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e1.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePCN@cellulase\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1.39\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.18\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.77\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.22\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e1.2\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePCN@cellulase/Glu\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.17\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.7\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePCN/Glu@cellulase\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e0.84\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.10\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.11\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e1.4\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe reuse stability of immobilized cellulase preparations was investigated in a batch type reactor for 10 cycles. Among the tested immobilized cellulases, the covalently immobilized PCN/Glu@cellulase retained 80% of its initial activity, while PCN@cellulase/Glu and PCN@cellulase retained 72% and 50% of their initial activities, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). These results highlight that PCN, particularly when modified through covalent attachment, provides a durable and reusable platform for enzyme immobilization, offering great potential for cost-effective and sustainable industrial bioprocesses. Celulase immobilized onto carbon coated Fe\\u003csub\\u003e3\\u003c/sub\\u003eO\\u003csub\\u003e4\\u003c/sub\\u003e nanoparticles protected 80% of its initial activity after 9 reuses (\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e). Rashid et al. modified nickel nanoparticle with 3-APTES to generate free -NH\\u003csub\\u003e2\\u003c/sub\\u003e group onto the support surface and immobilized cellulase from Aspergillus \\u003cem\\u003eniger\\u003c/em\\u003e by adsorption followed by glutaraldehyde crosslinking. After 10 reuses, the immobilized cellulase retained 84% of its initial activity after ten reuses (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e). C\\u003csub\\u003e3\\u003c/sub\\u003eN\\u003csub\\u003e4\\u003c/sub\\u003e-NS@CRL retained 72% of the initial activity after 10 reuses (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e). Shangguan et al. reported that CALB covalently bound to g-C\\u003csub\\u003e3\\u003c/sub\\u003eN\\u003csub\\u003e4\\u003c/sub\\u003e maintained 65% of its initial activity after 9 cycles (\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e).\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIn this study, PCN synthesized from melamine was successfully employed as a sustainable and metal-free support for cellulase immobilization. Three different immobilization strategies - simple adsorption, adsorption followed by glutaraldehyde crosslinking, and covalent binding via glutaraldehyde activation - were comparatively evaluated. All immobilized cellulase preparations retained their catalytic activity and exhibited improved thermal stability compared to the free enzyme. In particular, the covalently bound PCN/Glu@cellulase demonstrated the highest stabilization factor, extended half-life and better reusability, highlighting the effectiveness of covalent immobilization in enhancing enzyme performance. These findings confirm that PCN provides a promising platform for enzyme immobilization due to its biocompatibility, high surface area, and abundance. The results open up new perspectives for the application of PCN-based biocatalysts in sustainable bioprocesses, especially for biomass conversion and other industrial applications requiring robust and reusable cellulase systems.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eCredit author statement\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNuri Gulesci:\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eConceptualization\\u003cstrong\\u003e,\\u0026nbsp;\\u003c/strong\\u003eMethodology, Writing \\u0026ndash; original draft. Orhan Altan\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eConceptualization\\u003cstrong\\u003e,\\u0026nbsp;\\u003c/strong\\u003eMethodology, Data curation, Formal analysis. Ali Toprak:\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eConceptualization\\u003cstrong\\u003e,\\u0026nbsp;\\u003c/strong\\u003eMethodology, Data curation, Formal analysis.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eMustafa Serkan Yalcin: Methodology, Data curation, Formal analysis\\u003cstrong\\u003e. \\u0026nbsp;\\u003c/strong\\u003eRamazan Bilgin:\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eWriting \\u0026ndash; review \\u0026amp; editing.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eDeniz Yildirim: Methodology, Writing \\u0026ndash; original draft, Writing \\u0026ndash; review \\u0026amp; editing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe author declares that they have no conflict of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis research received no external funding.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eData will be made available on reasonable request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAssis Modenez, I., Sastre, D. E., Moraes, C., F. and, \\u0026amp; Marques Netto, C. G. C. (2018). Influence of glutaraldehyde cross-linking modes on the recyclability of immobilized lipase B from \\u003cem\\u003eCandida antarctica\\u003c/em\\u003e for transesterification of soy bean oil. \\u003cem\\u003eMolecules\\u003c/em\\u003e, \\u003cem\\u003e23\\u003c/em\\u003e, 2230.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAhmad, R., \\u0026amp; Khare, S. K. (2018). Immobilization of \\u003cem\\u003eAspergillus niger\\u003c/em\\u003e cellulase on multiwall carbon nanotubes for cellulose hydrolysis. \\u003cem\\u003eBioresource Technology\\u003c/em\\u003e, \\u003cem\\u003e252\\u003c/em\\u003e, 72\\u0026ndash;75.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAlag\\u0026ouml;z, D., Toprak, A., Yildirim, D., T\\u0026uuml;kel, S. S., \\u0026amp; Fernandez-Lafuente, R. (2021). 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A two-step method for the synthesis of magnetic immobilized cellulase with outstanding thermal stability and reusability. \\u003cem\\u003eNew Journal Of Chemistry\\u003c/em\\u003e, \\u003cem\\u003e45\\u003c/em\\u003e, 6144\\u0026ndash;6150.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"applied-biochemistry-and-biotechnology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"abab\",\"sideBox\":\"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)\",\"snPcode\":\"12010\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12010/3\",\"title\":\"Applied Biochemistry and Biotechnology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Cellulase, immobilization, polymeric carbon nitride, stability\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7657582/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7657582/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003ePolymeric carbon nitride (PCN) attracted considerable attention in recent years due to its semiconducting properties. In addition to its semiconducting properties, it is renowned as a support material due to its layered structure and donor groups such as terminal amines and triazine units. In this study, PCN was employed for the first time as a novel support material for the immobilization of cellulase, a key enzyme used in many industrial applications. Cellulase from \\u003cem\\u003eAspergillus sp\\u003c/em\\u003e. was immobilized onto PCN in three methods. In the first way, cellulase was adsorbed on PCN (PCN@cellulase). In the second way, PCN@cellulase was crosslinked with glutaraldehyde (PCN@cellulase/Glu). Finally, the primary amino group of PCN was modified with glutaraldehyde and the cellulase was immobilized on this support by covalent attachment (PCN/Glu@cellulase). Each cellulase preparation was individually assessed for its optimal pH, temperature conditions, heat stability, and enzyme kinetics. The optimal pH was 5.5 for all cellulase preparations, while the optimal temperature was 45\\u0026deg;C for free cellulase and 55\\u0026deg;C for all immobilized cellulase preparations. Thermal stability of PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase increased by 2.1-, 2.7-, and 3.7-fold, respectively, compared to the free cellulase. PCN/Glu@cellulase showed 1.4-fold higher catalytic efficiency than the free cellulase and retained 80% of its initial activity after 10 reuses. These results indicate that the use of metal-free, nitrogen-rich PCN, synthesized from abundant and low-cost melamine, aligns with the principles of green chemistry and offers a sustainable alternative to traditional immobilization supports.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Layered polymeric carbon nitride as a green support for cellulase immobilization: Improved stability, activity, and reusability\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-10-14 13:09:26\",\"doi\":\"10.21203/rs.3.rs-7657582/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2025-10-01T07:24:07+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-10-01T06:43:03+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"Applied Biochemistry and Biotechnology\",\"date\":\"2025-09-20T01:47:11+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Applied Biochemistry and Biotechnology\",\"date\":\"2025-09-19T06:29:54+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"applied-biochemistry-and-biotechnology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"abab\",\"sideBox\":\"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)\",\"snPcode\":\"12010\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12010/3\",\"title\":\"Applied Biochemistry and Biotechnology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"78a0e6d0-6bfc-41a9-91ec-af2d61b90209\",\"owner\":[],\"postedDate\":\"October 14th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-01-05T16:04:39+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7657582\",\"link\":\"https://doi.org/10.1007/s12010-025-05552-2\",\"journal\":{\"identity\":\"applied-biochemistry-and-biotechnology\",\"isVorOnly\":false,\"title\":\"Applied Biochemistry and Biotechnology\"},\"publishedOn\":\"2026-01-04 15:58:22\",\"publishedOnDateReadable\":\"January 4th, 2026\"},\"versionCreatedAt\":\"2025-10-14 13:09:26\",\"video\":\"\",\"vorDoi\":\"10.1007/s12010-025-05552-2\",\"vorDoiUrl\":\"https://doi.org/10.1007/s12010-025-05552-2\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7657582\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7657582\",\"identity\":\"rs-7657582\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}