Sustainable green approach to synthesize Fe3O4/α-Fe2O3 nanocomposite using waste pulp of Syzygium cumini and its application in functional stability of microbial cellulases

Synthesis of nanomaterials following green routes have drawn much attention in recent years due to the low cost, easy and eco-friendly approaches involved therein. Therefore, the current study is focused towards the synthesis of Fe3O4/α-Fe2O3 nanocomposite using waste pulp of Jamun (Syzygium cumini) and iron nitrate as the precursor of iron in an eco-friendly way. The synthesized Fe3O4/α-Fe2O3 nanocomposite has been extensively characterized through numerous techniques to explore the physicochemical properties, including X-ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, Ultraviolet-Vis spectroscopy, field emission scanning electron microscope, high resolution transmission electron microscope and vibrating sample magnetometer. Further, efficiency of the Fe3O4/α-Fe2O3 nanocomposite has been evaluated to improve the incubation temperature, thermal/pH stability of the crude cellulase enzymes obtained from the lab isolate fungal strain Cladosporium cladosporioides NS2 via solid state fermentation. It is found that the presence of 0.5% Fe3O4/α-Fe2O3 nanocomposite showed optimum incubation temperature and thermal stability in the long temperature range of 50–60 °C for 15 h along with improved pH stability in the range of pH 3.5–6.0. The presented study may have potential application in bioconversion of waste biomass at high temperature and broad pH range.


Scientific Reports
| (2021) 11:24371 | https://doi.org/10.1038/s41598-021-03776-w www.nature.com/scientificreports/ beverages industries. Production of sugar is carried out via substrate hydrolysis by these enzymatic group which include three sub-enzymes categorized as β-1,4-endoglucanase (EC 3.2.1.4), β-1,4-exoglucanase (EC 3.2.1.91), and β-d-glycosidase (EC 3.2.1.21) 3 . Synergic actions of all three enzyme components are required for the efficient hydrolysis of cellulosic substrate 4 . In general, the reported temperature and pH range for the enzymatic hydrolysis of cellulosic substrate are found to be 45-50 °C and acidic range, respectively. But, these hydrolysis conditions may results in low yields of sugars, partial or incomplete hydrolysis of cellulose, require high enzymes loading, and easily prone to the microbial contamination. In this context, production and development of thermostable and pH stable cellulase enzyme can be a potential alternative to overcome these limitations in a sustainable way 5 . Thermostable and pH stable cellulases show good stability even over their optimum working temperature and pH conditions. An enzyme can be considered to be thermostable or pH stable when it holds half-life at relatively higher temperatures or pH than that of optimum value for a longer duration. These temperature and pH stable enzymes are highly demanding in biomass to biofuels industries 6 . Cellulolytic enzymes which possess such thermo and pH stable properties are notably found in fungal strains and therefore are always preferred to produce cellulase enzyme over the bacterial strains due to the high cellulolytic index 7,8 .
For improving the thermal and pH stability of enzymes, nanomaterials have been well explored which act as catalyst owing to their unique physicochemical properties including high surface reaction and strong adsorption ability [9][10][11][12] . Additionally, the large surface area to volume ratio of nanomaterials facilitates multipoint attachment for the enzyme molecules and leads better immobilization of enzymes which is mainly because of the impeded unfolding of protein molelecules. In this way, stability of enzyme can be significantly improved in terms of better thermal and pH stability for longer time 13 . Though nanomaterial have tremendous potential to enhance the stability and efficiency of cellulase enzymes, their synthesis cost and strategies involved therein seems to be main issues in sustainable enzymes production which can greatly influence the economical production of biofuels technology.
A number of plant species have been recently studied to synthesize variety of nanomaterials. For example, Acorus calamus extract to synthesize cerium oxide NPs 14 , Delonix elata leaf extract to synthesize tin oxide NPs, 15 , Deverra tortuosa extract to synthesize zinc oxide NPs 16 , root extract of Kniphofia foliosa to synthesize titanium oxide NPs 17 , extracts of Impatiens balsamina and Lantana camara to synthesize silver NPs 18 , Mimosa tenuiflora extract to synthesize gold NPs 19 ,Carica papaya leaf extract to synthesize iron oxide NPs 20 , Syzygium aromaticum extract to synthesize nickel ferrite NPs 21 , Amaranthus blitum leaves extract to synthesize silver ferrite NPs 22 and Vernonia amygdalina leaf extract to synthesize reduced graphene oxide 23 have been reported. Further, among different types of nanomaterials iron based nanostructures have shown their tremendous potential to elevate the enzyme stability which can be further exploited to improve the biofuels production when employed as catalyst [24][25][26] . On the other hand, among variety of plant extracts Syzygium cumini has been well explored as the potential reducing agents to synthesize different types of nanomaterials 27,28 . In a recent study our group has reported synthesize of Fe 3 O 4 NPs using waste seeds of Syzygium cumini and explored its application as catalyst to improve the thermal and pH stability of crude cellulase obtained from Emericella variecolor NS3 which was further employed for the hydrolysis of sugarcane bagasse 8 .
Thus, inspired by our earlier study on the potential application of waste seeds extract of Syzygium cumini as a reducing agent, in the present work we explored the utilization of waste pulp extract of Syzygium cumini to synthesize Fe 3 O 4 /α-Fe 2 O 3 nanocomposite. In this work, ripe waste pulp extract of Jamun (Syzygium cumini) has been selected for the synthesis of Fe 3 O 4 /α-Fe 2 O 3 NCs, because it is a rich source of sugars and different organic compounds which acts as a potential reducing agent. In addition, since the waste pulp (fruit) of Syzygium cumini is renewable and easily available material can be exploited for a low cost and green synthesis of nanomaterials. The synthesized Fe 3 O 4 /α-Fe 2 O 3 NCs has been extensively characterized by different techniques to analyze the physicochemical properties. Moreover, the impact of Fe 3 O 4 /α-Fe 2 O 3 NCs has been investigated on incubation reaction temperature, thermal and pH stability of crude cellulase enzyme obtained from Cladosporium cladosporioides NS2 following the solid state fermentation (SSF).

Results
Characterizations of the synthesized product. Information about the phase formation has been confirmed through the powder X-ray diffraction (XRD) pattern ( Fig. 1(i)). The analysis of the XRD pattern explores that the synthesized product consists of two different forms of iron oxides which includes Fe 3 O 4 (magnetite) and α-Fe 2 O 3 (hematite) phase. Further, presence of different functional groups in waste pulp extract (WPE) of Syzygium Cumini has been analyzed through the Fourier transform infrared spectroscopy (FT-IR) spectrum recorded in the range of 4000-400 cm −1 (Fig. 1(ii)). The obtained results explored that the WPE of Syzygium Cumini consists of various functional groups e.g. amine, alkenes, phenyl ring/alkyl halides, thiocarbonyl and aliphatic esters. In addition, formations of the iron oxides phases have been reconfirmed by the Raman spectroscopy which also explored the formation of Fe 3 O 4 and α-Fe 2 O 3 phases (Fig. 2(i)). In addition, formation of these iron oxide phases has been also supported by the presence of metal-oxygen bonds observed below 600 cm −1 . The Ultraviolet-Vis (UV-Vis) spectrum was also recorded to probe the optical band gap and the obtained results suggest that the prepared nanoparticles are semiconducting in nature ( Fig. 2(ii)). In order to get the information about the surface morphology, elemental compositions, particle size, and particle shape, the prepared sample was extensively characterized through field emission scanning electron microscope (FE-SEM) and high resolution transmission electron microscope (HR-TEM) techniques and results are shown in Figs. 3 and 4, respectively. The obtained results explored that the synthesized particles are polycrystalline and possessed some mesoporous characteristics. On the other hand, magnetic properties of the synthesized product analyzed through the vibrating sample magnetometer (VSM) technique suggest that the nanoparticles are superparamagnetic in nature at room temperature (Fig. 5).  (Fig. 6b).

Effect of Fe 3 O 4 /α-Fe 2 O 3 nanocomposite on pH stability of cellulases enzyme.
The impact of nanocomposite on enzymatic activity has been investigated at different pH to determine the stability (Fig. 6c). Results clearly explore that the Fe 3 O 4 /α-Fe 2 O 3 nanocomposite treated enzyme holds 100% activity in the acidic medium i.e. pH, 3.5-6.0, while possessed its half-life at pH 8.0. It is worthy to mention that the control enzyme (without nanocomposite treatment) showed 100% relative activity only in the range of pH 4.0-4.5 and showed its half-life at pH 7.5.    29 . In this process, firstly iron metal salt (ferric nitrate) disassociates in to cations and anions where cations tend to form the hydroxyl complexes. Once there is saturation in the formation of hydroxyl complexes further growth of crystallite take place with the association of oxygen species. The organic compounds present in the WPE of Syzygium Cumini donates electrons to metal cations and acts as the reducing agent meanwhile it may also serve as the capping agent. Thereafter, heat treatment which plays a critical role leads to the formation of iron oxide nanoparticles 30 . Scheme 1 depicts the overall process and possible mechanism in the formation of iron oxide nanocomposites. The XRD pattern of the synthesized sample is shown in Fig. 1(i).     In order to probe the presence of various functional groups in WPE of Syzygium Cumini, the extracted sample was characterized through the FT-IR spectroscopy and results are presented in Fig. 1(ii). Presence of numerous vibrational peaks can be seen in the FT-IR spectrum where a broad peak in the range of 3551-3225 cm −1 is attributed to the O-H stretching and C-O stretching (carboxylic acid) 33 . The band observed at ~ 2938 cm −1 is corresponding to C-H stretching, a small intensity peak appeared at ~ 2353 cm −1 is related to the C=C stretching whereas the peak recorded ~ 1733 cm −1 could be correlated to the -C=O stretching of the aliphatic esters. In addition, various peaks corresponding to the different functional groups are also observed including 1636 cm −1 (aromatic ring, C=C stretching/amine groups, N-H stretching), 1415 cm −1 (alkenes aromatic, C=C stretching), 1257 cm −1 (C=N stretching), 1072 cm −1 (thiocarbonyl, C=S stretching), 820-870 cm −1 (phenyl ring/alkyl halides, C-H bending), and 640 cm −1 (alkynes, C-H bending) 34  www.nature.com/scientificreports/ Raman spectroscopy indeed is a very powerful technique and plays a vital role to determine the combination of iron oxides phase. Since iron oxides exhibit certain phase transitions which form different crystal structure e.g. Fe 3 O 4 , γ-Fe 2 O 3 , and α-Fe 2 O 3 it is imperative to perform the Raman analysis to know the exact forms of iron oxide nanoparticles. On the basis of the group theory analysis, spinel types of crystal structure (Fe 3 O 4 ) should exhibit five Raman active vibrational modes. These vibrational modes can be represented as A 1g + Eg + 3T 2g . On the other hand, in case of α-Fe 2 O 3 phase group theory suggests that there should be seven Raman active vibrational modes which can be represented as 2A 1g + 5E g 36 . Raman spectrum as shown in Fig. 2(i) exhibits seven peaks in the range of 175-850 cm −1 . The most intense peak appeared at 677 cm −1 could be assigned to Eg mode whereas peak ~ 299 cm −1 is attributed to the A 1g mode and confirms the formation of Fe 3 O 4 phase. This observation is consistent with the earlier reported value observed in the case of spinel type crystal structure 37 . In addition, presence of α-Fe 2 O 3 phase was also confirmed by the presence of several peaks corresponding to different vibrational modes including 224 cm −1 [A1g (1) The optical property of synthesized Fe 3 O 4 /α-Fe 2 O 3 NCs has been investigated through the UV-Vis spectrum recorded in the range of 300-700 nm. It is noticed that the UV-Vis spectrum exhibits a peak ~ 350 nm. Further, optical band gap has been calculated through the Tauc plot and presented in Fig. 2(ii). From the Tauc plot the direct band gap has been calculated to be ~ 2.73 eV. The obtained value is in good agreement with earlier studies 38 . The morphology and elemental compositions of the synthesized nanocomposite was confirmed by the FE-SEM technique (Fig. 3). FE-SEM micrographs show that the nanoparticles are granular in shapes which are uniformly distributed over the entire region of the micrograph. The grain size of particles can be seen in the range of 20-30 nm. In addition, elemental mapping of the corresponding micrograph was also done, revealing uniform distribution of the Fe and O elemental and having wt% compositions of 62% and 32%, respectively. TEM micrographs of the nanoparticles suggest that particles are mesoporous in nature and particles size are in the size of 5-10 nm (Fig. 4). The SAED pattern shows the presence of co-centric rings along with a number of bright spots which suggests that the formed nanocomposite is polycrystalline in nature. Moreover, in the HR-TEM micrographs well defined lattice fringes can be clearly seen which support crystalline properties of the nanoparticles.
The magnetic properties of Fe 3 O 4 /α-Fe 2 O 3 nanocomposite could be analyzed through the VSM measurements and results are shown in Fig. 5. The magnetic properties have been measured by the M-H graph obtained by applying an external magnetic field of 10 KOe. The M-H loop suggests that the prepared sample exhibits superparamagnetic characteristics. This superparamagnetic property is mainly due to the presence of very small size of the nanoparticles which possesses single magnetic domains 39 . In addition, it may be noted that the Fe 3 O 4 phase was mainly responsible to display the superparamagnetic property of the Fe 3 O 4 /α-Fe 2 O 3 nanocomposite because α-Fe 2 O 3 phase shows weak magnetic properties at room temperature 40 . The saturation magnetization at the maximum applied filed was found to be ~ 19.75 emu/g. This value was found to be significantly lower than that of bare Fe 3 O 4 nanoparticles which is attributed to the simultaneous presence of α-Fe 2 O 3 phase and in fact this form of iron oxide nanoparticles have very low contribution to attain the overall saturation magnetization value i.e. ~ 19.75 emu/g 41 .
Incubation temperature range of 50-65 °C is commonly recorded as the optimum condition for the cellulase enzyme activity, and has been reported in several studies 42,43 . The obtained results in the present study showed three temperatures 50, 55 and 60 °C as the optimum value for the enzyme activity in presence of Fe 3 O 4 /α-Fe 2 O 3 nanocomposite when compared to the control enzyme which shows only 50 °C as the optimum condition. The improved incubation temperature at three different temperatures in case of nanocomposite treated cellulase may be due to the conjugation of enzyme with the nanocomposite which helps to avoid the protein unfolding and thus enhanced the stability of the enzyme for a longer duration 44,45 . In addition, such types of higher temperature tolerance ability of nanocomposite treated enzymes can have large scale industrial application due to the improved potency to survive in the reaction medium for a longer time.
Further, due to the high immobilization/conjugation and catalytic property, Fe 3 O 4 /α-Fe 2 O 3 nanocomposite is likely to act as a shield to enhance the stability of enzymes for a longer duration at moderate temperatures along with improved activity at relatively higher temperature but for a shorter duration 46 . In a study, cellulase immobilized on Fe 3 O 4 NPs showed stability at 60 °C up to 5 h which retained its 80% relative activity 47 . Though, both the cellulase enzyme system i.e. control and Fe 3 O 4 NPs immobilized enzyme showed their highest activity and stability at 60 °C, Fe 3 O 4 bound cellulase was more stable up to 80 °C but, control enzyme could not preserve its activity. As per the explanation given by these authors, optimum incubation temperature is likely to enhance the activation energy of the enzyme molecules and therefore, offers adequate interaction with the substrate which could be further improved with the immobilization made by using nanoparticles. Song et al., also reported improved thermal stability of β glucosidase-A and cellobiohydrolase-D enzymes when immobilization was done on superpamagnetic NPs 48 . Moreover, the immobilization was helpful to exhibit thermal stability in a wider temperature range as compared to control system. It is also noticed that the types of immobilization exert a significant influence to determine the activity of cellulase enzyme. And the enzyme immobilized on TiO 2 NPs via co-valent bonding exhibited superior activity (at 75 °C) as compared to physical-adsorption 49 . In a study, Cherian et al., reported that the cellulase enzyme immobilized on MnO 2 NPs demonstrated superior stability for a longer duration even at higher temperature as compared to bare enzyme system 50 . More importantly, a sharp decline in the enzymatic activity could be avoided in case of MnO 2 NPs immobilized enzyme as compared to the free enzyme and therefore such types of enzymes can be of high potential for the bioconversion processes. Bohara et al., also reported that the celulase enzyme immobilized on cobalt ferrite NPs via surface functionalization exhibited relatively better thermal stability as compared to free enzyme 51 54 . In all the above studies, incubation temperature and thermal stability have been reported nearly in the common range. Additionally, 50 and 60 °C were found to be very common thermal stability range in all the investigations as discussed above, whereas the thermal stability testing range was also made between 40 and 80 °C. On the other hand, the results obtained in the present study focused on the long duration sustainability of the Fe 3 O 4 /α-Fe 2 O 3 nanocomposite treated crude enzyme system at moderate temperature range of 50-60 °C along with the relatively higher thermal stability testing range which is 40-90 °C as compared to reported literatures. Thus, such types of enzyme system might have potential application in various industries indulging bioprocessing of enzymes. This kind of enzyme system which has ability to hold its stability for such a long duration can be of great importance in the cellulosic waste bioconversion process and this can lead to the higher sugars production and consequently better yield of biofuels is achieved. Enzymes which show their higher stability may offer several unique properties like fast reaction rate, higher substrate to product conversion efficiency and thus higher yield of the product can be achieved. Moreover, enzymatic hydrolysis medium using such types of enzyme systems would be non-sustainable and exhibit better tolerance ability towards the contaminants which might spoil the reaction 59 . Moreover, it has been confirmed through various studies that nanomaterials have great potential to enhance the stability of enzymes up to a great extent. Therefore, Fe 3 O 4 /α-Fe 2 O 3 nanocomposite treated cellulase developed in the present study may have tremendous potential in the biofuels industries. Additionally, enhanced stability of nanoparticles treated cellulase in the higher acidic range suggests it's numerous industrial scope including paper and pulp industries, bioprocessing, juice industries, as well as in the biofuels industries. In addition, properties of nanoparticles (e.g. shape, size and morphology) significantly contribute toward the interaction of nanoparticles with the enzyme and thereby improve the pH stability 60,] 61 . The mutual interaction of nanoparticles with enzyme may provide better stability in a broader acidic medium as compared to the untreated enzyme and make it suitable for various industries. In a recent study, pH stability was performed between the pH 4.0-8.0 for 1 h using Fe 3 O 4 immobilized cellulase and results were compared with the free cellulase enzyme as the control system 47 . It was found that the immobilization on Fe 3 O 4 NPs was helpful to elevate the stability and the cellulase showed its stability at pH 6.0 whereas control showed the same at pH 5.0. Thus, apart from temperature, pH plays a significant role to determine the enzyme activity. It should be noted that, pH shift and its stability significantly rely on the enzyme and the physicochemical properties of the immobilizing substrate (due to the presence of different functional groups) which lead conformational changes while changed in the pH value 53 . Additionally, shifting towards the higher pH range in case of NPs immobilized enzyme is because of the accumulation of higher net charge of the magnetic NPs that conjugates with the cellulase. Similar observations have been also reported where enzyme immobilized on superparamagnetic NPs exhibited more sensitivity towards the different pH medium compared to free cellulase 48 . Li el al., reported better pH (3.0-6.0) stability of cellulase enzyme immobilized on Fe 3 O 4 @SiO 2 as compared to free enzyme 55 . Nevertheless, over the pH 6.0, stability was recorded to be lower in case of Fe 3 O 4 @SiO 2 immobilized enzyme. Similarly, compared to the free enzyme, relatively higher stability in the pH range of 4.0-8.0 has been reported in case of MnO 2 NPs immobilized enzyme 50 . In a study, it was found that though, both the systems i.e. free cellulase and the cellulase immobilized on carbon nanotubes exhibited their optimum activity at pH, 30., the activity of immobilized enzyme was superior than that of free enzyme 62 . Huang et al., investigated pH stability of Fe 2 O 3 / Fe 3 O 4 nanocomposites immobilized cellulase in the range of pH 4.0 to 6.0 with the pH difference of 0.5 and found the stability of nanocomposite immobilized cellulase at pH 5.0 whereas free cellulase exhibited its stability at pH 4.0 43 . Additionally, results explored the fact that the nanocomposite provides a type of support and improves the mechanical stability of the enzyme at higher acidic range as compared to the control enzyme. Further, in the study of Abbaszadeh & Hejazi, Fe 3 O 4 nanoparticles immobilized celluase and control enzyme both, showed the maximum enzyme activity at pH 3.0 and stability in the pH range of 2.0-6.0 57 . These observations explained the acidic habitat of the enzyme for the full growth and maximum enzyme production. In the study of Kumari et al., cellulase immobilized on magnetic nanoparticles were tested for the pH stability in the pH range of 2.0-12, and pH 8.0 was found to be optimum whereas enzyme retained its half-life at pH 12 58 . Binding/conjugation of cellulase on the surface of nanoparticles which provide improved mechanical strength is the basics concept of the pH stability as observed in the present study which is well supported by numerous studies as discussed above. Moreover, in the present study high stability of enzyme has been recorded in the broader pH range of 3.5-6.0 nanocomposite as the catalyst has been evaluated at a concentration 0.5%, on the incubation temperature, thermal and pH stability of the cellulase enzyme. The Fe 3 O 4 /α-Fe 2 O 3 nanocomposite treated crude cellulase showed optimum incubation temperature and thermal stability in the long temperature range of 50-60 °C for 15 h and pH stability in the range of 3.5-6.0. The present study may have potential application in the bioconversion of waste biomass at high temperature and broad pH range. Nevertheless, it is noticed that the different physicochemical properties of the nanomaterials such as their types (e.g. different metals and their oxides forms), size, shape and morphology may significantly contribute to exhibit varying stability (thermal, pH and long duration stability) of the cellulase enzyme. Therefore, rigorous investigation should be made on the effect of different shape, size and morphology of the same nanomaterials to explore the efficiency of the enzyme in different environment (pH, thermal and long term stability). In addition, efficiency of the enzyme may further be improved with the help of covalent bonding by the surface functionalizations of nanoparticles. In these ways, efficiency of the enzyme system can be greatly improved which can be of great potential for the numerous applications at industrial level.

Materials and methods
Chemicals and utilized substrate. Required chemical and sugarcane bagasse (SCB) waste employed for the enzyme production were arranged from the local market of Varanasi, (U.P.), India. Physicochemical treatments of SCB for the enzyme production were done as per the recent study reported by our group 8 . Ripe waste pulp of Syzygium cumini was collected from the local garden of the Department of Chemical Engineering and Technology IIT (BHU), Varanasi, India. The obtained waste pulp was properly washed by using 90% ethanol and double distilled water 4-5 times, subsequently strict Sun and oven drying at 50 °C until the complete dryness of pulp was performed. Thereafter, a homogenous powder was obtained by grinding dry pulp waste.

Preparation of waste pulp extracts (WPE) of Syzygium cumini. Dry 5 g of WPE powder was mixed
in to 100 mL of DD water, shaken for 10 min and then boiled at 100 °C for 20 min. Further, extract was allowed to cool down and filtered, subsequently used for the preparation of Fe 3 O 4 /α-Fe 2 O 3 nanocomposite.

Synthesis of Fe 3 O 4 /α-Fe 2 O 3 nanocomposite through green route. Preparation of Fe 3 O 4 /α-Fe 2 O 3
nanocomposite has been done by following the modified method as per the earlier study 8 . In the present approach, to synthesize Fe 3 O 4 /α-Fe 2 O 3 nanocomposite, firstly 1 M solution (50 mL) of ferric nitrate was prepared. Thereafter, WPE and ferric nitrate solution were mixed in to 2:1 ratio with a continuous magnetic stirring till we observed the color of the solution to be brownish. Thereafter, mixture was incubated for 10 min for settling down and then centrifuged to collect the precipitate. The obtained precipitate was rigorously washed with DD water and dried in an oven at 55 °C consequently calcination was done at 300 °C in a preheated furnace for 10 min to obtain the final product.
Characterization studies. Physicochemical properties of the synthesized product has been analyzed by different techniques including powder X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, UV-Vis spectroscopy, filed emission scanning electron microscope (FE-SEM), high resolution transmission electron microscope (HR-TEM), and vibrating sample magnetometer (VSM).. Fungal culture, enzyme production and enzyme assays. The labs isolate fungal culture Cladosporium cladosporioides NS2 (KT160360) was used for the cellulase enzyme production following the solid state fermentation (SSF). A detail method about the process parameters to produce cellulase enzyme can be found in the earlier study 8 . In the present study, enzyme activity has been analyzed in terms of Filter paper (FP) activity 63 . In addition, reducing sugar concentration has been analyzed by using dinitrosalicylic acid (DNS) method 64 . Effect of Fe 3 O 4 /α-Fe 2 O 3 nanocomposite on optimum reaction temperature, thermal and pH stability of cellulase enzyme. The optimum reaction temperature for FP activity was determined at the temperature between 45 and 80 °C at an interval of 5 °C for 1 h in presence of Fe 3 O 4 /α-Fe 2 O 3 NCs (at 0.5% concentration) and compared with control. The thermal stability of cellulase enzyme was studied at different temperatures and pH in presence of Fe 3 O 4 /α-Fe 2 O 3 NCs. Further, thermal stability of the enzyme was probed by pre-incubating the enzyme at different temperature over the range of 50-90 °C for 0-15 h. To perform the pH stability test, crude enzyme was tested in different reaction buffers of different pH range 3.0-10 for 1 h at a pH interval of 0.5 8 .
Statistical analysis. Experiments were performed in triplicate whereas means and standard deviation have been calculated by using excel software. In addition, analysis of the experimental data was done by variance (ANOVA) [SPSS (version 16)]. Turkey's test has been also employed to calculate the significance of the difference between the treatment means 8 . License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.