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. 2023 Sep 4;33(5):1163–1175. doi: 10.1007/s10068-023-01422-x

Characterization of cellulase immobilized by different methods of entrapment and its application for carrot juice extraction

Bhaskar Jyoti Kalita 1, Nandan Sit 1,✉
PMCID: PMC10908674  PMID: 38440682

Abstract

In the present study, cellulase has been immobilized by two different methods of entrapment viz. encapsulation in calcium alginate and matrix entrapment in agar. The calcium alginate encapsulated beads showed an immobilization efficiency of 92.11% and agar entrapped cubes showed an immobilization efficiency of 97.63%. The free cellulase was found to show optimum activity at 50 °C and pH 4, had a Km of 39.29 mg/mL, Vmax of 0.50 μmol/min. The calcium alginate encapsulated beads showed optimum activity at 50 °C, and pH 8, had a Km of 72.28 mg/mL and Vmax of 1.32 μmol/min. The agar entrapped cubes showed optimum activity at 60 °C, and pH 4, had a Km of 13.08 mg/mL, and Vmax of 0.38 μmol/min. The immobilized cellulases could be used for 5 cycles after which their activity deteriorated. The immobilized as well as the free enzyme were effective in increasing the yield of carrot juice.

Keywords: Cellulase immobilization, Entrapment, Characterization and application

Introduction

Cellulose from agricultural and forestry residues, industrial processing of agricultural materials (Sulyman et al., 2020), municipal waste, woody plants etc. (Zhang et al., 2016) gets dumped in our surroundings in enormous quantities everyday leading to space constraint besides unpleasant odor. These cellulosic matters can serve as inexpensive raw materials for the process of chemical or enzymatic conversion (Andriani et al., 2011) to different utilizable products such as enzymes, fuel, organic acids, ethanol, and other chemicals (Sanchez et al., 2014). Enzymatic hydrolysis is a suitable process for utilizing cellulose (Ingle et al., 2017) as it is a clean process and the reaction conditions like temperature, pH, etc. can be controlled (Zhang et al., 2016). It is made possible by the use of enzyme cellulase which cleaves the β-1,4-glycosidic bonds of cellulose to release glucose (Andriani et al., 2011).Cellulase is a complex system of enzymes basically composed of three components, endoglucanase (EC 3.2.1.4) that acts on amorphous sites and cleaves internal bonds, exoglucanase or cellobiohydrolyase (EC 3.2.1.91) that acts on exposed ends to generate disaccharide or tetra-saccharides, and β-glucosidase or cellobiase (EC 3.2.1.21) that cleaves disaccharides into glucose (Pandey and Negi, 2020). Cellulase is obtained from different sources and each of them exhibit unique features like specific pH optima, solubility depending on the amino acid composition, thermal stability, substrate specificity etc. (Sulyman et al., 2020). A method to reuse the same enzyme multiple number of times can help reduce the cost of enzyme processing. Cellulase obtained from fungal genera of Trichoderma and Aspergillus has already been commercially produced for industrial use (Sulyman et al., 2020).

Industrial application of enzymes is faced with various limitations like low stability, difficult recovery, poor reusability (Ahmed and Khare, 2018), inactivation by organic solvents, sensitivity to high temperature and pH (Andriani et al., 2011), and most importantly high cost of production (Sulyman et al., 2020). These limitations and drawbacks can easily be overcome by immobilizing the enzyme (Borges et al., 2014). The overall objective of enzyme immobilization is enhancement of the stability of enzyme-related processes, and to enable a continuous process through the reuse of enzymes (Sanchez et al., 2014). Immobilization of enzymes onto solid carriers has proven to be an effective solution for stabilization under operational conditions, besides making the process economically feasible (Sojitra et al., 2017). This has attracted the attention of various users towards it and resulted in an increased industrial demand.

The immobilization method and immobilized carriers are the most important factors affecting the biocatalyst properties (Borges et al., 2014). An enzyme carrier should preferably be chemically stable, physically strong, and cost effective (Zhang et al., 2016). Numerous matrices have been used for cellulase immobilization that includes calcium alginate (Plahuta and Raspor, 1996), alginate-chitosan (Sanchez et al., 2014), vermiculite clay (Duman et al., 2020), chitin, chitosan, (Viet et al., 2013), polyacrylamide, agar–agar (de Oliveira et al., 2018) and by forming cellulase aggregates (Khorsidi et al., 2014) etc.

Encapsulation is an immobilization technique which can be defined as the process by which the enzyme is enclosed physically or chemically within semi-permeable membrane (Abd Rahim et al., 2013). The enzymes in encapsulation are adsorbed by weak interactions on solid support via ionic interactions, van der Waals forces and hydrogen bonds (Ingle et al., 2017). Entrapment on the other hand synthesizes a network of polymers around the enzyme (Homaei et al., 2013) which forms a permeable membrane allowing substrate and products to pass through but retaining the enzyme within the network (de Oliveira et al., 2018).

Calcium alginate encapsulation is the most commonly used immobilization process owing to its easy formulation, non-toxicity, biocompatibility, low cost, resistance towards microbial attacks (Abd Rahim et al., 2013), heat stability and ability to form hydrogels at room-temperature (Pandey and Negi, 2020). Alginates forms gels with most di- and multivalent cations but with calcium ions they form thermally stable and biocompatible hydrogel at room temperature making it an ideal immobilizing agent and thus has been of interest in most applications (Mahajan et al., 2010). A mixture of cellulase and sodium-alginate on dropping over the calcium chloride solution, replaces the Na+ ions of Na-alginate by Ca2+ ions of CaCl2 forming spherical Ca-alginate beads (Andriani et al., 2011).

Agar is a complex polysaccharide obtained from the cell wall of red-algae, and an inert, non-toxic, temperature tolerant and biocompatible matrix making it a favorable immobilizing material for application in pharmaceutical, food and biotechnological industries (Sattar et al., 2018). Agar has been used to immobilize or entrap cells in the form of spherical beads, blocks, and membranes (Mahajan et al., 2010). The Gel entrapment method maintains the 3D structure of enzyme and is cost-effective, therefore desirable (Imran et al., 2018).

Carrot juice is believed to possess therapeutic properties boosting our immunity, cleansing our liver by removing fats and bile, reducing blood pressure, reducing risk of heart diseases and fighting anemia and improving eye health etc. (Kaur and Sharma, 2013). It is also a rich source of antioxidants (Purkiewicz et al., 2020). Enzymatic extraction of carrot juice has been reported to increase the yield besides maintaining its nutritional properties (Khandare et al., 2011). Other properties of the extracted juice like sugar content, viscosity, clarity, TSS, color etc. also shows a positive effect on using enzymes for juice extraction (Kaur and Sharma, 2013).

A comparative study between free cellulase, calcium alginate encapsulated cellulase beads and agar entrapped cellulase cubes has not been reported so far. Hence this study will provide a comparison between the two types of immobilization methods and their efficiency. The use of immobilized cellulase in particular and cellulase in general for the extraction of carrot juice and its effect on the juice properties has also been scantily reported. So, this study is likely to provide useful insight into the process of application of immobilized enzymes i.e., cellulase for carrot juice extraction. Therefore, in the present study we immobilized cellulase in calcium alginate beads and agar cubes and both the immobilized forms of enzyme were characterized for different parameters of reaction conditions and stability against free cellulase. The results obtained were compared and used to determine the effects of immobilization on cellulase. Next the immobilized cellulases were tested for their ability to enhance the yield of juice from raw carrot and its effect on other properties of extracted juice.

Materials and methods

Materials

Cellulase (aq. sol.) from Aspergillus sp. used for the experiment was purchased from Sigma Aldrich, Carboxymethyl cellulose (CMC) sodium salt from Himedia, and 3,5, dinitro-salicylic acid (DNS) from SRL. All other chemicals used were of analytical grade supplied by Zenith India Pvt. Ltd. Carrots was purchased from the local market and used for juice extraction.

Cellulase immobilization

The immobilization of cellulase was carried out by adopting a previously reported method as followed by Pandey and Negi (2020) with slight modifications. Sodium alginate in conc. of 1%, 2% and 3% was dissolved in distilled water (DW) over a magnetic stirrer for the preparation of beads. Cellulase (1 mL) was diluted with distilled water (4 mL) and poured into the sodium alginate solution (1:5) and stirred for few more minutes. The alginate-enzyme mixture was rested for 30 min to eliminate air bubbles. After this, the alginate-enzyme mix was slowly sprayed into 0.2 M ice cold calcium chloride (CaCl2) solution and stored at 4 °C for few hours. The calcium alginate beads formed were then washed over a sieve twice with 50 mM sodium citrate buffer (pH 4.8) and then weighed and stored (in sodium citrate buffer) for further uses. A set of blank beads (without enzyme) were also prepared using the above steps and stored in the same way.

Agar cubes were prepared by boiling different concentrations of agar (1%, 2% and 3%) in DW. The enzyme (1 mL) diluted with DW (4 mL) was poured into warm agar solution (1:5), mixed and poured into a petri dish and left undisturbed for at least 1 h. After this, the agar layer was cut with a sharp knife/blade into cubes of around 10 mm size and washed twice over a sieve with 50 mM sodium citrate buffer (pH 4.8) and then weighed and stored for further use. Blank agar cubes (without enzyme) were also prepared and stored in the same way (Mahajan et al., 2010).

Immobilization efficiency

The immobilization efficiency is defined as the amount of protein immobilized within the matrix (Pandey and Negi, 2020). The residual protein in the wash solutions from the encapsulated beads and entrapped cubes including the blanks were estimated using Lowry’s method (Lowry et al., 1951) with few modifications. The residual wash solutions (5 mL) of each matrix were taken and an equal amount of 10% trichloroacetic acid (TCA) was added to it, mixed and kept in freezer for 15 min. The diluted free enzyme (5 mL) was also taken and treated similarly. The cold mixtures were then centrifuged (7000 rpm at 4 °C) for 20 min. The supernatant was discarded, and the precipitate was dissolved in 1 mL of 2 N NaOH solution. And then protein estimation was done.

Enzyme activity assay of cellulase

The enzyme activity of the free and immobilized cellulase was determined by Miller’s method using 3,5-dinitro-salicylic acid (DNS) reagent. The suitably diluted (1 mL cellulase diluted with 4 mL DW) free enzyme (1 mL), encapsulated Ca-alginate beads and entrapped agar cubes of equivalent protein content were taken in separate tubes. Carboxymethyl cellulose (9 mL of 1% CMC) prepared in 50 mM Na-citrate buffer (pH 4.8) used as substrate was added to each tube and incubated at 50 °C for 30 min. The enzymatic reaction was terminated by exposing it to boiling water bath for 5 min. 1 mL of the above solutions were taken and 3 mL dinitro-salicylic acid (DNS) reagent added to it and heated in boiling water bath for 10 min. The solution was allowed to cool and 3 mL DW added to it and used to determine the cellulase activity using glucose as standard (Saha et al., 2018; Pandey and Negi, 2020). One unit of activity is defined as the amount of enzyme producing 1 μmol glucose equivalent per minute under optimum reaction conditions (Sadhukhan et al., 1993).

Characterization of free and immobilized cellulase

Optimal temperature

The suitably diluted free cellulase (1 mL) (as mentioned for enzyme activity), encapsulated bead (1 g) and entrapped agar cube (1 g) were taken in separate tubes. To each tube was added 9 mL of 1% CMC (prepared in 50 mM citrate buffer pH 4.8) and incubated at 20–90 °C at an interval of 10 °C for 30 min. After this, 1 mL solution from each tube was transferred to another tube and the activity of the free cellulase and immobilized cellulase were calculated as described above (Sulyman et al., 2020).

Optimal pH

The change in activity of the free cellulase, encapsulated cellulase beads and entrapped cellulase agar cubes at different reaction pH was also estimated. The different enzymes (1 mL or 1 g) were allowed to react with 9 mL of 1% CMC (prepared in 50 mM citrate buffer) at different pH of 2, 4, 6, 8, 10, and 12 for 30 min at 50 °C. After incubation, the same methodology as for the thermal treatment was followed and enzyme activity of the free cellulase and immobilized cellulase were calculated (Sulyman et al., 2020).

Kinetic parameters

The change in activity of the different cellulase on reaction with different concentrations of CMC was studied by reacting 1 mL or 1 g of enzyme with CMC of different concentrations ranging from 2 to 20 mg/mL prepared in citrate buffer of pH 4.8 at 50 °C for 30 min. After incubation, the same methodology as for the activity determination was followed and enzyme activity of the free cellulase and immobilized cellulase were calculated. The Michaelis Menten constants Km and Vmax were calculated from the Lineweaver–Burk graph plotted from the results obtained (Sulyman et al., 2020).

Reusability

The ability to reuse the cellulase encapsulated beads and cellulase entrapped agar cubes for multiple number of cycles was also studied. The cellulase immobilized beads and agar cubes were used for the digestion of 9 mL of 1% CMC (in 50 mM citrate buffer of pH 4.8) at 50 °C for 30 min. The digested solution (1 mL) was transferred to another test tube and the reaction stopped by boiling. The remaining substrate was discarded while the alginate beads and agar cubes were preserved. The beads and agar cubes were washed with water and used for a 2nd cycle of digestion of CMC. Similar steps were repeated for 5 cycles and the activity estimated from the solution after each cycle (Sojitra et al., 2017).

Cellulase stability study

Temperature

The thermal stability of free cellulase enzyme (FE), encapsulated cellulase beads and entrapped cellulase agar cubes was studied by incubating 1 mL FE, 1 g beads and 1 g agar (without the substrate) in citrate buffer (pH 4.8) at temperatures ranging from 20 to 90 °C for 1 h and then immediately storing them in refrigerator overnight. The residual activity of the enzymes was estimated on next day by the DNS method (de Oliviera et al., 2018) against control samples of the different cellulases without temperature treatment.

pH

The stability against different pH of free cellulase, encapsulated cellulase beads and entrapped cellulase agar cubes was studied by exposing 1 mL FE, 1 g beads and 1 g agar (without the substrate) to citrate buffer of different pH ranging from 2 to 12 for a period of 24 h at room temperature. The residual activity of the different cellulase was estimated on the next day using the DNS method (Andriani et al., 2011) against controls of the different cellulases without pH incubation.

Storage (Time)

The storage stability of free cellulase, encapsulated cellulase beads and entrapped cellulase agar cubes was estimated by storing the cellulases for a period of 12 days under refrigeration in citrate buffer (without substrate) and measuring the residual activity of the enzymes at an interval of every 3 days (Sojitra et al., 2017) taking the activity of different cellulases at 0 day as their 100% activity.

Application for carrot juice extraction

The free cellulase, Ca-alginate encapsulated cellulase beads and agar entrapped cellulase cubes were used for pre-treatment of blended raw carrot to estimate its ability for enhancing the juice extraction. The sample preparation and enzyme treatment for carrot juice extraction was done according to Kaur and Sharma (2013) with modifications. The encapsulated cellulase bead (1 g), along with entrapped cellulase agar cubes and free cellulase of equivalent protein content were each added to 100 g carrot pulp blended in a kitchen grinder and incubated at 40 °C for 60 min. A control with 100 g carrot pulp without enzyme treatment was also analyzed under similar conditions. The control and treated carrot pulps were squeezed by hand and filtered through a muslin cloth to extract the juice and each yield was measured. The extracted juice was exposed to hot water (85 °C) for 5 min to inactivate the enzymes and centrifuged to settle down the suspended particles (Qin et al., 2005). Various physicochemical properties of the extracted carrot juice were then analyzed to determine the effect of enzyme treatment.

Study of the physicochemical properties

Total soluble solid of carrot juice was estimated using a hand-held refractometer, clarity was determined in “MULTISKAN Skyhigh” UV–vis spectrophotometer, Thermo Scientific, USA at a wavelength of 660 nm; titrable acidity was estimated by titration against 0.1 N sodium hydroxide solution and expressed as percent citric acid. Reducing sugar was estimated by DNS method as mentioned above and viscosity was measured using DV-79 Digital Viscometer. The color was estimated using UltraScan VIS Hunter Lab, USA.

Statistical analysis

The least significant difference was determined by Duncan’s multiple range test (DMRT) using IBM SPSS statistics 26 software. The significance level was obtained at p ≤ 0.05.

Results and discussion

Immobilization efficiency

The residual protein content is a measure of the immobilization efficiency of cellulase enzymes. The residual protein in the wash solutions from the different concentrations of sodium alginate beads and agar cubes (Fig. 1) were compared with the protein content of the free enzyme to choose the best immobilization efficiency (Abd Rahim et al., 2013; Zhou et al., 2010). Lower residual protein in the wash solution implies a better immobilization efficiency. The immobilization efficiency was calculated using the formula (Pandey and Negi, 2020):

Immobilizationefficiency=CiVi-CfVfCiVi×100

where Ci = Concentration of protein in free enzyme, Vi = Volume of free enzyme used for immobilization, Cf = Concentration of residual protein in the wash solution, Vf = Volume of wash solution

Fig. 1.

Fig. 1

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Cellulase immobilized in (A) calcium alginate beads and (B) agar matrix

The immobilization efficiency of the different immobilization matrices was: 1% Ca-alginate—72.76%, 1% agar—89.42%, 2% Ca-alginate—80.80%, 2% agar—91.74%, 3% Ca-alginate—92.11%, and 3% agar—97.63%. From the above comparison, the beads of 3% calcium alginate and cubes of 3% agar matrix were found to have the best immobilization efficiency. The lower immobilization efficiency at lower matrix concentration may be a result of larger pore size making them fragile (Andriani et al., 2011). As the concentration of Na-alginate or agar increases, the strength of the immobilizing layer increases due to higher number of bond formation and reduction of pore size. Saha et al. (2018) and Sattar et al. (2018) also observed best immobilization efficiency for 3% alginate and 3% agar respectively. It may be due to acquiring appropriate hardness for optimum immobilization and prevention of leakage. A still higher concentration of immobilizing matrix may restrict the exchange of enzyme and substrate due to excessive viscosity and hence not preferred here.

Cellulase activity

The enzyme activity under the given reaction conditions for free cellulase was found to be 0.211 U (μmol/mL/min) while the equivalent activity of calcium alginate encapsulated cellulase bead was found to be 0.215 U and equivalent activity of agar entrapped cellulase cubes was 0.213 U. The specific activity of free enzyme was calculated as 0.58 U/mg of protein. The specific activity of calcium alginate beads was 0.59 U/mg of immobilized protein and of entrapped agar cubes was 0.58 U/mg immobilized protein respectively. Immobilization yield defined as the ratio of enzyme activity of immobilized enzyme to the enzyme activity of free enzyme (de Oliviera et al., 2018) expressed as percentage (%) provides a measure of the extent of residual activity after immobilization in a matrix.

Immobilizationyield%=ActivityofimmobilizedenzymeActivityoffreeenzyme×100

The immobilization yield of encapsulated Ca-alginate beads was found to be 102.21% and of entrapped agar cubes was found to be 100.98%. This result indicates that both the matrix was able to provide an immobilization yield ≥ 100% which is a desired characteristic (Lei et al., 2008). The high immobilization yield can be reasoned due to attaining appropriate stability by the cellulase within the matrix after immobilization and prevention in loss of activity. Another reason for a ≥ 100% yield may also be due to concentration of more protein within a few beads or cubes.

Effect of temperature on the activity of cellulase

The activity of the free cellulase was found to be maximum at 50 °C while it showed at least 85% relative activity at other temperatures (Fig. 2A). This is an indication that the cellulase enzyme being used is temperature tolerant and not much affected by changes in temperature. The cellulase activity for encapsulated calcium alginate beads was also found to be highest at 50 °C, while for entrapped agar cubes the highest activity was observed at 60 °C. The enhancement of optimum reaction temperature for entrapped agar cubes may be a result of increased rigidity of immobilized cellulase on binding to the agar matrix. For the reaction full contact of substrate molecules and active sites on enzymes was made possible by increased temperature. A similar increase in activity of cellulase immobilized in magnetic nanoparticle composites at a higher temperature had been reported (Han et al., 2018). Andriani et al. (2011) too reported a slight increase in optimal temperature of alginate encapsulated cellulase from 50 to 60 °C in comparison with the free cellulase. While Duman et al. (2020) found that the optimum temperature of immobilized cellulase to be the same as that observed for free cellulase. Another study also reports the optimal temperatures for the immobilized cellulase to be the same, higher, or lower than for the native cellulase (Sanchez et al., 2014). The activity of immobilized cellulase declined to 67% relative activity for encapsulated bead at 90 °C while for entrapped agar cubes it came down to 78% relative activity at the same temperature. The immobilized cellulase exhibited a marginal decline in its activity in comparison to the free cellulase. It may be due to conformational changes in protein structure with increasing temperature (Huang et al., 2020). High temperature may also result in leaching out of the cellulase from the matrix. Moreover, the matrix will hinder the free exchange of substrate for reaction and the resultant products as compared to free enzyme which may result in reduction in activity compared to free cellulase. Free commercial xylanase had showed higher resistance to heat at 75 °C than the immobilized xylanase (Sanchez et al., 2014). In addition, other immobilized enzymes like vermiculite-immobilized alkaline protease, bentonite and sepiolite immobilized catalase also displayed similar character (Duman et al., 2020).

Fig. 2.

Fig. 2

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Effect of (A) temperature and (B) pH on the activity of cellulose. (Where FE = Free cellulase; Bead = Calcium alginate encapsulated cellulase bead; Agar = Agar entrapped cellulase cubes)

Effect of pH on the activity of cellulase

Cellulase is sensitive to the change in pH of its reaction medium. The free cellulase and agar entrapped cellulase showed optimum activity at pH 4, while the calcium alginate encapsulated cellulase beads showed optimum activity at pH 8 (Fig. 2B). The optimal pH remained the same on agar entrapment while it moved to alkaline region on encapsulation in Ca-alginate. Cellulase might have developed some kind of interaction with the alginate matrix that provides it stability in the alkaline environment and a shift in its optimum reaction condition. Various previous works on immobilization have reported such shift of optimum pH to alkaline range. Duman et al. (2020) has stated the shift in optimal pH of enzymatic reaction towards a more alkaline pH caused by the displacement of the H+ ion of the microenvironment around the immobilized enzyme. The same reason had been proposed by Han et al. (2018) for a shift in optimum pH of magnetic nanoparticle composite immobilized cellulase. Cellulase immobilized on vermiculite showed optimum pH of 7 against an optimum pH of 5 for free cellulase at 50℃ (Duman et al., 2020). Khorsidi et al. (2014) also observed immobilized cellulase to be more active in alkaline conditions than the free enzyme. The process of immobilization usually resulted in changes in optimum pH. It is believed that this phenomenon is related to the charged surfaces of the beads and enzymes, which could alter the pH of the entrapped enzyme and affect its nature (de Oliveira et al., 2018). The cationic and anionic characteristics of the surfaces of the beads that localize the enzymes induces microenvironment charges, which subsequently changes the nature of the active enzyme (Abd Rahim et al., 2013). As stated in previous literatures the interaction of calcium alginate and cellulase on immobilization must have altered or modified the active site of cellulase to remain stable at alkaline conditions which results in shifting of the optimum pH of cellulase to a higher level. The immobilized cellulase retained ≥ 70% relative activity at all pH under study, except for the extreme limits of pH 2 and pH 12 where activity dropped. The free cellulase lost activity as it moved further from its optimum pH retaining just 27% relative activity at pH 12 while encapsulated cellulase bead retained 41% relative activity and agar entrapped cellulase retained 66% relative activity at the same pH. The free enzyme’s structure must have been affected by a more acidic or alkaline pH while the immobilization matrix protected the cellulase against a sudden pH drop in the environment which helped it retain its activity for a wider pH range. Therefore, both the immobilization methods served the purpose of tolerance to change in pH in relation to the free enzyme.

Effect of substrate concentration on cellulase activity

With the increase in substrate concentration, the activity of free cellulase, Ca-alginate encapsulated cellulase beads and agar entrapped cellulase cubes also increased. This data was used to determine the enzyme kinetics of the different forms of cellulase. The free cellulase had a Km value of 39.30 mg/mL (Fig. 3A), 72.29 mg/mL for the encapsulated beads (Fig. 3b), and 13.08 mg/mL for the entrapped agar cubes (Fig. 3C). The Km value of encapsulated cellulase beads was higher than the free cellulase depicting a lower affinity of the encapsulated cellulase for the substrate (Huang et al., 2020). Larger Km for immobilized cellulase was also reported for magnetic nanoparticle composite cellulase (Han et al., 2018). The work reported by Sanchez et al. (2014) has also found the Km for immobilized cellulase to be higher as compared to the native form. The interaction between substrate and alginate encapsulated cellulase might be affected by the spherical shape of the beads which requires more substrate for the reaction to take place and thus a higher Km. On the other hand, the agar entrapped cellulase showed a lower Km value than the free cellulase depicting it to have a higher affinity for the substrate. The flat surface of agar cubes may play a role here to easily attach the substrate with the entrapped cellulase and hence more strong interaction. Cellulase immobilized in vermiculite too showed a lower Km value than the free cellulase. This might be caused by a reduced steric barrier, altered enzyme diffusion, or altered enzyme flexibility. It indicates the active site of the immobilized cellulase probably bound the substrate more strongly than free cellulase in the transition state (Duman et al., 2020). It had been stated in a review of various enzymes that compared with free enzymes most immobilized enzymes commonly showed lower activity and in general higher Michaelis constant (Km) because of the relative difficulty in accessing the substrate (Homaei et al., 2013).

Fig. 3.

Fig. 3

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Lineweaver–Burk plots of (A) free cellulase (B) Ca-alginate encapsulated cellulase and (C) Agar entrapped cellulase

The Vmax of the free cellulase was 0.50 μmol/min, 1.32 μmol/min for the encapsulated cellulase beads, and 0.38 μmol/min for the entrapped agar cubes. In our research alginate encapsulated cellulase beads showed higher Vmax than free cellulase. High Vmax could be due to the result of more exposed active sites caused by positive changes in immobilized enzymes. Here again the spherical shape of beads may be a reason as the substrate has to cover a shorter distance for the reaction with the encapsulated enzyme. Duman et al. (2020) also observed a higher Vmax value for vermiculite immobilized cellulase than the free cellulase. Increased Vmax was an indication of increased stability of the cellulase after immobilization (Andriani et al., 2011) indicating a faster reaction rate and better catalytic yield (Duman et al., 2020). On the other hand, agar entrapped cellulase showed lower Vmax than free form. Lowering of Vmax for entrapped cellulase compared to free cellulase on immobilization in agar was also reported in the work of Sattar et al. (2018). This may be a result of restricted diffusion of substrate through the matrix to the enzyme active sites, thus lowering the reaction rate (Alhassan et al., 2021). As stated above the flat surface of agar cubes might help in strong attachment of enzyme–substrate complex and hence a lesser reaction rate. A lower Vmax for immobilized cellulase in nanohybrid was found in a study which have been reasoned due to formation of a concentration gradient inside the matrix resulting in slow hydrolysis rate (Saha et al., 2018).

An increase in both Km and Vmax values for immobilized enzymes have been reported for commercial enzymes like pectinase and polygalacturonase etc. (Andriani et al., 2011). A reduction in both Km and Vmax for immobilized enzymes as observed for agar entrapment was also reported by Alhassan et al. (2021) for alginate encapsulated cellulase. Both these effects, as stated above, probably are linked with the shape of immobilization matrix. The change in kinetic parameters was attributed to a change in the substrate accessibility to the active sites of enzymes resulting from diffusional limitations, steric hindrances, or structural changes in enzymes due to immobilization (Sanchez et al., 2014). Bead size, pore size, and enzyme loading per bead were also probable reasons for limiting the diffusion of the substrate into the enzyme (Andriani et al., 2011). Km and Vmax are different for different organisms due to genetic variations among micro-organisms (Sulyman et al., 2020).

Catalytic efficiency (Vmax/Km) of an enzyme measures the catalytic performance of an enzyme i.e., the amount of enzyme required to catalyze a reaction (Duman et al., 2020). Higher catalytic efficiency is a desired characteristic for enzymes. The present research found the catalytic efficiency (Vmax/Km) of immobilized cellulase to be more than the free enzyme and the values were 2.30/min for free enzyme, 3.28/min for beads, and 5.28/min for agar. Similar results have been reported by Ahmad and Khare (2018) where catalytic efficiency of bio nanoconjugate was found to be higher than free cellulase.

Reusability study

The immobilized cellulase was tested for its residual activity after repeated use. The activity of the encapsulated cellulase beads and entrapped cellulase agar cubes in the first cycle was considered as being 100% activity. The encapsulated cellulase beads showed approximately 73% activity after the second cycle while entrapped cellulase agar cubes had 71% residual activity after the second cycle of use (Fig. 4). In the third cycle of use, encapsulated beads retained nearly 53% activity while agar cubes showed 60% residual activity. After the fourth cycle encapsulated beads had 38% residual activity and entrapped agar cubes retained 44% activity with respect to the initial activity. After the fifth cycle of reuse encapsulated beads retained just 18% activity while entrapped agar retained 29% residual activity. The rapid decline in immobilized enzyme activity might be due to inactivation and leakage of cellulase caused by repeated washing or conformational change in enzyme structure (Huang et al., 2020; de Oliveira et al., 2018). It was seen that there was a sharp loss between the first and second cycles and then gradually the gap of loss in activity decreased. This may be due to a sudden heat shock initially within a short time, leading to loosening up of matrix pores, but in the next cycles the matrix was able to cope with the shock and the amount of loss decreased. Another reason might be due to protein denaturation resulting from the separation, washing, and filtration steps. The loss in activity might also be due to the repeated encounter of the substrate with the immobilized cellulase active site causing changes in the conformation of the active site (Duman et al., 2020). β-glucosidase immobilized on polyacrylic resin and glyoxyl-agarose had been reported to remain stable till the third cycle of reuse and then lose its activity to around 25% after five cycles (Borges et al., 2014). In another study cellulase immobilized in calcium alginate was also found to lose its activity below 10% after the fourth cycle. A probable reason may be the binding strength of the matrix and cellulase gets weakened by the recycling of the immobilized cellulase causing a loss in activity (Andriani et al., 2011). The incubation temperature to which the matrices were treated might also play a role in loosening the binding between matrix molecules.

Fig. 4.

Fig. 4

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Reusability of immobilized cellulase (Where Bead = Calcium alginate encapsulated cellulase bead; Agar = Agar entrapped cellulase cubes)

Thermal stability of cellulase

The stability study for free and immobilized cellulase treated at different temperatures, found free cellulase to retain its stability better relative to the immobilized forms. Free cellulase retained greater than 80% of its initial activity at all temperatures (Fig. 5). While in the case of encapsulated cellulase beads, exposure to the different temperatures gradually brought down the residual activity to 70% at 90 °C while entrapped cellulase agar cubes retained 58% activity at 90 °C after incubation for 1 h. This can be thought of as an effect of increasing temperature on the immobilizing matrix which probably widens up the pores size leading to flow out of enzymes. Similar results have been reported for cellulase immobilized on amino silica where the thermal stability of cellulase decreased on immobilization (Kitaoke et al., 1989). β-glucosidase immobilized on polyacrylic resin activated by carboxyl groups showed three times lower thermal stability than free commercial enzyme (Borges et al., 2014). Andriani et al. (2011) also reported a decrease in the stability of immobilized cellulase as compared to the free form upon increase in temperature. A mixture of suitable hardening agents with the matrix may serve the purpose better.

Fig. 5.

Fig. 5

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Stability of free and immobilized cellulase at different (A) Temperature, (B) pH, (C) storage period. (where FE = Free cellulase; Bead = Calcium alginate encapsulated cellulase bead; Agar = Agar entrapped cellulase cubes)

pH stability of cellulase

The pH stability of free cellulase too was found to be better compared to its immobilized counterparts. Free cellulase remained stable on incubation for 24 h at different pH with a relative activity above 90% throughout (Fig. 5). The calcium alginate encapsulated beads gradually lost their activity and above pH 8 a sharp decrease in relative activity was observed decreasing to 53% at pH 12. The Ca-alginate matrix, although stable at pH 8 for a short time, could not withstand pH higher than pH 8 for an elongated time which might have caused it to dissociate leading to a drastic loss in activity. On the other hand, the agar entrapped cubes were relatively stable with the residual activity decreasing gradually to 83% at pH 12. Agar must be more resistant to pH change than alginate thus could withstand alkaline pH for 24 h. Saha et al. (2018) also observed a sharp decline in immobilized cellulase nanoparticles activity at alkaline pH. Li et al. (2019) also reported a decrease in pH stability of immobilized cellulase after attaining an optimum point. A probable reason for reduced stability might be reduced matrix strength at higher pH with increasing time. However, Kitaoke et al. (1989) reported that immobilized cellulase adsorbed on amino-silica and free cellulase showed almost similar pH stability.

Storage stability

The residual activity of free cellulase, calcium alginate encapsulated cellulase beads and agar entrapped cellulase cubes gradually decreased with increasing storage time (Fig. 5). The free cellulase retained 89% of initial activity after 12 days of storage in refrigerated conditions at pH 4.8. The stability of cellulase encapsulated alginate beads decreased to approximately 60% after storage for 12 days. On the other hand, agar entrapped cellulase cubes retained about 85% residual activity after 12 days of storage under the same conditions. Thus, agar entrapped cellulase and free cellulase showed almost similar stabilities on storage under refrigerated condition while calcium alginate encapsulated cellulase lost a significant amount of activity. Calcium alginate matrix was active for 6 days after which the matrix strength might have deteriorated due to the constant exchange of storage buffer through it. Another probability might be loss of calcium ions from the Ca-alginate beads making it unstable. Therefore, on long storage Ca-alginate beads lost significant activity. Agar was stable in the storage buffer which prevented it from losing much activity. Calcium alginate immobilized cellulase has been found to lose 30% activity after 2 days, 45% after 6 days and 90% after 12 days when stored at 4 °C (Andriani et al., 2011). de Oliviera et al. (2018) also reports a loss of 32.3% activity for immobilized cellulase after 12 days storage at 4 °C. Another reason for the decreasing stability of cellulase may be that the active sites of enzymes deteriorate when stored in solution for longer time periods (Sattar et al., 2018).

The degree of stabilization of an enzyme depends on the micro-organism from which it originates, as well as the type of anchorage of the enzyme to the activated support, temperature, and inactivation pH, amongst other factors (Borges et al., 2014).

Application for carrot juice extraction

Juice yield

The yield of juice from carrot was found to increase on treating it with cellulase. Free cellulase treated carrot pulp was found to yield 5.64% more juice than the control, while Ca-alginate encapsulated bead treated pulp increased juice yield by 6.78% against a 9.03% increased yield by agar entrapped cellulase cubes. There was a significant (p ≤ 0.05) increase in juice yield after incubation with free and immobilized cellulase compared to the control sample (Table 1). As can be seen from Table 1, although there was a significant (p ≤ 0.05) difference in yield of juice from control and all the cellulase treated carrot pulp the difference in yield after treatment with various forms of cellulase was not significant. Free cellulase yielded 62.33 mL juice while Ca-alginate encapsulated bead yielded 63.00 mL juice and agar entrapped cellulase yielded 64.33 mL juice against a yield of 59.00 mL from the control sample. This implies that the free cellulase, Ca-alginate encapsulated cellulase and agar entrapped cellulase had similar activity towards juice extraction from carrot pulp. This might be due to diffusional limitations of the enzyme samples to the whole carrot pulp as it got absorbed at the point of application as continuous agitation was not provided although the pulp was stirred periodically. The increase in juice yield was due to the breakdown of cellulose, constituting the cell walls of carrot which results in extraction of water content of the cells. Reports of enzymatic hydrolysis of cell wall resulting in increased juice yield from various fruits and vegetables are available (Sharma et al., 2017; Kaur and Sharma, 2013).

Table 1.

Physicochemical parameters of carrot juice extracted using different immobilized cellulase

Sample Yield (%) TSS (° Brix) Clarity (OD) Titrable acidity (g/100 g) citric acid Reducing sugar (mg/g) Viscosity (mPa.s) Colour
L a b
Control 59.00 ± 1.00a 5.93 ± 0.06a 0.38 ± 0.05b 0.08 ± 0.003a 11.96 ± 1.73a 1.14 ± 0.06c 27.16 ± 0.01a − 0.46 ± 0.02a 2.89 ± 0.14a
Free cellulase treated 62.33 ± 0.57b 6.03 ± 0.06b 0.09 ± 0.004a 0.10 ± 0.003b 14.72 ± 0.15b 1.11 ± 0.17ab 28.23 ± 0.45b − 0.29 ± 0.01a 4.44 ± 0.47b
Ca-alginate encapsulated cellulase bead treated 63.00 ± 2.00b 6.10 ± 0.00b 0.099 ± 0.01a 0.10 ± 0.003b 13.85 ± 0.13b 1.12 ± 0.11bc 28.00 ± 0.16b − 0.17 ± 0.02a 4.52 ± 0.16b
Agar entrapped cellulase cube treated 64.33 ± 1.15b 6.03 ± 0.06b 0.12 ± 0.007a 0.10 ± 0.003b 14.08 ± 0.48b 1.09 ± 0.15a 27.80 ± 0.13b − 0.09 ± 0.02a 4.46 ± 0.15b
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*Different alphabets depict significant difference in values at p ≤ 0.05 level of significance

Characterization of extracted juice

The TSS of the carrot juice extracted by different forms of cellulase was found to significantly (p ≤ 0.05) increase, as compared to the control sample (Table 1). Control carrot juice exhibited a TSS of 5.93°Brix against free cellulase exhibiting a TSS of 6.03°Brix, Ca-alginate encapsulated cellulase bead a TSS of 6.10°Brix and agar entrapped cellulase cubes exhibiting a TSS of 6.03°Brix respectively. Increased TSS may be a result of the broken cell components after hydrolysis that gets mixed up to the solid already present in carrot juice increasing the TSS. The absorbance of cellulase treated carrot juice decreased significantly (p ≤ 0.05) as compared with the control sample. Free cellulase and Ca-alginate encapsulated cellulase beads had an OD of 0.09 and agar entrapped cellulase cubes had an OD of 0.12 while the control had an OD of 0.38. This implies that the free cellulase and both the immobilized cellulase extracted juice exhibited better clarity than the control juice. The haze or cellulolytic fibers present in juice gets hydrolyzed by cellulase resulting in a clear solution. The acidity of cellulase extracted juice was also found to increase compared to the control. All forms of cellulase extracted carrot juice had an acidity expressed in terms of citric acid of 0.10 g/100 g against 0.08 g/100 g for the control. On statistical analysis this value was found to be significantly (p ≤ 0.05) different. Increased acidity for treated juice may be a result of an increase in organic acids produced during hydrolysis of cell wall of carrot. This also causes a decrease in pH of the clarified juice. The reducing sugar produced on juice extraction after cellulase treatment was found to be significantly (p ≤ 0.05) higher for both free cellulase and the immobilized forms. Free cellulase had a reducing sugar content of 14.73 mg/g, Ca-alginate encapsulated cellulase beads a reducing sugar content of 13.85 mg/g, and agar entrapped cellulase cubes a reducing sugar content of 14.08 mg/g while control juice had a reducing sugar content of only 11.96 mg/g. This may be a result of conversion of cellulose to glucose during the enzyme treatment. The viscosity of cellulase extracted juice and control did not differ much, however the difference was found to be statistically significant (p ≤ 0.05). The viscosity decreased for the treated carrot juice with respect to the control. Control sample exhibited a viscosity of 1.14 mPa.s which decreased in free cellulase treated juice to 1.11 mPa.s in Ca-alginate encapsulated cellulase bead treated juice to 1.12 mPa.s and in agar entrapped cellulase cube treated juice to 1.09 mPa.s. The decrease in viscosity can be a result of the breakdown of the cell constituents after cellulase treatment leading to free flow of juice. An increase in TSS, clarity, titrable acidity, reducing sugar and reduction in viscosity on pre-treatment with cellulase and other enzymes have been also reported by several other researchers (Sharma et al., 2017; Kaur and Sharma, 2013). The main reason put forth for higher TSS, clarity, titrable acidity, reducing sugar and lower viscosity of cellulase treated carrot samples than the control sample is the breakdown of the cell walls of carrot by the enzyme (Kaur and Sharma, 2013).

Color measurements

The color of the different extracted carrot juice was expressed in terms of L, a, and b values and from the values obtained the immobilized cellulase treated carrot juice was found to have increased “L”, “a” and “b” value (Table 1). The difference in color of the control and cellulase treated juice was found to have significant (p ≤ 0.05) difference. Increased “L” value depicts more brightness which is a result of clarity of treated juice and increase in “a” depicts tendency to move towards red hence extraction of carotene pigments for cellulase treated juice. On the other hand, increase in “b” depicts a move towards yellow color which may be related to clarity. An increase in “L” is a positive sign as brightness is a better color for clear juice. Enzymatic extraction was reported to improve the color of carrot juice. The heat applied to carrot juice for enzyme inactivation also affected the color change. Studies have found increased “L” and “a” value for enzymatically treated carrot juice at initial stages (Kaur and Sharma, 2013).

From the above observations it was found that calcium alginate and agar provided a decent efficiency in terms of enzyme loading and are efficient matrices for immobilization. Calcium alginate encapsulated cellulase had optimum activity at the same temperature as free cellulase, i.e., 50 °C, while agar entrapped cellulase had optimum activity at higher temperature of 60 °C that makes agar entrapped cellulase better suited to industries requiring temperature resistant catalysts. On the other hand, alginate encapsulated cellulase had optimum pH 8 against agar entrapped and free cellulase’s optimum pH 4 making Ca-alginate encapsulated cellulase suitable for industries like the textile industry (Alhassan et al., 2021) working in alkaline conditions. Also, Ca-alginate has high Km and high Vmax meaning more substrates can be converted into product in a shorter time at faster rate. While agar entrapped cellulase has lower Km and lower Vmax indicating more binding with substrate and thus lesser reaction rate than the free cellulase that can be used for unstable compounds. In terms of reusability both the immobilized cellulase could be used multiple times though with loss of considerable activity in each step which needs to be studied further. Thermal stability of Ca-alginate encapsulated beads was higher than the entrapped agar cubes and can be used at its optimum temperature with 80% activity against entrapped agar having a 70% activity at the optimum temperature. Similarly, Ca-alginate beads had 83% activity at its optimum pH and entrapped agar cubes 98% activity at optimum pH conditions. In terms of storage stability agar entrapped cellulase was similar to free cellulase in residual activity and had above 85% activity after 12 days but encapsulated cellulase beads gradually lose activity and can be used for 6–7 days with about 70% activity. These characteristics make both the immobilized forms of cellulase suitable for repeated industrial use. Both the immobilized cellulase were found to increase juice yield from carrot when used for pre-treatment and positively affect the physicochemical properties of juice. Thus, after immobilization, cellulase retained the catalytic activity and properties similar to the free form and can be suitably used for hydrolysis. Further improvements in the characteristics of immobilization of cellulase to get better stability, product yield, longer storage, reusability, etc. besides reducing the process cost need to be studied and developed.

Author contributions

BJK: Methodology, validation, Formal analysis, investigation, Writing-original draft; NS: Conceptualization, validation, writing-review and editing, visualization, supervision, project administration.

Funding

No external funding was received to carry out the present work.

Data availability

The authors declare that data will be made available on request.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

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