Introduction

Cellulosic material is an abundant renewable source that can serve as a substrate for production of chemicals and fuel ethanol by chemical or enzymatic conversion. Utilization of cellulose as a nutrient source requires the enzyme cellulase, which cleaves β-1,4-glycosidic bonds in the polymer for release of glucose units [1, 2].

The main problems associated with use of cellulase on an industrial scale include the difficulty of their separation from the solution and their inactivation by organic solvents and extreme pH or temperature. Thus, immobilization can be considered as the way out of these problems. For many industrial applications, enzymes and cells can be immobilized, via very simple and cost-effective protocols, in order to be re-used over very long periods of time [3]. The use of an immobilized enzyme permits its recovery and reuse, resulting in an economically feasible process [3–5].

Cellulase has been immobilized by several physical and chemical methods, including cross-linking [6], copolymerization [7], fiber ultra-filtration [8, 9], aqueous two-phase systems [10], and by use of an enzyme carrier, such as non-porous ultrafine silica particles [11]. Enzymes can be immobilized using a variety of natural and synthetic supports. The choice of support and/or technique is dependent on the nature of the enzyme, its substrate, and its application.

Entrapment of enzyme in calcium alginate is an important method for immobilization. Alginates are commercially available as water-soluble sodium alginates. Entrapment within calcium alginate is recognized as a rapid, nontoxic, inexpensive, and versatile method for immobilization of enzyme as well as cells [12]. Alginate is a natural polysaccharide derived from marine plants and its basic structure consists of linear unbranched polymers containing β-(1, 4)-linked d-mannuronic acid (M) and α-(1, 4)-linked l-guluronic acid (G) residues. It can form thermally stable and biocompatible hydrogel in the presence of calcium cations [13, 14]. The use of alginate as an immobilizing agent in most applications rests in its ability to form heat-stable strong gels which can develop and set at room temperatures. Alginate forms gels with most di- and multivalent cations. Monovalent cations and Mg2+ ions do not induce gelation while ions like Ba2+ and Sr2+ will produce stronger alginate gels than Ca2+. However, it is the alginate gel formation with calcium ions which has been of interest in most applications [15].

The present research focused on immobilization of partial purified cellulase from a new strain of Bacillus subtilis, TD6, isolated from Takifugu rubripes fish using calcium alginate as a support material. Various parameters, including pH maximum, temperature maximum, and concentration of sodium alginate and calcium chloride solution were observed. As the purpose of immobilization, repeated use of the immobilized cellulase was also studied with storage stability.

Materials and methods

Microorganism

Bacillus subtilis TD6, obtained from Takifugu rubripes gut, was used in this study. It was maintained on CMC medium composed of (g l−1): Carboxymethyl cellulose, 10; K2HPO4, 4; Na2HPO4.2H2O, 4; MgSO4.7H2O, 0.2; CaCl2, 0.001; FeSO4.7H2O, 0.004; Tryptone, 2 at pH 6.0.

Preparation of crude enzyme

About 100 ml of culture medium was dispensed into 300 ml conical flasks and sterilized at 121 °C for 15 min. The flask was then inoculated with Bacillus subtilis TD6 and incubated at 45 °C 100 rpm for 3 days. The cultures were centrifuged 10,000×g for 20 min. Following collection of the culture filtrate, the enzyme solution was tested based on the DNS method.

Enzyme assays

CMCase activity assay was determined for estimation of endoglucanase activity, as given below [16]. Through the determination of the amount of reducing sugars liberated from CMC solubilized in 50 mM sodium citrate buffer, pH 4.8. This mixture was incubated for 30 min at 50 °C and the reaction was stopped by the addition of DNS solution. The treated samples were boiled for 5 min, cooled in water for color stabilization, and the optical density was measured at 540 nm. The cellulase activity was determined by using a calibration curve for glucose. One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of glucose per minute.

Determination of protein content

The SMARTTM BCA Protein Assay Kit for standard assay was used for protein determination. The purple-colored reaction product of this assay is formed by chelation of two molecules of BCA with one cuprous ion. This water soluble complex exhibits a strong absorbance at 562 nm.

Ammonium sulfate precipitation

The enzyme solution was partially purified using ammonium sulfate precipitation up to 80% saturation. The precipitate fraction of enzyme was then re-dissolved in a 0.05 M sodium phosphate buffer and dialyzed against the same buffer.

Immobilization of cellulase

The partially purified enzyme solution was mixed with sodium alginate solution in a 1:1 ratio. The cellulose–alginate mixture was added drop-wise into calcium chloride solution with continuous shaking at 4 °C. The beads thus formed were washed 3–4 times with deionized water, and, finally, with sodium phosphate buffer 5 mM pH 6.0.

Optimization of working parameters

Concentration of sodium alginate was 0.5–3% and calcium chloride concentration was 0.1–0.3 M. The pH range 5, 6, 7, and 8 was chosen to observe the optimum pH for immobilized CMCase. Temperatures selected for the immobilization were 30, 40, 50, 60, and 70 °C.

For thermal and pH stability, the immobilized enzyme was incubated at various temperatures (30, 40, 50, 60, and 70 °C) and pH (5, 6, 7, and 8) for 6-h. The residual activity was determined based on DNS method [16].

Results and discussion

CMCase was precipitated at 40 to 80% ammonium sulfate saturation, with the highest specific activity of 1.48 U/mg obtained at 60% saturation (Fig. 1).

Fig. 1
figure 1

Ammonium sulphate precipitation profile of CMCase from B. subtilis TD6

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Effect of various concentrations of sodium alginate and calcium chloride

Various concentrations of sodium alginate were used for acquisition of beads with greater stability. As drops of cellulose–alginate mixed with CaCl2 solution increased, Na+ ions of sodium alginate were replaced by Ca2+ ions of the CaCl2 solution, which finally resulted in formation of Ca-alginate beads [17].

The enzyme specific activity after immobilization was found to be maximum at 2% (w/v) sodium alginate concentration, which is 61% of free enzyme specific activity (Fig. 2). Maximum leakage of enzyme from beads was observed to occur at 0.5% (w/v) sodium alginate. This occurrence might be the result of the larger pore sizes that fragile the Ca–alginate beads. At 3% (w/v) sodium alginate concentration, the activity of the enzyme was found to be low, which might be due to the decrease of pore size, resulting in hindered penetration of the substrate to the beads.

Fig. 2
figure 2

CMCase activity of immobilized enzyme based on free enzyme (control) at various concentrations of sodium alginate

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The concentration of calcium chloride for formation of the capsule of the enzyme also varied in order to acquire stable beads capable of maximum specific activity. In this work, 2% (w/v) of sodium alginate was used. CaCl2 at a concentration of 0.15 M was found to retain the highest activity of immobilized CMCase (Fig. 3). At this concentration, the activity of immobilized CMCase was found to be 60% of free CMCase (control). Other research using different enzymes also reported a decrease in activity of alkaline protease when the concentration of CaCl2 is increased [18]. They observed that the pH of CaCl2 solution changed with its concentration, which might affect the activity of the immobilized enzyme.

Fig. 3
figure 3

CMCase activity of immobilized enzyme based on free enzyme (control) at various concentrations of calcium chloride

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Effect of temperature and pH on immobilized CMCase

The immobilized enzyme was assayed at various temperatures and pHs. Optimum temperature of immobilized CMCase was found to be 60 °C, higher than that of free CMCase (50 °C), which indicated that the support material retained the tertiary structure of the enzyme at higher temperature (Fig. 4). Although the maximum temperature was 60 °C, the immobilized enzyme was considered more stable at 50 °C, at which 89% of its activity was retained after a 6-h incubation period. On either side of this point, lowered stability was displayed by the immobilized CMCase, nontheless sufficient activity was present at 70 °C during a 6-h incubation period (Fig. 5).

Fig. 4
figure 4

Effect of various temperatures (°C) on CMCase activity of immobilized enzyme based on free enzyme

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Fig. 5
figure 5

Thermostability of immobilized CMCase produced by B. subtilis TD6

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The effect of various pH on the immobilized enzyme activity was reported in Fig. 6. The optimum pH for immobilized CMCase was known to be 6.0, respectively. The surface of the beads has been reported to have a cationic and anionic nature. The charged surface of beads and enzyme produces a charged microenvironment, which might affect the nature of active enzyme and alter the pH of entrapped enzyme [19]. Nonetheless, in this study, the surface of beads had no effect on the active enzyme; therefore, the optimum pH of free and immobilized enzyme remained the same.

Fig. 6
figure 6

Effect of various pHs on CMCase activity of immobilized enzyme on the basis of free enzyme

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The pH stability of immobilized CMCase after a 6-h incubation period at 25 °C indicated that the enzyme retained approximately 90% of its specific activity after incubation at a pH value of 6.0 (Fig. 7). Although maximum stability of the immobilized enzyme was found at pH 6.0, the enzyme was found to be throughly stable at pH 5.0–7.0, where it retained more than 80% of the maximum activity for a 6-h incubation period.

Fig. 7
figure 7

pH stability of immobilized CMCase produced by B. subtilis TD6

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Kinetic constant of immobilized enzyme

The K m and V max values of immobilized CMCase calculated from a Lineweaver–Burk plot were 2.9 mg/ml and 32.1 μmol/min/mL, respectively. These values were higher compare to free CMCase (2.4 mg/mL and 29.94 μmol/min/mL). The increase in K m of entrapped enzyme might be due to inaccessibility of the substrate to the enzyme due to limitation in diffusion of substrate into enzyme beads, which is affected by bead size, pore size, and enzyme loading per bead. The increase in the V max, compared with that of the native enzyme, may be attributed to increased stability of the enzyme after immobilization.

Another research study using a different enzyme also reported an increase in both K m and V max values of commercial immobilized pectinase [20]. Buga et al. [21] also reported the same finding using immobilized polygalacturonase. These findings are consistent with the findings of this study.

Reusability of immobilized enzyme

The activity immobilized CMCase was evaluated using a repeated batch process in order to observe the reuse of the immobilized enzyme (Fig. 8). After the fourth cycle, the enzyme showed 80 and 45% for the second and third cycles, respectively. After the fourth cycle, the remaining activity was less than 10%. This decrease might due to leakage of enzyme from beads along with the process and washing steps at the end of each cycle.

Fig. 8
figure 8

Reusability of immobilized CMCase

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Storage stability of immobilized CMCase

The immobilized CMCase was stored at 30 and 4 °C for 15 days for determination of the stability of immobilized enzyme. The enzyme was more stable at a temperature of 4 °C, which showed 30% loss of activity after 48 h, 45% after 6 days, and 90% after 12 days. Significant loss in enzyme activity of immobilized enzyme was observed at 30 °C, which showed 85% loss of activity after the second day and no activity on the third day.

Conclusion

The enzyme has been partially purified using ammonium sulphate precipitation and immobilized using sodium alginate. The immobilized CMCase showed optimum activity at pH 6.0 and 60 °C, respectively. It has been found to be stable at pH 5.0–7.0 and at 40–60 °C. This work has demonstrated that immobilized CMCase can be used through at least three catalytic cycles.