Immobilization of Dextranase Using Anionic Natural Polymer Alginate as a Matrix for the Degradation of a Long-Chain Biopolymer ( Dextran )

Alginate is an inexpensive, nontoxic, valuable biopolymer utilized in the study for the immobilization of commercially applicable biocatalyst dextranase. Dextranase was immobilized by an entrapment method, and alginate hydrogel spheres were synthesized after optimizing several parameters. A sodium alginate concentration of 4.0% was noticed to be suitable along with a calcium chloride concentration of 0.2 molar after providing a curing time of 20 minutes. After comparing the characteristics of the entrapped enzyme with those of the soluble one, it was observed that the characteristics were more or less the same except for the change in reaction time which was noticed to be prolonged in the case of entrapped dextranase while the change in temperature and pH optima was not observed. The variation in Vmax and Km values of dextranase after entrapment was also noted. However, after extensive stability examination studies, it was found that dextranase became more stable after entrapment; as a result, it retained more than 50% of its original activity at elevated temperature even after exposure for about 2.0 hours. The reusability of dextranase was up to 7.0 cycles after performing catalytic activity under constant condition.


Introduction
In recent years, nonsynthetic polymers have dominated over the synthetic polymers and gained pronounced commercial application in many fields because of their intrinsic properties [1].Among several polymers, polysaccharides obtained from plants, microbes, and seaweeds are of utmost importance [2].Alginate is a natural heteropolysaccharide composed of α-L-guluronic and β-D-mannuronic acids.The main sources of this polymer are brown algae and microbes.The presence of high amount of guluronic acid results in the formation of more resistant gel [3,4].Alginate has many commercial applications; however, its special features attract the attention of researchers due to its use in biomaterial sciences.It is utilized in the preparation of nanoparticles for proficient release of substances.The polymer is also used for the entrapment of a biocatalyst, resulting in successful recycling of the enzyme [5,6].In the current study, it is used as a matrix for the entrapment of the industrially important biocatalyst dextranase.
Dextranase is a biocatalyst belonging to the hydrolase family and possesses vast commercial applications in food and medicine as well as in biotechnological processes [7][8][9][10][11][12].Although highly active forms of dextranase have been isolated from several different microbial sources, still some hurdles are associated with the applicability of the enzyme at a large scale.In order to minimize these difficulties, a selective immobilization method using a suitable matrix is adapted to stabilize the enzyme under a harsh industrial environment.Immobilization not only enhanced the functionality of the enzyme but also reduced the cost by providing ease for separating the enzyme from the reaction mixture and utilizing it for the next batch process [13].Among various methods of immobilization, entrapment is considered the most effective for encapsulating an enzyme in alginate in the form of hydrogel spheres.It is a simple, inexpensive procedure that does not alter the structural configuration of the enzyme [14].
Owing to such vast application of alginate and dextranase, the current study is designed to synthesize calcium alginate spheres by immobilizing dextranase.A number of entrapment parameters were optimized to synthesize the stable sphere system.Along with these, the characteristics of dextranase after entrapment in alginate spheres were examined and compared with the characteristics of the soluble unentrapped enzyme.

Materials and Methods
2.1.Producer of Dextranase.Among three thermophilic strains isolated from hydrothermal spring, Bacillus megaterium KIBGE-IB31 (GenBank: KF241867) was selected as a producer based on screening of bacterial strains for the production of dextranase [15].

Enzyme Preparation.
The optimized medium for the production of dextranase is composed of dextran (15.0 g L -1 ), yeast extract (1.0 g L -1 ), tryptone (10.0 g L -1 ), sodium chloride (3.0 g L -1 ), magnesium sulphate (0.2 g L -1 ), and potassium dihydrogen phosphate (1.0 g L -1 ).Inoculum (10%) of bacterial culture was transferred into sterile medium, and after 24 hours of fermentation at 60 °C, cells were separated from fermented broth by centrifugation at 40,000×g for 15 minutes at 4 °C.The enzyme was precipitated from a cell-free filtrate (CFF) by ammonium sulphate salt saturation (40%).Precipitates of dextranase were collected after overnight incubation at 4 °C, solubilized in sodium phosphate buffer (100 mM, pH 7.0), and desalted using PD-10 desalting.Enzyme activity was carried out, and total protein of collected dextranase was utilized for further experiments.The production of the enzyme as well as the preparation of partially purified dextranase has been described earlier [15].

Enzyme Assay and Protein
Concentration.The Nelson-Somogyi assay [16,17] was performed for the estimation of the enzyme activity of soluble and entrapped dextranase.Soluble precipitates of dextranase (100 μl) were incubated with 1 ml dextran solution (30.0 g L -1 , 10 kDa in 100 mM sodium phosphate buffer of pH 7.0) at 50 °C for 5 minutes.The catalytic activity of dextranase entrapped in alginate was determined by placing 0.5 g of alginate beads having dextranase with 1 ml dextran solution (30.0 g L -1 , 10 kDa) at 50 °C for 5 minutes, and reducing sugar release during reaction was quantified with the help of the standard curve of maltotriose.One unit of dextranase is defined as "the amount of enzyme required to release 1.0 μmol of maltotriose per minute under the standard assay conditions." The concentration of protein was determined using Lowry's method [18] with the standard calibration curve of bovine serum albumin (BSA).
2.4.Enzyme Entrapment in Sodium Alginate Spheres.Entrapment was initiated by proper mixing of partially purified dextranase (1894.3U mg -1 ) with sodium alginate solution (40.0 g L -1 in 100 mM sodium phosphate buffer of pH 7.0) in an equal ratio.For the polymerization of alginate in the form of spheres, the amalgam was dropped through a syringe into the chilled calcium chloride solution of 0.2 M with continuous stirring that resulted in the synthesis of calcium alginate spheres having entrapped dextranase.The same protocol was followed to prepare control alginate spheres having buffer instead of the enzyme.Both spheres of control and entrapped dextranase were washed with buffer and utilized for further experiments.
2.5.Optimization of Entrapment Parameters.Various concentrations of sodium alginate (10.0-70.0g L -1 ) and calcium chloride (0.1-0.5 M) were optimized to determine suitable concentration for the polymerization of alginate spheres as well for the proper entrapment of the enzyme.Stability of beads was also improved by optimizing the curing time of alginate spheres from 5.0 to 30.0 minutes.
The entrapment yield of dextranase was calculated by applying the following formula: Entrapment yield % = activity of entrapped dextranase activity of soluble dextranase × 100 1 2.6.Characterization of Soluble and Entrapped Dextranase.
The reaction time of soluble and entrapped dextranase was determined by letting both forms of the enzyme react with a dextran solution of 30.0 g L -1 at 50 °C from 5.0 to 40.0 minutes.The samples were drawn from the reaction mixture with time intervals of 5.0 minutes, and the enzymatic assay was carried out.Temperature maxima for the soluble as well as entrapped enzyme were also optimized by performing enzymatic reaction at temperature ranging from 40 °C to 90 °C.Similarly, optimum pH required by soluble and entrapped dextranase was also investigated at a pH range from 5.0 to 8.0.To determine maximum velocity and substrate-binding affinity of the soluble and immobilized enzyme towards a substrate, varying concentrations of dextran from 5.0 mg mL -1 to 50.0 mg mL -1 were used.
V max and K m were also calculated by the Lineweaver-Burk plot (Lineweaver-Burk, 1934).

Stability of Soluble and Entrapped
Dextranase.Thermal stability of both forms of dextranase was noted by incubating the enzyme at different temperatures from 50 °C to 80 °C.The catalytic assay was performed after every 30 minutes till 120 minutes of each temperature.The stability of dextranase after storage at 4 °C and 30 °C was analyzed by performing the assay every day for a period of time.
Stability of dextranase was also studied in the presence of different surfactants including Triton X-100, Tween 80, Tween 20, and sodium dodecyl sulphate (SDS).Soluble dextranase and entrapped dextranase were incubated with 5.0 mM solution of these surfactants in a 1 : 1 ratio for about 30 minutes.The enzyme activity was carried out as described earlier.
2.8.Reusability of Dextranase.For the continuous utilization of the enzyme, the activity of the entrapped enzyme was carried out repeatedly.After every cycle, the alginate spheres 2 International Journal of Polymer Science were washed with respective buffer and used again to perform catalytic function.The enzyme activity of the first cycle was considered 100%.
2.9.Scanning Electron Microscopy (SEM).Surface morphology of alginate spheres having the entrapped enzyme and control spheres without the enzyme was also examined using a scanning electron microscope (JSM 6380A Jeol, Japan) of different magnification powers (1000x, 2500x, and 10000x) operated at 10.0 kV.Both samples were sputter coated in an autocoater (model JFC-1500 Jeol, Japan) with gold (Au) after drying and targeted up to 300 Å.

Results and Discussion
3.1.Optimization of Entrapment Parameters 3.1.1.Sodium Alginate Concentration.Calcium alginate spheres were prepared using an ionotropic gelation method [19].As alginate solution is extruded dropwise in calcium chloride solution, gel formation started due to cross-linking reaction of the divalent cations [20].It has been previously reported that the pore size of the alginate spheres is dependent on the concentration of sodium alginate [21].Therefore, varying concentrations of sodium alginate (10 to 70 g L -1 ) were examined to synthesize the efficient alginate spheres with the entrapped enzyme.It was noticed that a sodium alginate concentration of 40 g L -1 resulted in the formation of mechanically stable spheres with entrapped dextranase having a 70% entrapment yield (Figure 1(a)).Alginate spheres prepared using low concentration of sodium alginate were soft and friable and resulted in leakage of entrapped dextranase because of high porosity.On the other hand, alginate spheres synthesized using high concentration of alginate caused less penetration of the substrate (dextran) due to the small pore size of the gel.Therefore, a sodium alginate concentration of 40 g L -1 was selected for the formation of functionally active alginate spheres with the entrapped enzyme.
3.1.2.Calcium Chloride Concentration.Along with concentration of sodium alginate, calcium chloride concentration is also crucial for the synthesis of a stable alginate sphere system.Consequently, concentration of calcium chloride from 0.1 to 0.5 M was also optimized to increase the immobilization yield.The results indicated that 0.2 M concentration of calcium chloride was appropriate for proper entrapment of dextranase and a three-fold increase in the entrapment yield was observed (Figure 1(b)).However, as the ionic strength of calcium chloride increases, the entrapment yield was gradually decreased which could be due to the change in pH of the calcium chloride solution [22].
3.1.3.Curing Time.Optimization of curing time is an important phenomenon during alginate sphere synthesis as the hardness of the sphere depends upon it which resulted in the formation of firm alginate spheres [23].Therefore, in the current study, immobilization efficiency is noticed at different curing times (5.0 to 30 minutes) and it was 3 International Journal of Polymer Science observed that stable spheres were synthesized at 20 minutes curing time (Figure 1(c)).At low curing time, complete gel formation did not occur and large pore size spheres were formed; as a result, dextranase was easily percolated from the matrix.However, prolonged curing time did not cause any improvement in the efficiency due to continuous hardness of spheres.

Topological Studies of Calcium Alginate Spheres.
The hydrogel spheres of calcium alginate without enzyme entrapment were uniform in size and transparent and designated as a control (Figure 2(a)).However, the hydrogel spheres change from being transparent to having a color after entrapment of dextranase, and these spheres are the spheres being tested (Figure 2(b)).In order to examine the structural changes in alginate hydrogel spheres after entrapment of dextranase, both the control spheres and the alginate spheres being tested were analyzed using SEM.The micrographs of the surface structure of control spheres showed smooth surface morphology (Figure 2(c)) whereas spheres with the entrapped enzyme showed slight texture difference in comparison with control spheres, and the surface of spheres being tested exhibited a porous structure due to gap formation (Figure 2(d)).

Characterization of Soluble and Entrapped Dextranase
3.3.1.Reaction Time.Entrapment of dextranase within a microenvironment influences the characteristics of the biocatalyst.Hence, enzyme kinetics was studied to compare the characteristics of entrapped dextranase with its soluble counterpart.It was observed that enzyme catalysis reaction time was prolonged from 5.0 minutes for soluble dextranase to 20.0 minutes for entrapped dextranase (Figure 3(a)).The increased reaction time is the time required by the substrate (dextran) to reach the active site of entrapped dextranase by penetrating through the alginate matrix.An increase of 10.0 minutes in the reaction time of entrapped dextranase from the soluble dextranase is also reported [24].

Reaction
Temperature.The variation in optimum temperature of entrapped dextranase was also examined, and for this purpose, enzyme-substrate reaction was carried out at various temperatures (40 °C to 90 °C).It was found that catalytic activity of dextranase after entrapment in alginate spheres was performed at optimum temperature (50 °C) the same as that of soluble dextranase.However, the stability of entrapped dextranase seemed to be higher as compared to that of the soluble enzyme.Dextranase entrapped in hydrogel spheres retained 65% activity even at 80 °C (Figure 3(b)).In most of the cases, optimum temperature of the biocatalyst after immobilization shifted to high temperature, but in some cases, it remains unchanged.The similar results were also reported by [25].

Reaction pH.
The effect of pH on the activity of dextranase after entrapment was estimated at different pH ranging from 5.0 to 9.0.It was examined that pH optima of dextranase after entrapment remain the same but the pH stability of entrapped dextranase was enhanced with reference to soluble dextranase (Figure 3(c)).The catalytic activity of soluble as well as entrapped dextranase gradually declines after an optimum pH of 7; however, the entrapped enzyme retained 73% of its activity as compared to its soluble 4 International Journal of Polymer Science counterpart which results in the loss of more than 50% of its activity at the same pH.

Enzyme Kinetics.
In order to examine the steady-state kinetics of dextranase after entrapment in alginate hydrogel spheres, varying concentrations of the substrate (dextran) were used to carry out catalytic activity.The data was generated after performing experiments, and graphs were plotted using the calculated values in GraphPad Prism (version 6.0) by nonlinear regression (curve fit).The maximum rate of soluble dextranase was observed with a V max value of 5191 μmol ml -1 and a K m value of 4.38 mg ml -1 with standard errors of 116.4 μmol ml -1 and 0.49 mg ml -1 , respectively, with a determination coefficient (R 2 ) of 0.9717.However, the V max value of dextranase decreased after entrapment into the spheres with a calculated value of 4732 μmol ml -1 with a standard error of 90.34, and the K m value of entrapped dextranase varied from the value of its soluble counterpart with a twofold increase of 9.466 mg ml -1 having a standard error of 0.654.The same phenomenon after immobilization was also reported by El-Tanash, and the reason suggested for the increased K m value was the low availability of the substrate for the active site of immobilized dextranase while a decrease in velocity of reaction represented a decrease in flexibility of dextranase after entrapment [26].Previously, it was also reported by another author that entrapment did not cause a pronounced effect on the kinetic characteristics of the enzyme [27].

Stability of Soluble and Entrapped Dextranase
3.4.1.Thermal Stability.The stability of the enzyme at high temperature is essential to fulfill industrial criteria; therefore, the thermal stability of dextranase after entrapment was examined by keeping the entrapped enzyme in alginate spheres at temperatures ranging from 50 °C to 80 °C for 2.0 hours with an interval of 60 minutes.The stability of dextranase, being a thermozyme, was pronounced at extreme temperature as compared to the enzyme isolated from mesophilic bacteria.However, the stability of dextranase after entrapment was significantly improved compared to that of the soluble thermozyme.It has been previously computed that the thermal stability of the entrapped biocatalyst is enhanced due to the stabilization in 3D structure after entrapment [28].The soluble enzyme reduced more than 30% of its original activity at temperature above 50 °C while entrapped dextranase retained 65% residual activity at 80 °C even after 2.0 hours (Figures 4(a) and 4(b)).The improvement in thermal stability of the biocatalyst after entrapment in alginate hydrogel has also been reported by Milovanović et al. [29].The residual activity of dextranase entrapped in hydrogel spheres was determined daily.It was noticed that entrapment enhanced the storage life of the biocatalyst and entrapped dextranase remains active for a long duration at both temperatures compared to soluble dextranase.But the dextranase leaching from the hydrogel sphere during storage contributes a major role in decreased dextranase activity.From Figure 4(c), it can be concluded that dextranase entrapped in alginate spheres retained 82% activity at 4 °C after 24 days.On the other side, the activity was declined during the same time period when alginate spheres were stored at 30 °C.The storage stability of soluble dextranase seemed to be higher than that of the entrapped enzyme, but in this case, the reason behind the lower activity could be due to dextranase leaching from the spheres during storage.

Operational Stability.
Reusability of the biocatalyst is a very important factor from an economical perspective.Alginate spheres were washed after every cycle with buffer and used again for the next reaction.The entrapped dextranase retained its activity even after 7.0 cycles (Figure 5(a)).However, it was reported that amylase entrapped in alginate gel showed its activity up to 10 cycles with a residual activity of 35% [22].Soluble dextranase could not possess this recycling capabilities; therefore, the immobilized biocatalyst gets a superior position due to recycling efficiency.

Dextranase Stability in the Presence of Surfactants.
The influence of different surfactants on the activity of dextranase before and after entrapment was examined.It was observed that Tween 80 and Tween 20 slightly had an effect on the activity of soluble dextranase.However, Triton X-100 and SDS exhibited inhibitory action on soluble dextranase, and more than half of the activity decreases in the presence of these surfactants.The same inhibitory action of SDS even at the very amount was also reported in the case of dextranase from Streptococcus mutans [30].On the contrary, when immobilized dextranase was incubated with these surfactants after entrapment in alginate spheres, marked improvement in dextranase activity was noticed (Figure 5(b)).The similar effects were also reported by other authors on the immobilized enzymes [31,32].

Conclusions
Carbohydrate polymer alginate was efficiently utilized for the entrapment of the important biocatalyst dextranase.Hydrogel spheres of calcium alginate were prepared having

6
International Journal of Polymer Science dextranase encapsulated within them.Different entrapment parameters were applied to investigate the mechanical strength of the synthesized alginate spheres.After that, enzyme kinetics was also studied and compared with that of dextranase's soluble counterpart.It can be concluded that entrapment is a simple but effective method of immobilizing an enzyme; however, certain immobilization parameters must be taken into consideration in order to prepare functionally active spheres.

Figure 1 :
Figure 1: Influence of entrapment parameters on the synthesis of alginate spheres and entrapment of dextranase with them: (a) optimization of sodium alginate concentration; (b) optimization of calcium chloride concentration; (c) determination of curing time.All values are shown as mean of three independent experiments (n = 3).

Figure 3 :
Figure 3: Comparison of characteristics of entrapped dextranase with those of the soluble enzyme: (a) effect on reaction time after entrapment; (b) effect of temperature on entrapped dextranase; (c) effect of change in pH after entrapment in alginate spheres.All values are shown as mean of three independent experiments (n = 3).

Figure 4 :
Figure 4: Stability studies of dextranase entrapped in alginate spheres: (a) thermal stability of soluble dextranase at different temperatures for different time intervals; (b) stability of entrapped dextranase at high temperature for varying time intervals; (c) storage stability of dextranase after entrapment at 4 °C and 30 °C.All values are shown as mean of three independent experiments (n = 3).

Figure 5 :
Figure 5: (a) Recycling efficiency of dextranase entrapped in the calcium alginate sphere system under specified condition.(b) Effect of different surfactants on the activity of soluble as well as entrapped dextranase.All values are shown as mean of three independent experiments (n = 3).