Catalytic Properties and Immobilization Studies of Catalase from Malva sylvestris

Catalase was partially puri�ed from Malva sylvestris L. and immobilized onto chitosan. en, its catalytic properties were investigated. (NH4)2SO4 precipitation and dialysis were performed in the extracted enzyme. Further puri�cation was performed with sephadex G-200 column. Kinetic studies of the puri�ed enzyme activity were measured and characterized. e inhibitory effects of KCN, NaN3, CuSO4, and EDTA onM. sylvestris L. catalase activity were observed except NaCl. Furthermore,M. sylvestris L. catalase was immobilized covalently with glutaraldehyde onto chitosan particles. e pH and temperature optima as well as the changes in the kinetics (Km, Vmax) of the immobilized and free M. sylvestris L. catalase were determined. e Km value for immobilized catalase (23.4mM) was higher than that of free enzyme (17.6mM). Optimum temperature was observed higher than that of the free enzyme. e optimum pH was the same for both free and immobilized catalases (pH 7.50). Immobilized catalase showedhigher storage and thermal stabilities than free catalases. Free catalase lost all its activitywithin 60 dayswhereas immobilized catalase lost 45% of its activity during the same incubation period at 4○C. e remaining immobilized catalase activity was about 70% aer 8 cycles of batch operations.

Catalase has also very high industrial importance with its industrial applications in removal of hydrogen peroxide used as oxidizing, bleaching or sterilizing agent and in the analytical �eld as a component of hydrogen peroxide [23][24][25].Using soluble enzyme is restricted because of certain properties like nonreusability, instability, high cost, and denaturation.On the other hand, immobilized enzymes improve thermostability, operational stability, recovery, reusability, high purity, and high product yields in their industrial applications [26].
Malva sylvestris L. leaves are used as a food and also as a traditional medicine [37].Furthermore, previous studies have been reported that M. sylvestris L. leaves have powerful antioxidant properties including high radical-scavenging activity [38].ese properties of M. sylvestris L. leaves are very important for an antioxidant enzyme such as catalase.ere is no available information on the basic aspects of the catalase enzyme from M. sylvestris L. puri�cation and its immobilization process in literature.
e aim of this work was to investigate the isolation and characterization of catalase from Malva sylvestris L. leaves and then, immobilization of this plant catalase on glutaraldehydepretreated chitosan �lms.e resultant immobilized catalase was characterized, and its thermal and operational stability, catalytic properties and reusability aspect were compared to free catalase.Immobilization of Malva sylvestris L. plant catalase will improve its thermostable, operational, reusable properties compare to that of its free counterpart.ese properties of immobilized plant catalase will give more advantages than soluble-free catalase.us, the immobilized catalase might have useful applications in food and textile industries in removal of hydrogen peroxide used as oxidizing, bleaching, or sterilizing agents.

E�traction and Puri�cation
. Malva sylvestris L. catalase enzyme extraction was prepared as described in a previous work [15].Fieen g of M. sylvestris L. was obtained from local Sakarya region.Aer that samples were added to 100 mL 50 mM sodium phosphate buffer (pH; 7.0) with 0.3 g polyvinylpolypyrolidone (PVPP).e mixture was homogenized with blender.en, the �ltrate was centrifuged at 14.000 g for 30 min and supernatant was collected [39].Extraction was fractionated with (NH 4 ) 2 SO 4 , solid (NH 4 ) 2 SO 4 was added to the supernatant to obtain 80% saturation.e mixture was centrifuged at 14,000 g for 30 minutes and the precipitate was dissolved in a small amount of phosphate buffer and then dialyzed at 4 ○ C in the same buffer for 24 h with three changes of the buffer during dialysis.e dialyzed enzyme extract was centrifuged and loaded onto sephadex G 200 column (1 × 10 cm) previously equilibrated with extraction buffer and washed with the same buffer to remove unbound proteins.e eluate was used as the CAT enzyme source in the following experiments.

Determination of Protein
. e amount of CAT was performed according to method of Bradford with bovine serum albumin as standard [40].

Preparation of Chitosan
Beads.ree grams of chitosan powder were suspended into 99 mL of distilled water by stirring for 10 min.One milliliter of glacial acetic acid was then added, and stirring was allowed for 3 h at room temperature.e solution was �ltered and dried.Some 2.0 wt.% NaOH aqueous solution was added to neutralize the acetic acid in the chitosan �lm.e �lms were repeatedly washed with deionized water and �nally dried again.

Reinforcement of Chitosan
Beads by Glutaraldehyde Treatment.e crosslinking was carried out by immersing the dried chitosan into 0.05% (w/v) glutaraldehyde solution in 0.05 M cold phosphate buffer (pH 7.0) for 1 h.e brownish reinforced beads were washed several times by 0.05 M cold phosphate buffer (pH 7.0) to remove the excess of glutaraldehyde [41].

Immobilization of Catalase into Chitosan Beads.
Chitosan beads were mixed with 2 mg/mL Malva sylvestris L. catalase solution in 0.05 M phosphate buffer (pH 7.0) for 5 h with slight stirring and washed at 4 ○ C. en, the beads dried in a vacuum incubator at room temperature and stored at 4 ○ C. e amount of immobilized enzyme was estimated by subtracting the amount of protein determined in the supernatant aer immobilization from the amount of protein used for immobilization.

Activity Assays of Catalase.
Catalase (CAT) activity was determined at 25 ○ C according to Aebi [42].e reaction mixture contained 40 mM H 2 O 2 in a 50 mM phosphate buffer pH 7.0 and 0.1 mL enzyme in a total volume of 3 mL.CAT activity was estimated by decreased in absorbance of H 2 O 2 at 240 nm.Activities were carried out at optimum conditions.Approximately 100 mg of catalase immobilize chitosan beads were mixed 10 mL of 10 mM H 2 O 2 solution in 0.05 M phosphate buffer (pH 7.0) 25 ○ C. Aer 5 min, the reaction was determinated by removal of the chitosan beads from the reaction mixture.e absorbance of the reaction mixture was determined, and the immobilized catalase activity was calculated.e kinetic constant was determined using Lineweaver-Burk plot by initial reaction rates of free or immobilized enzyme catalase.
2.�.In�uence of p�.e activity assays were carried out over the pH range 3.00-9.00.Reaction rates of free and immobilized enzyme preparations depending on pH were investigated using 50 mM acetate buffer pH 3.5, 4.0, and 5.0 50 mM phosphate buffer at pH 6.0, 7.0, 7.5, 8.0, and 9.0.Activity of pH pro�les was determined at various pH in 10 mM H 2 O 2 solution at 25 ○ C.

2.1�
. In�uence of Temperature.e effect of temperature on enzyme activity was investigated in the range of 4-70 ○ C for both free and immobilized catalases.Activity of temperature pro�les was determined at indicated temperatures in 10 mM H 2 O 2 solution (pH 7.5).

Storage Stability of Free and Immobilized
Catalase.e activity of free and immobilized catalase was investigated at 25 ○ C and 4 ○ C in 50 mM phosphate buffer (pH 7.5).
2.12.Reuse of the Immobilized Catalase.e residual activity of the immobilized enzyme was determined under standard assay conditions.e same immobilized enzyme was reused aer it was thoroughly washed.

Results and Discussion
3.1.�uri�cation of Catalase.Typical results of the partial puri�cation procedure of CAT from Malva sylvestris L. are summarized in Table 1.Aer the enzyme extraction from M. sylvestris L., the extracted enzyme solution was reacted with several (NH 4 ) 2 SO 4 (0-20%, 20-40%, 40-50%, 50-60%, and 60-80% saturated solutions) to purify CAT by altering its solubility and to �nd out the almost completely precipitation of CAT with maximum activity.In conclusion, CAT activity of the precipitate of 30-60% (NH 4 ) 2 SO 4 saturation was found to be 53.8%puri�cation, and this saturation was used all the extraction processes.
Aer ammonium sulfate precipitation, the dialyzed enzyme extract was loaded onto Sephadex �200 gel �ltration column and eluted with 50 mM phosphate buffer (pH 7.0).As a result, CAT enzyme was puri�ed 123.6 fold with a yield 42.7%.

Effect of Inhibitors on Catalase
Activity.e percentage of the catalase activity was determined with different concentrations of inhibitors (Figure 1).KCN, EDTA, sodium azide (NaN 3 ), copper sulphate (CuSO 4 ) and NaCl were used for the inhibition studies.Only 5.2% of catalase activity was retained in the presence of 0.5 mM sodium azide which was also observed by Kandukuri [14].In presence of 3 mM EDTA, 55% activity was retained.EDTA inhibited the enzyme activity because it was known a metal chelator that could have complex with iron ion in the active site of catalase [37].NaCl had almost no effect to M. sylvestris L. catalase.KCN also inhibited the enzyme activity 80% in 3 mM concentration.

Immobilization of Malva sylcestris L. Catalase on Chitosan
Beads.Enzymes have been used in several industrial processes for a long time.Textiles, food, animal feed, detergent, pulp and paper are among the industries that use enzymes.Catalase has been used especially in the textile industry as a bleaching material [35,36].Many studies on immobilization of catalase have been reported [43,44].
In this study, we investigated immobilization of M. sylvestris L. catalase onto chitosan beads with gluteraldehyde.ere is no report yet about M. sylvestris L. catalase immobilization in the literature.
e catalase was incubated with glutaraldehyde-pretreated chitosan beads for 5 hours.e Michaelis constants, Km and Vmax, for free and immobilized catalase were deter- mined using H 2 O 2 as substrate.e amount of bound enzyme and kinetic parameters were shown in Table 2.
e Km values of free and immobilized enzyme were found to be 17.6 mM and 23.4 mM (Table 1).e Km value of immobilized enzyme was higher than that of free enzyme, which meant immobilized enzyme had less affinity to its substrate.Change in the affinity of the enzyme to its substrate was probably caused by structural changes in the immobilization procedure of the enzyme and by lower accessibility of the substrate to the active site of the immobilized catalase enzyme.

Effect of pH on
Catalase.e effect of pH on the free and immobilized enzyme for hydrogen peroxide degradation was investigated in the pH range between 3.00 and 9.00 in acetate or/and phosphate buffers at 25 ○ C. e results are presented in Figure 2. At the end of the time, the activity measurements of the enzymes were made under the optimum assay conditions (Figure 2).As it was shown from the �gure, pH stabilities of the both enzymes had some similarities.Both free and the immobilized catalase gave an optimum at pH 7.50; however, the immobilized catalase gave a much broader pH stability than the free enzyme.is suggested that immobilized enzyme was less sensitive to pH changes than free enzyme.

Effect of Temperature on
Catalase.Temperature pro�les of free and immobilized catalase are shown in Figure 3. Optimum temperature was found at about 25 ○ C for free and 40 ○ C for immobilized catalase.Figure 3 showed that the activity of immobilized catalase was more stable than the free catalase around 25-40 ○ C. e activity loss of immobilized enzyme was less than the free enzyme for higher temperature.Chitosan support might have a protecting effect at the high temperatures at which enzyme deactivation takes place.Immobilization of catalase in chitosan beads can cause an increase in the enzyme rigidity which is commonly re�ected  by increase in stability towards denaturation by raising the temperature [45,46].at 4 ○ C and the activity measurements were carried out for a period of 60 days (Figure 4).e free enzyme lost all of its activity within 35 days.On the other hand, immobilized catalase lost 45% of its activity during the incubation period (Figure 4).Free and immobilized catalase were stored in a phosphate buffer (50 mM, pH 7.0) at 25 ○ C and the activity measurements were carried out for a period of 60 days.When the temperature was raised to 25 ○ C (Figure 5), there was a signi�cant decrease in the activity of the enzyme over a 3 days period.Aer 3 days, only 15% of the activity was remained.
Immobilized enzyme was preserved %96 activity aer 3 days.At 25 ○ C, the immobilization process provided an advantage in the stability of enzyme over free enzyme activity, especially at longer durations.It was shown that immobilized enzyme could be reused (Figure 6).e activity of immobilized enzyme remained 50% aer it was reused for ten times.e results con�rmed that the stability of immobilized enzyme was good.

Conclusions
Catalase enzyme form Malva sylvestris L. was successfully characterized and immobilized onto chitosan with glutaraldehyde.Our work showed that the catalase enzyme was  �rst time isolated and characterized from Malva sylvestris L. leaves as a plant source.en, this plant catalase successfully immobilized onto chitosan material.us, the plant enzyme was gained strength against thermal denaturation by this successful immobilization of plant catalase on chitosan beads.It was found that thermal stability and storage stability of immobilized catalase were better than that of free catalase.It is possible to increase the degradation of hydrogen peroxide and can be used many times in the processes.Our results show that the immobilization of Malva sylvestris L. catalase on chitosan makes the enzyme more useful in degradation of hydrogen peroxide in various industrial applications such as food and textile.

F 1 :
Effects of various compouns on CAT from Malva sylvestris L.

3. 6 .F 4 :
Storage Stability and Reusability.Free and immobilized catalase were stored in a phosphate buffer (50 mM, pH 7.0) Storage stability at 4 ○ C.
T 1: Partial puri�cation of catalase from Malva sylvestris L.