Immobilization of a - Amylase from Locale Bacteria Isolate Bacillus Subtilis ITBCCB148 with Diethylaminoethyl Cellulose (DEAE-Cellulose)

Determination of Optimum Temperature of Enzyme Before and After Immobilization

%  activity of enzyme before and after immobilization at various pH’s is shown in Fig. 1. The optimum temperature of the purified and immobilized enzyme was found to be the same, which is 60°C. Similar previous studies also reported that the chemical modification and immobilization process did not always cause a change in the optimum temperature.^4,15,16 Although the optimum temperature did not change, however, the stability of the enzyme has increased due to the immobilization process, especially at higher temperatures, 70-80^oC. The activity of purified enzyme at 65^oC was 91.1%, while the immobilized enzyme has activity of 97.4%. At higher temperature, 75 and 80^oC, the immobilized enzyme has activity of 78.4 and 71.7%, respectively, while the purified enzyme has activity of only 9.9 and 2. 8%, respectively. This is because the immobilization process has caused the rigidity of the immobilized enzyme to increase; as a result it was more tolerant of temperature.

Figure 1: Optimum temperature of enzyme before and after immobilization

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Determination Of Kinetic Data

The graphs of determination of K_M and V_max values are shown in Figures 2 and 3.

Figure 2: Lineweaver-Burk graph of purified α-amilase

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Figure 3: Lineweaver-Burk graph of immobilized a-amilase

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Based on the Lineweaver-Burk graph in Figure 2, the V_max value of purified enzyme was 204 mmol mL^-1 min.^-1 and the K_M value was 4.08 mg mL^-1 substrate, whereas the immobilized enzyme has V_max and K_M values of 42.4 mmol mL^-1 min^-1, and 14.8 mg mL^-1 substrate, respectively (Figure 3). These results indicated that the V_max value of purified enzyme was very much different to that of the immobilized enzyme. The sharp decrease of the V_max value in the immobilized enzyme was due to the steric hindrance of the insoluble immobile matrix, so the substrate interaction was hindered. The K_M value of purified enzyme also showed discernible value to that of the immobilized enzyme. This is because the effect of the immobile substance caused the immobilized enzyme affinity toward substrate to decrease, so the activity was also sharply decreased. The decrease of enzyme activity before and after the immobilization process is presented in Table 1.

Table 1: The decrease of enzyme activity by the immobilization process with DEAE-Celullosa

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The great decrease of enzyme activity in the immobilization process was similar to data previously reported where â-amylase, which was modified by silica, was decreased by 57%.⁴ Gaertner and Puigserver have also reported similar observation on the enzyme activity of trypsin which was modified by PEG, where the immobilized trypsin was decreased 10 times compared to the non modified one.¹⁷ This effect also caused the decrease of autolysis rate for the modified enzyme, as a result the enzyme stability was also increased.

The Enzymatic Conversion of Amylum to Glucose Using the Immobilized Enzyme with Repeat Use

The enzymatic conversion of amylum to glucose using immobilized enzyme is shown in Figure 4.

Figure 4: The use of immobilized enzyme repeatedly

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From Figure 4 it is clear that the immobilized enzyme still has high activity even after it has been used 6 times. Its activity was approximately 51.9%. This result illustrated that the use of immobilized enzyme was still effective for enzymatic conversion of amylum to glucose, although in the process of immobilization, there was activity loss of about 52.6%.

Thermal stability of the enzyme The residual activities of the purified and immobilized enzymes were determined by incubating each enzyme for 60 minutes at 60°C. At certain interval times, the activity of each enzyme was determined. The graph of thermal stability tests of the enzymes is shown in Figure 5.

Figure 5: Thermal stability presented as graph of residual activity (%) of enzyme before and after immobilization at 60°C and pH 9.0 vs time

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Figure 5 showed residual activity of each enzyme at 60°C and pH 9.0 for 60 minutes. The purified enzyme has residual activity of 16.1 %, whereas the immobilized enzyme has 28.6%. This data informed us that the immobilized enzyme has thermal stability higher than that of the purified one.

The Constant of Thermal Inactivation Rate (k_i), Half-Life (t_1/2), and Echange Due to Denaturation

The values of constant of thermal inactivation rate (k_i), half-life (t_1/2), and energy change due to denaturation of purified and immobilized enzymes are presented in Table 2.

Table 2: The energy change due to denaturation (ΔGi), half-life (t½), and ki values of enzyme before and after immobilization

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The half-life of immobilized enzyme was increased 1.5 times higher compare to the purified one, while the activity recovery was 47.7%. Stahl reported that thermal stability of enzyme was determined by finding the half-life of the enzyme.¹⁸ The increase of half life of the immobilized enzyme used in this research was about the same as with other data reported by others where the half-life of the enzyme was increased from 12 hours to 25 hours, while the enzyme activity recovery was 21%¹⁹. The modified enzyme with modification degree of 68% was able to increase the half life of the enzyme to 60 hours and activity recovery of 13%. The result showed that the immobilized enzyme was able to increase the half life of the enzyme similar to other data¹⁸, however, the activity loss of the immobilized enzyme with DEAE-Cellulose was much better.

The ΔG_i of the immobilized enzyme was increased compared to that of the purified one. The low G_i value indicated that the enzyme was more flexible in solution which caused the denaturation of the enzyme which will occur more easily and have much higher activity, and vice versa.

It can be summarized that the immobilization process of α-amylase obtained from locale bacteria isolate B. subtilis ITBCCB148 with diethylaminoethyl Cellulose (DEAE-Cellulose) was successfully able to increase the stability of the enzyme at higher temperature, although the thermal stability was only increased 1.5 times. The repeat use of immobilized enzyme was also by far better than that of the purified enzyme. Attempts to increase stability of the enzyme used in this research will also be done with other immobile substances such as carboxy methy celullose (CMC) or local immobile substances such as bentonite and zeolite, two local clay minerals, in Indonesia.