The Chemical Modification of Cellulase Obtained from Bacillus subtilis ITBCCB148 With Dimethyladimipidate

Abstract

Cellulase obtained from Bacillus subtilis ITBCCB148 has successfully been isolated and purified. The native enzyme was modified with dimethyladipimidate (DMA) to increase the stability of the enzyme. The native and modified enzymes were characterized including determination of optimum temperature, optimum pH and thermal stability. The activity of the cellulase was determined based on Mendels method and the protein content was determined based on Lowry method. The results showed that the native enzyme has optimum temperature of 55 o C and optimum pH of 5.5. The thermal stability for 60 minutes at temperature of 55 o C indicated that the native enzyme has k i = 0.034 min.t -1 , t 1/2 = 20.4 min., and Δ Δ Δ Δ ΔG i = 100.9 kJ/mol. The modified enzyme with modification degrees of 64.5; 69.5 and 82% have optimum temperature of 55 o C and optimum pH of 6. The thermal stability for 60 minutes at temperature of 55 o C of the modified enzyme with modification degrees of 64.5; 69.5 and 82% have k i values of 0.024; 0.021 and 0.022 min. -1 , t 1/2 values of 28.9; 33.0 and 31.5 minutes, and Δ Δ Δ Δ ΔG i values of 101.9; 102.3 and 102.2 kJ/ mol, respectively. The modification with DMA has successfully increased the thermal stability of the modified enzymes between 1.4 -1.6 times compared to that of the native enzyme. The decrease of ki values, increase of half-lives and Δ Δ Δ Δ ΔG i indicated that the modified enzymes were more stable compared to the native enzyme.

Stability enhancement of Bacillus subtilis ITBCCB148 originating -amylase by immobilization using chitin

“…Calculation of k i of α-amylase as a result of purification and immobilization was done using the kinetic equation of the first order inactivation [24].…”

“…Based on Figure 7, it can be calculated that the value of half-life (t ½ ) of the immobilized enzyme is 346.50 min, while the t ½ of the native enzyme is 28.88 min, thus t ½ of immobilized enxyme has increased 12 times. The longer the half-life of the enzyme, the better its stability [28,29], while, in addition, a decrease in the inactivation rate constant (k i ) in immobilized enzymes indicates that there is a decrease in the denaturation rate of proteins (enzymes). Half-life indicates the time at which the enzyme activity is reduced to 50% of its original activity.…”

“…A decrease in the value of k i is estimated due to the condition of enzymes that is less compatible with water, leading to reduced protein unfolding and increased stability of the enzyme [28,29].…”

“…It was also observed that the immobilized enzyme displayed thermal stability as high as 2-3.8 times more than that of the enzyme without immobilization. To improve the stability of purified αamylase obtained from B. subtilis ITBCCB148, researches have been carried out by Yandri et al [11,12], by immobilizing the enzyme on chitosan and bentonite matrices, and chemically modifying using dimethyladipimidate [13]. Another potential enzyme immobilizing material is chitin.…”

Peningkatan Kestabilan Enzim Α–amilase Dengan Penambahan Gliserol

“…Menurut Mozhaev and Martinek (1984), stabilisasi enzim yang berasal dari mikroba mesofilik merupakan cara yang lebih disukai, karena isolasi langsung dari mikroorganisme termofilik memiliki kelemahan, yaitu memerlukan perancangan bioreaktor maupun metode pemrosesan baru (Janecek, 1993). Beberapa cara yang dapat digunakan untuk meningkatkan stabilitas enzim adalah penggunaan zat aditif, modifikasi kimia, amobilisasi dan penggunaan rekayasa protein (Illanes, 1999;Yandri et al, 2011;Yandri et al, 2012;Yandri et al, 2013;Yandri et al, 2014;Yandri et al, 2015;Yandri et al, 2018;Yandri et al, 2020).…”