The Chemical Modification of Protease Isolated from Locale Bacteria Isolate Bacillus subtilis ITBCCB148 with Nitrophenolcarbonate-Polyethylene Glycol (NPC-PEG)
Abstract:The research aims to study the effect of chemical modification on the stability of protease enzyme from a local bacteria isolate B. subtilis ITBCCB148 with NPC-PEG. The result showed that the native enzyme has optimum pH and temperature of 6.5 and 60C, respectively. The stability test of the native protease at pH 6.5 and temperature 60C for 360 minutes produce the following results: the residual activity of 5.75%, t½= 84.5 min., k i = 0.0082 min. . The modified enzyme with NPC-PEG (33%, 42%, and 75%), showed… Show more
“…The result showed that the enzyme activity was maximum (180±0.06 unit/ml) at pH 7 and minimum (120±0.02 unit/ml) at pH 4 ( Figure 6). Similar results were obtained for the optimum pH for enzymatic activity of other Bacillus sp: pH 7.5 for B. subtilis ITBCCB 148 (Yandri et al, 2008), Bacillus sp. HS08 (Huang et al, 2006) and Bacillus sp.…”
Section: Effects Of Ph On Protease Activitysupporting
Protease-producing bacterium Bacillus cereus SU12 was isolated from oyster Saccostrea cucullata. Fifteen strains of bacteria were isolated from oyster S. cucullata and screened for secretion of protease on casein agar plates. Among them, SU12 isolate was selected due to its high enzyme production capacity and was identified as B. cereus SU12 on the basis of its morphological, biochemical and 16S rDNA properties. Media and cultivation conditions were studied to optimize bacterial growth and protease production which includes different carbon and nitrogen sources, in addition to different factors such as incubation time, pH, temperature, NaCl concentrations. At pH 7, temperature 40°C and 2.5% NaCl concentration, carbon source such as starch and beef extract as nitrogen source, the protease activity was maximum. Extracellular protease was isolated, purified to 8.73 fold by diethyl aminoethyl (DEAE) ion-exchange chromatography and its specific activity was determined to be 886.56 U/mg and the sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) showed a single band for the purified enzyme, with an apparent molecular weight of 66 kDa. These findings suggest that the scope for the use of B. cereus SU12 strain as suited organism for the industrial production of the extracellular protease enzyme.
“…The result showed that the enzyme activity was maximum (180±0.06 unit/ml) at pH 7 and minimum (120±0.02 unit/ml) at pH 4 ( Figure 6). Similar results were obtained for the optimum pH for enzymatic activity of other Bacillus sp: pH 7.5 for B. subtilis ITBCCB 148 (Yandri et al, 2008), Bacillus sp. HS08 (Huang et al, 2006) and Bacillus sp.…”
Section: Effects Of Ph On Protease Activitysupporting
Protease-producing bacterium Bacillus cereus SU12 was isolated from oyster Saccostrea cucullata. Fifteen strains of bacteria were isolated from oyster S. cucullata and screened for secretion of protease on casein agar plates. Among them, SU12 isolate was selected due to its high enzyme production capacity and was identified as B. cereus SU12 on the basis of its morphological, biochemical and 16S rDNA properties. Media and cultivation conditions were studied to optimize bacterial growth and protease production which includes different carbon and nitrogen sources, in addition to different factors such as incubation time, pH, temperature, NaCl concentrations. At pH 7, temperature 40°C and 2.5% NaCl concentration, carbon source such as starch and beef extract as nitrogen source, the protease activity was maximum. Extracellular protease was isolated, purified to 8.73 fold by diethyl aminoethyl (DEAE) ion-exchange chromatography and its specific activity was determined to be 886.56 U/mg and the sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) showed a single band for the purified enzyme, with an apparent molecular weight of 66 kDa. These findings suggest that the scope for the use of B. cereus SU12 strain as suited organism for the industrial production of the extracellular protease enzyme.
“…S17110, Bacillus sp. HS08 (Huang et al 2006 ) and Bacillus subtilis ITBCCB 148, (Yandri et al 2008 ) pH 8.0 for Bacillus cereus KCTC 3674, (Kim et al 2001 ). Fig.…”
The aim of the study was to identify new sources of substrate from agro-industrial waste for protease production using Bacillus sp., a local bacteria isolated from an agro-waste dumping site. The strain was identified as Bacillus sp. BT MASC 3 by 16S rRNA sequence followed by phylogenic analysis. Response surface methodology-based Box–Behnken design (BBD) was used to optimize the variables such as pH, incubation time, coffee pulp waste (CPW) and corncob (CC) substrate concentration. The BBD design showed a reasonable adjustment of the quadratic model with the experimental data. Statistics-based contour and 3-D plots were generated to evaluate the changes in the response surface and understand the relationship between the culture conditions and the enzyme yield. The maximum yield of protease production (920 U/mL) was achieved after 60 h of incubation with 3.0 g/L of CPW and 2.0 g/L of CC at pH 8 and temperature 37 °C in this study. The molecular mass of the purified enzyme was 46 kDa. The highest activity was obtained at 50 °C and pH 9 for the purified enzymes.
“…Some previous researches performed with immobilization and chemical modification have shown that the enzymes obtained have significantly increased their stability against pH and temperature compared to the native enzyme (Yandri, et al, 2008(Yandri, et al, , 2010a(Yandri, et al, , 2010bGermain dan Crichton, 1988;Cordt, et al, 1992;Steadman, et al, 1991;Skerker dan Clark, 1987;Rani, et al, 2007;Reshmi, et al, 2007).…”
This paper describes the stability increase of α-amylase obtained from Bacillus subtilis ITBCCB 148 by immobilization process using carboxymethyl cellulose (CM-Cellulose) as the immobile matrix. To achieve this aim the enzyme was purified by the following steps: fractionation with ammonium sulphate, dialysis, ion exchange column chromatography with CM-cellulose and molecule filtration column chromatography with Sephadex G-100. The purified enzyme was then immobilized with CM-Cellulose. The result showed that the immobilization with CM-cellulose on α-amylase obtained from B. subtilis has successfully increased the thermal stability of the native enzyme. The thermal stabilities of the modified enzyme were increased 3.67 times compared to the native enzyme. The decrease of ki value, the increase of half-life and ΔGi values showed that the modified enzymes were more stable than the native enzyme.
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