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Immobilization and Performance Evaluation of alpha-Amylase BmaN2 on Silica Propyldiethylenetriamine-Glutaraldehyde Matrix (a) Analytical Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung, Indonesia Abstract alpha-Amylase (E.C.3.2.1.1) is an enzyme that works specifically to hydrolyze alpha-1,4-glycosidic bonds in starch into glucose, oligosaccharides, and maltotriosadextrin units, which are raw materials for various industries. Starch processing with alpha-amylase has the least negative effects when compared to the use of non-enzyme chemicals. However, the starch processing industry requires an abundant amount of α--amylase because it cannot be used repeatedly. Immobilization is one of the techniques that can be used to allow repeated use of the enzyme. In this study, alpha-amylase from Bacillus megaterium NL3 (BmaN2) was immobilized on propyldiethylentriamine-glutaraldehyde silica matrix. This study also aimed to evaluate the performance of immobilized BmaN2 using starch solution. The matrix to immobilize BmaN2 was based on silica synthesized from tetraethylorthosilicate (TEOS) precursor by Stober method. The synthesized silica was further modified using N-1-(3-trimethoxysilpropyl)diethylentriamine (N-TMSPen)-glutaraldehyde. The success of silica synthesis was determined by infrared (IR) spectroscopy, i.e. the presence of absorption at wave number 1093 cm-1 (Si-O-Si vibrations) indicates that silica has been successfully synthesized. Typical vibrations of N-H at 1400 cm-1 and 3421 cm-1 indicate the successful modification of silica has been modified with propyldiethylentriamine. The C=O vibration at 1654 cm-1 indicated the successful modification of silica with glutaraldehyde, while the successful immobilization of the BmaN2 enzyme was marked by the presence of C=N vibration at 1544 cm-1. In addition to the results of the IR spectrum, the success of silica modification is determined from the color change of the matrix from white to brownish yellow also gives an indication of the success of the modification carried out. Based on SEM images, the morphology of silica, propyldiethylentriamine silica, and propyldiethylentriamine-glutaraldehyde silica is spherical. The presence of nitrogen atoms detected in Energy Dispersive X-ray Spectroscopy (EDS) also support the data on the success of the modification carried out. Based on Scanning Electron Microscopy (SEM) images, the morphology of silica, propyldiethylentriamine silica, and propyldiethylentriamine-glutaraldehyde silica is spherical. Characterization with Particle Size Analyzer (PSA) gave the average size of propyldiethylentriamine silica as 1174.6 nm, propyldiethylentriamine-glutaraldehyde silica as 1551.9 nm, and immobilized BmaN2 enzyme as 1174.6, 1551.9, and 5312 nm, respectively. In determining the enzyme performance test, the optimum conditions for free BmaN2 were pH 6, 30 minutes reaction time, and reaction temperature of 30 oC. For the immobilized BmaN2, the optimum conditions were pH 6, 90 minutes reaction time, and reaction temperature of 60 oC. The optimum starch solution concentration for immobilized BmaN2 was 2% m/v and free BmaN2 was 1% m/v. The Michaelis Menten constant (KM) values for immobilized and free BmaN2 were 1.55 and 2.23, respectively. These results show that the affinity of the enzyme in immobilized BmaN2 towards starch solution is higher than that of free BmaN2. Regarding the stability of BmaN2, the immobilized BmaN2 still has activity after being used repeatedly ten times and after being stored for two months in the refrigerator (0 oC). The results of this study indicate that silica modified with propyldiethylentriamine-glutaraldehyde can be used to immobilize BmaN2 so that it has the potential to be applied to the industrial world. Keywords: alpha-Amylase, immobilization, silica, N-TMSPen, glutaraldehyde Topic: Biokimia Pangan |
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