PERFORMANCE EVALUATION OF IMMOBILIZED ?-AMYLASE BMAN2 ON SILICA-APTES-GLUTARALDEHYDE MATRICES

• Main Author: Daniel Despen Sihombing, Zico
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/72884
• Institution: Institut Teknologi Bandung

The use of enzymes in the industrial world has often been applied as a biocatalyst, this is evidenced by the high number of industrial enzymes used which reached 12.46 billion USD in 2022. ?-Amylase (E.C.3.2.1.1) is one of the enzymes that is often used in the processing industry starch. Industrial production costs increase because enzymes are generally used only once in high enough quantities. Enzyme immobilization is one way to overcome this problem. This study aims to immobilize the crude ?-amylase extract from Bacillus megaterium NL3 (BmaN2) covalently on 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde modified silica matrix. Covalent immobilization of the enzyme is intended to obtain BmaN2 which is more resistant to extreme conditions. Synthesis of the silica matrix was carried out by the Stober method with the precursor tetraethylorthosilicate (TEOS), isopropanol as a solvent, water as a hydrolyzing agent, and ammonia as a catalyst. Based on the infrared spectrum (IR), silica has a vibrational absorption band of Si–O–Si siloxane bonds at 1093 cm–1 and O–H silanol at 3441 cm–1 indicating that silica was successfully synthesized. Then, the silica obtained was modified by adding APTES. IR characterization showed absorption bands of primary N–H bond vibrations at 3404 cm–1 and 1635 cm–1 and C–N bond vibrations at 1400 cm–1 which indicated that the modification of silica with APTES was successful. Silica-APTES which was successfully synthesized was then re-modified with glutaraldehyde. The success of the modification was indicated by the presence of a C=O vibrational absorption band at 1637 cm–1 with a color change from white to reddish-beige. Self condensation between APTES particles (without silica) did not occur as evidenced by the fact that the color of the solution did not change to become cloudy when synthesized. BmaN2 immobilization was carried out by contacting the enzyme with the synthesized matrix. The success of immobilization is determined based on the infrared spectrum and performance determination. Infrared spectral characterization showed the presence of a C=N vibrational absorption band at 1533 cm–1 which indicated that immobilization by covalent binding was successful. The average particle diameter obtained from the Particle Size Analyzer (PSA) characterization for silica-APTES, silica-APTES-glutaraldehyde, and immobilized BmaN2 were 937.3; 1060.9; and 2338.7 nm. The higher particle diameter also indicates successful modification and immobilization. Then, based on the determination of atomic percent by Energy Dispersive Spectroscopy (EDS) it can be seen that along with the modification of silica with APTES and glutaraldehyde, the atomic percent of carbon and nitrogen is also higher, the same thing happens to BmaN2 immobilized on silica-APTES-glutaraldehyde. This increase in atomic percent indicates that matrix modification and BmaN2 immobilization into the matrix were successful. Determination of enzyme performance showed that the enzyme had an optimum pH of 6 for free and immobilized BmaN2, optimum reaction time of 30 minutes for free BmaN2 and 90 minutes for immobilized BmaN2, optimum reaction temperature at 30 °C for free BmaN2 and 60 °C for immobilized BmaN2, and the optimum concentration of starch solution is 1% w/v for free BmaN2 and 2% w/v for immobilized BmaN2. Differences in the optimal performance state for free BmaN2 and immobilized BmaN2 can occur due to conformational changes in the 3-dimensional structure of the enzyme. Kinetic calculations show that immobilized BmaN2 (Km=1.62 mg/mL) has a stronger starch binding affinity than free BmaN2 (Km=2.22 mg/mL). The use of immobilized BmaN2 up to 10 times still leaves a specific activity of 12.46% and is stable for use up to 60 days of storage.