CHARACTERIZATION OF W198F AND H141Q ?-AMYLASE RECOMBINANT MUTANT FROM BACILLUS MEGATERIUM NL3 WITH RAW STARCH ACTIVITY TEST AND BIOINFORMATICS ANALYSIS

• Main Author: Pramudya, Reivaldi
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/55120
• Institution: Institut Teknologi Bandung

Starch is a source of carbohydrates for the human body. Starch is produced from photosynthesis which is stored for a long time. Starch-producing crops such as rice, corn and potatoes have been widely cultivated by humans. Currently, starch is widely used in industries such as the food, textile, pharmaceutical and paper industries. Starch is generally degraded into oligosaccharides which can then be utilized according to their needs. Due to its semicrystalline nature, starch granules must be broken down to easily hydrolyze them through a heating process called gelatinization. The starch hydrolysis process can be carried out using acid and heating, but many industries have switched to using enzymatic reactions because they are more environmentally friendly, work specifically, and are needed in small amounts. ?-Amylase (EC 3.2.1.1) is an enzyme that catalyzes the hydrolysis of starch by breaking ?-1,4-glycosidic bonds. ?-Amylase is produced by many organisms, one of organism is Bacillus megaterium NL3 which lives in Lake Kakaban, East Kalimantan, Indonesia. These organisms produce 3 types of ?-amylase, BmaN1, BmaN2, and BmaN3. BmaN2 has the ability to degrade raw starch so that hydrolysis products can be obtained without gelatinization. In addition, BmaN2 does not have an additional domain, starch binding domain (SBD), which is generally owned by raw starch degrading enzymes which function to bind to the substrate and help the ?-amylase penetration. However, it is estimated that BmaN2 has residues that play a role in the binding of the substrate called the surface binding site (SBS), Tyr101, His141, Trp179, and Trp198. The purpose of this study was to determine the role of the two residues estimated as SBS, His141 and Trp198 through the activity test of raw starch and bioinformatics analysis. In previous research, mutations were carried out in the genes coding for histidine and tryptophan to produce two BmaN2 mutants, W198F and H141Q, where the genes encoding all types of bman2 were inserted into the pET30a expression vector. All types of BmaN2 are produced in E. coli BL21 (DE3) as dissolved proteins. The purification of BmaN2 was carried out by metal affinity chromatography of Co- NTA. Wild type BmaN2 has specific activity on 1% (b/v) dissolved substrate at 21.21 U/mg. The specific activity of BmaN2 W198F and H141Q mutants on 1% of the substrate decreased by 1.17% and 59.78%, respectively. In addition, the activity of BmaN2 H141Q on 3% (b/v) wheat and potato starch in time variation has a random pattern when compared to wild type BmaN2 and BmaN2 W198F which have regular patterns such as the Michaelis-Menten curve. The docking result between the analog substrate (DAF ligand), which was obtained from ?-amylase from Hordeum vulgare, showed a lost interaction between ?-amylase and the substrate when His141 was replaced with glutamine. On the other hand, the interaction between ?-amylase and the substrate was maintained when Trp198 was replaced with phenylalanine. Based on these two types of analysis, His141 is a strong candidate as SBS, but other methods are still needed to further confirm the role of the two residues, such as dissociation constant analysis using the Surface Plasmon Resonance (SPR).