BIOCHEMICAL AND BIOINFORMATICS APPROACH TOWARDS THE ROLES OF TRP198 AND HIS141 RECOMBINANT BACILLUS MEGATERIUM NL3 ????-AMYLASE

• Main Author: Pratiwi, Septiani
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/55385
• Institution: Institut Teknologi Bandung

Starch is a polymer of carbohydrates comprising of amylose and amylopectin. Both polymers consist of D-glucose linked by ?-1,4-glycosidic and some additional ?-1,6-glycosidic bonds forming amylopectin branches. Compisition ratio of amylopectin and amylose could affect the physicochemical characteristics and the semicrystalline structure of starch. The presence of amylose causes the formation of a single helix structure with a hydrophobic cavity, thus starch’s low solubility in water. Raw starch needs to be gelatinized for it to be hydrolysed by enzymes. Gelatinization of raw starch requires heating of the solution. On the other hand, not all enzymes are having high termal stability. An alternative would be the use of Raw Starch Degrading Amylase (RSDA) which has the ability to hydrolize starch without gelatinization. Generally, RSDA has addition domain called Starch Binding Domain (SBD) which plays a role in carbohydrate binding. ?-Amylase produced by Bacillus megaterium NL3 (BmaN2) has the ability to degrade a raw starch whitout the presence of SBD. The non-existence of SBD was compensated by the presence of Surface Binding Site which is predicted to be provided by residues His141 and Trp198. His141 is located on the molecular tweezers used to retain the bound carbohydrate. Trp198 located at the loop of A domain serves as the gate for the starch into the enzyme’s catalysis site. The aim of this research is to determine the role of His141 and Trp198 in the enzyime’s starch binding process using biochemical and bioinformatics approaches. Studies on the structure and activity of BmaN2 is done by performing site-directed mutagenesis to get BmaN2/H141K and BmaN2/W198A mutants using a recombinant plasmid constructed from a previous study. Recombinant wild type of BmaN2 and its mutants are expressed and produced by Escherichia coli BL21 (DE3) in 25 oC for 4 hours of incubation. The production of protein utilizes 0,1 mM isopropyl-?-D-1-thiogalactopyranoside as an inducer. Crude protein extract is collected from lysis using homogenizer and sonicator in lysis buffer pH 8 (50 mM NaH2PO4, 300 mM NaCl, protease inhibitor, dan 10 mM imidazole). Protein purification is done by cobalt and nickel affinity chromatography with the addition of lysis buffer without protease inhibitor and imidazole concentration variation of 50 mM, 75 mM, and 100 mM. Expression analysis indicated by the presence of ~61,5 kDa BmaN2 showed a decrease in expression efficiency for the BmaN2/H141K mutant and was dominantly aggregated. It shows the mutation on His141 into Lys141 could effect protein’s stability. BmaN2/W198A mutant showed a 52% decreased on soluble starch hydrolysation activity and doesn’t show huge difference from the wild type enzyme for wheat and potato starch degradation activity. The activity showed a regular pattern and follows Michaelis-Menten curve. The starch product visualized by Scanning Electron Microscope (SEM) shows the enzyme’s ability to degrade raw starch. Bioinformatics analyses by acarbose molecular docking showed the presence of hydrophobic interaction with the two BmaN2 residues. Carbohydrate binding score probability only showed a necessary role of His141 in starch binding. Based of both observations, Trp198 mutation into Ala198 is believed to provide a substrate specificity residue which has the ability to recognize specific substrate types. The H141K mutant on the other hand, is believed as the surface binding site. Hopefully, the capability of raw starch degradation by BmaN2 could expand opportunities for the use of BmaN2 as an alternative enviromentally friendly method in starch industry.