DETERMINATION OF ENZYMATIC ACTIVITY AND BIOPHYSICAL CHARACTERS OF BMAN2, BMAN2 W198A, AND BMAN2 W198F

• Main Author: Shifa Aulya Wildania, Alyn
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/72975
• Institution: Institut Teknologi Bandung

Starch is a polysaccharide consisting of amylose and amylopectin. The presence of amylose can form a hydrophobic cavity that makes starch difficult to dissolve in water, thus requiring a gelatinization process at temperatures above 60°C for enzymatic hydrolysis. One alternative in order to save industrial costs is by utilizing raw starch degrading amylase (RSDA), an amylase that has the ability to hydrolyse raw starch without the gelatinization step. Bacillus megaterium NL3 isolate from Lake Kakaban, East Kalimantan is known to produce one of the raw starch degrading ?-amylase, BmaN2, which does not have an additional starch-binding domain, but is believed to have residues that play a role in starch binding. Computational studies show that Trp198 (W198) residue of BmaN2 is suspected to play an important role in starch binding. Therefore, the aim of this research is to determine the activity and biophysical character of Trp198 residue on BmaN2 variants, namely, the wild type, W198A mutant, and W198F mutant, towards the binding of raw starch and soluble starch. BmaN2 was produced in E. coli BL21 (DE3) as a soluble protein. The protein solution was purified using Ni-NTA affinity chromatography and characterized using spectrofluorometric and DNS activity assay. The successful expression of BmaN2 was confirmed using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The expression and purification analysis of the protein using SDS-PAGE showed the presence of a band at ~61.5 kDa size, which corresponds to the size of BmaN2. The pure mutant BmaN2 activity assay using soluble starch substrate showed a deficiency of activity by 47% (W198A) and 48% (W198F) compared to the wild type. The characterization results using spectrofluorometric based on tryptophan emission showed that the three structures did not undergo significant changes. This was indicated by the relatively similar spectrum peak, which was at a wavelength of 340 nm. However, the fluorescence spectrum showed a decrease in emission intensity in BmaN2 W198A and W198F compared to the wild type, indicating an increase in tryptophan flexibility. In addition, the addition of starch solution caused a shift in wavelength from 340 nm to 355 nm (red-shifted) in both the wild type and mutant BmaN2, indicating a conformational change in BmaN2 due to interaction with starch. Tryptophan tends to be buried in a hydrophobic cavity, but when interacting with starch, the enzyme conformation becomes more open. Furthermore, the Circular Dichroism (CD) analysis showed the similarity of the secondary structure between the wild type and W198F mutant, while W198A showed a different secondary structure.