Determination of Enzymatic Activity and Biophysical Characters of BmaN2, BmaN2 W198A, and BmaN2 W198F
Alyn Shifa Aulya Wildania, Rindia Maharani Putri, Ihsanawati

Institut Teknologi Bandung

Abstract

Starch, consisting of amylose and amylopectin, requires gelatinization for enzymatic hydrolysis due to its hydrophobic cavity that hinders water solubility. To reduce industrial costs, raw starch degrading amylase (RSDA) can be used, eliminating the need for gelatinization. BmaN2, an alpha-amylase produced by Bacillus megaterium NL3, lacks an extra starch binding domain but possesses starch-binding residues. Computational studies highlight Trp198 (W198) residue as potentially crucial for starch binding. Thus, this research aims to investigate the activity and biophysical properties of BmaN2 variants wild type, W198A mutant, and W198F mutant, regarding their interaction with raw and soluble starch. BmaN2 was obtained as a soluble protein by expressing it in E. coli BL21 (DE3), followed by purification using Ni-NTA affinity chromatography. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) confirmed successful BmaN2 expression, showing a band at approximately 61.5 kDa, corresponding to BmaN2^s size. Activity assays using soluble starch substrate revealed reduced activity in the pure mutant BmaN2, with a deficiency of 47% (W198A) and 48% (W198F) compared to the wild type. Spectrofluorometric analysis based on tryptophan emission indicated minimal structural changes among the three variants, as evidenced by the similar peak at 340 nm. However, fluorescence spectra demonstrated decreased emission intensity in BmaN2 W198A and W198F, suggesting increased tryptophan flexibility. The addition of starch solution caused a red-shift in wavelength from 340 nm to 355 nm in both the wild type and mutant BmaN2, indicating conformational changes due to starch interaction. This interaction opens the enzyme^s conformation, exposing tryptophan residues initially buried in the hydrophobic cavity. Circular Dichroism (CD) analysis revealed similar secondary structures between the wild type and W198F mutant, while the W198A mutant exhibited a distinct secondary structure.

Keywords: alpha-amylase BmaN2, CD, raw starch, spectrofluorometric, substrate binding residues

Topic: Bioteknologi