ROLE OF TRYPTOPHAN179 OF &#945--AMYLASE BMAN2 IN STARCH BINDING
Muhammad Aqib Hanif, Reza Aditama, Dessy Natalia

Institut Teknologi Bandung

Abstract

Plants typically use starch as the origin of carbon and energy. Seeds, roots, tubers, and legumes are the most common sources of starch. Enzymatic hydrolysis of raw starch can be accomplished with the aid of &#945--amylase. Bacillus megaterium NL3 from Lake Kakaban in East Kalimantan, Indonesia produces &#945--amylase BmaN2 which belongs to the GH13 family. BmaN2 has the ability to hydrolyse raw starch into linear and branched oligosaccharides. In Silico study of BmaN2 revealed that Tyr101, His141, and Trp179 are predicted to play role as SBS (Surface Binding Sites). The purpose of this research was to determine the role of W179 residue in substrate binding. Therefore, to find out the role of W179, two variants of bmaN2 constructed as bmaN2 W179F and bmaN2 W179A by site directed mutagenesis of TGG into TTC to produce bmaN2 W179F and TGG into GCG to construct bmaN2 W179A. Site directed mutagenesis was performed by PCR using a recombinant plasmid template pET30a-bmaN2 carrying bmaN2 gene. The expression of bmaN2 WT, bmaN2 W179F, and bmaN2 W179A in Escherichia coli BL21(DE3) was induced by the addition of 0.1 mM IPTG at 25 &#8451- for 4 hours. SDS-PAGE analysis showed the presence of a protein with a molecular mass of 61.5 kDa. The specific activities for BmaN2 WT, BmaN2 W179F, and BmaN2 W179A were 1.61 U, 1.43 U, and 0.69 U, respectively. These results indicate that W179 of BmaN2 has an important role in substrate binding.

Keywords: &#945- -amylase, BmaN2, SBS, Site directed mutagenesis , tryptophan179

Topic: Bioteknologi

Plain Format | Corresponding Author (Muhammad Aqib Hanif)

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