CONSTRUCTION AND CHARACTERIZATION OF MUTANT BAQA Α-AMYLASE WITHOUT 34 HYDROPHOBIC AMINO ACID RESIDUES AT C-TERMINAL

• Main Author: ULPIYANA (NIM: 20511307) ; Pembimbing Dr. Dessy Natalia, AYRA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/18119
• Institution: Institut Teknologi Bandung

&#945;-Amylase (1,4-&#945;-D-glucan-glucanohydrolase, E.C 3.2.1.1) hydrolyzes an &#945;-1,4- <br /> <br /> <br /> <br /> <br /> glycosidic bond in starch to produce maltooligosaccharides and maltodextrin. &#945;- <br /> <br /> <br /> <br /> <br /> Amylases have many application in industries, such as in food, detergents, paper, and <br /> <br /> <br /> <br /> <br /> textiles industries. &#945;-Amylase from Bacillus aquimaris MKSC 6.2 (BaqA) contains <br /> <br /> <br /> <br /> <br /> 1539 nucleotides which encode 512 amino acid residues. BaqA is a member of new <br /> <br /> <br /> <br /> <br /> GH13 sub-family based on two consecutive tryptophan residues (W201 and W202) <br /> <br /> <br /> <br /> <br /> which might play a role in starch binding.The other member of this sub-family is <br /> <br /> <br /> <br /> <br /> Geobacillus thermoleovorans CCB_US3_US5 &#945;-amylase (GTA), which also has two <br /> <br /> <br /> <br /> <br /> tryptophan residues W204 and W205. <br /> <br /> <br /> <br /> <br /> The amino acid residues of BaqA has 50% similarity with the amino acid residues of <br /> <br /> <br /> <br /> <br /> GTA, while hydrophobic amino acid residues at C-terminal region of BaqA has 60% <br /> <br /> <br /> <br /> <br /> identity with hydrophobic C-terminal region of GTA. The truncated GTA without <br /> <br /> <br /> <br /> <br /> hydrophobic region has a compact structure of GTA which binds Ca2+ ion stronger, <br /> <br /> <br /> <br /> <br /> and has higher thermostability than its wild type counterpart. BaqA expression in E. <br /> <br /> <br /> <br /> <br /> coli BL21 (DE3) produces inactive &#945;-amylase as an inclusion body, which might be <br /> <br /> <br /> <br /> <br /> the presence of 34 hydrophobic amino acid residues at C-terminal. Hence this <br /> <br /> <br /> <br /> <br /> research was directed to study the role of the C-terminal region in the BaqA solubility <br /> <br /> <br /> <br /> <br /> and activity. To obtain BaqA mutant without the hydrophobic C-terminal region, <br /> <br /> <br /> <br /> <br /> PCR-based-site directed mutagenesis which converted GGA codon (Gly479) into <br /> <br /> <br /> <br /> <br /> stop codon (TAA) was performed.