Construction and Expression of a-Amylase from Bacillus megaterium NL3 (BmaN 1) Without 22 Hydrophobic Amino Acid Residues on C-Terminal

• Main Author: Aji Widhi Astuti, Siwi
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38575
• Institution: Institut Teknologi Bandung

Starch is a glucose homopolymer consists of amylose and amyl opectin. a.­ Amylase (EC. 3.2.1.1) is an enzyme with ability to hydrolyze starch at o.-1,4- glycosydic bond releasing linear and branched oligosaccharides. An a.-amylase BmaNl i s isolated from Baczllus megatenum NL3 in Kakaban Lake, Derawan Island, Indonesia. Expression of bmaN1 in Escherichia coli BL2l (DE3) resulting BmaN 1 in the form of inclusion bodies. Protein agregation is suspected caused by the presence of hydrophobic transmembrane helix in C-tenninal of protein. The aim of this research i s to construct and express a.-amylase mutant of BmaN I without 22 hydrophobic amino acid residues on C-terminal as a soluble protein. BmaNl mutation had been done on a codon GGG which codes Gly440 resulting a ;:,tvp .:.vdun TAG by PCR (Polymcia;:,c Chain Rcactiun ) technique. Mutant verification had been done by DNA Sequencing. Screening of one factor for bmaN 1LJ(' expression optimization had been do.ne is a variation of host cell E. cob BL21 (OE3) and E. coli ArcticExpress (DE3). Result shows that expression is optimum in E. coli BL21 (DE3) marked by high and consistent a.-amylase specific activity SDS-PAGF analy<;i<; re<;ult <;how<; that molecular weight of RmaNl ::.no BmaN1C respectively are -55 kDa and -49 kDa. a.-Amylase activity of BmaN I and BmaNIC crude extract are identified based on the amount of reducing sugar group resulted from hydrolysis of soluble and raw starch. a.-Amylase specific activity of BmaN1 and BmaN1C on soluble starch respectively are 22,92 U/mg and 80,23 U/mg. The degree of raw starch hydrol ysis of BmaNlC on sagoo, canna, wheat, and cassava respectively are 13,58%, 11 ,60%, 27,17%, dan 14,84%. Meanwhile, BmaN1 relatively has no hydrol ysis activity of raw starch on those substrates.