Perpustakaan Digital ITB

Recombinant cyclodextrin glycosyltransferase (rCGTase BA2-5a) is an enzyme naturally produced by Bacillussp. A2-5a and recombinantly produced by Escherichia coli BL21(DE3). This enzyme catalyzes the formation of cyclodextrins from starch and has a size of approximately 75 kDa. The determination of three-dimensional protein structures using cryo-electron microscopy (cryo-EM) generally requires a minimum protein size of 100 kDa, thus necessitating a strategy to increase the size of CGTase without affecting enzyme’s structure and activity. This study aimed to construct the recombinant plasmid pJx_mbp-cgtase encoding the MBP-rCGTase BA2-5a fusion protein and to examine the effect of Maltose Binding Protein (MBP) fusion on ?-cyclization activity. The mbp gene was inserted into the pJx_cgtase template vector using QuickStep Cloning, a restriction-ligation-free, megaprimer-based cloning method. The construct was confirmed through migration analysis, colony PCR, restriction analysis, and DNA sequencing. The recombinant plasmid was subsequently expressed in E. coli BL21(DE3) under IPTG induction, and the fusion protein was purified by affinity chromatography. The results showed that the fusion enzyme retained its ?-cyclization activity, producing ?-cyclodextrin as the dominant product from both soluble starch and pineapple stem starch substrates. The successful binding of the fusion protein to amylose resin confirmed that the MBP domain folded into a correct and functional conformation. This study demonstrates that MBP fusion can increase the size of rCGTase BA2-5a without interfering with its cyclization activity, using the same substrates in previous research which was soluble starch and pineapple stem starch.