Site-Directed Mutagenesis palI gene and Sucrose Isomerase Enzyme Assay from Klebsiella pneumoniae in Recombinant Escherichia coli

• Main Author: Sani Alia 10414034, Insi
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28070
• Institution: Institut Teknologi Bandung

<p align="justify">Isomaltulose is an isomer of sucrose that have lower glycemic index thus allowing slower release of energy to the body. Hence, isomaltulose can be used as an alternative sugar that is good for health, therapy for diabetics, or as a sweetener for energy drink. Isomerization process of sucrose into isomaltulose is catalyzed by sucrose isomerase (SI) that is produced by several organisms including Klebsiella pneumoniae. From the previous study of SI gene, palI gene that encode SI from K. pneumoniae AxMC have been cloned to expression vector pTXB1 and expressed in recombinant Escherichia coli BL21(DE3). In this study, RLDRDS region that determine product specificity of SI were mutated into RLDRYP based on Pantoea dispersa UQ68J SI sequence. Mutation were done using inverse PCR method and then E. coli DH5&#945; transformation was performed using heat shock method. Mutation was then confirmed with DNA sequencing. Sample plasmid that contain desired mutation was then used for the transformation of E. coli BL21(DE3) for expression and enzyme assay. DNS method was used to determine enzyme activity from cytoplasm, cell membrane, extracellular, and whole cell. Protein analysis was performed using SDS-PAGE. The sequencing result showed that the DNA was mutated, the amino acid sequence was changed from RLDRDS to RLDRYP. From SDS-PAGE result, a protein between 70-100 kDa was observed, in accordance with the weight of SI protein, 71,6 kDa. Enzyme assay showed that sucrose isomerase mutant convert sucrose with lower yield compared to SI non-mutant in every cell fraction. In conclusion, RLDRDS mutation lower sucrose conversion yield and thus decrease enzyme SI activity.<p align="justify">