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Y93F substitution of cyclodextrin glucanotransferase from bacillus sp. A2-5a and its enzyme characterization
Rostinawati T.a,b, Riani C.a, Elfahmi E.a, Sumirtapura Y.C.a, Retnoningrum D.S.a
a Laboratory of Pharmaceutical Biotechnology, Bandung Institute of Technology, School of Pharmacy, Bandung, Jawa Barat, 40132, Indonesia
b Laboratory of Microbiology, Faculty of Pharmacy, Padjadjaran University, Bandung, Jatinangor, Jawa Barat, 45363, Indonesia
[vc_row][vc_column][vc_row_inner][vc_column_inner][vc_separator css=”.vc_custom_1624529070653{padding-top: 30px !important;padding-bottom: 30px !important;}”][/vc_column_inner][/vc_row_inner][vc_row_inner layout=”boxed”][vc_column_inner width=”3/4″ css=”.vc_custom_1624695412187{border-right-width: 1px !important;border-right-color: #dddddd !important;border-right-style: solid !important;border-radius: 1px !important;}”][vc_empty_space][megatron_heading title=”Abstract” size=”size-sm” text_align=”text-left”][vc_column_text]© 2015 Asian Network for Scientific Information.In this recent study, Tyr93 of Bacillus sp. A2-5a (BA2-5a) cyclodextrin glucanotransferase (CGTase) was replaced with Phe residue by site-directed mutagenesis to study its role on the product specificity and kinetic properties. Molecular docking approach was also applied to acquire a detailed analysis of enzyme-substrate interaction. The Y93F was overproduced in Escherichia coli BL21 (DE3) and purified by Ni-NTA resin. The purified Y93F CGTase was 76.39 kDa based on 10% SDS-PAGE analysis and showed both β-cyclization and starch hydrolysis activities in a zymography assay. There was no major structural conformational change in the Y93F since, its optimum and stability of temperature and pH were the same as the wild type. The substitution did not alter the cooperativity property of the enzyme. Our work provided the new evidence that Y93F substitution caused no α-cyclodextrin (CD) formation in BA2-5a CGTase. In addition, the Y93F produced more β-CD and decreased its kcat and kcat/Km. In conclusion, Y93F substitution of BA2-5a CGTase alters the enzyme kinetic property and product specificity.[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Author keywords” size=”size-sm” text_align=”text-left”][vc_column_text]Bacillus sp,Cyclodextrin glucanotransferases,Enzyme characterization,Enzyme-substrate interactions,Escherichia coli bl21,Product specificity,Site directed mutagenesis,Y93F cgtase[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Indexed keywords” size=”size-sm” text_align=”text-left”][vc_column_text]Bacillus sp. a2-5a,Kinetic,Product specificity,Site-directed mutagenesis,Y93F cgtase[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Funding details” size=”size-sm” text_align=”text-left”][vc_column_text][/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”DOI” size=”size-sm” text_align=”text-left”][vc_column_text]https://doi.org/10.3923/biotech.2015.181.187[/vc_column_text][/vc_column_inner][vc_column_inner width=”1/4″][vc_column_text]Widget Plumx[/vc_column_text][/vc_column_inner][/vc_row_inner][/vc_column][/vc_row][vc_row][vc_column][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][/vc_column][/vc_row]