Abstract
The use of the cyclodextrin glucanotransferase (CGTase) of the US132 strain, which is an effective anti-staling agent, has been hampered by its high cyclization activity. Since that random mutagenesis using error-prone PCR is nowadays a method of choice for enzymes engineering, we have optimized this method by adjusting manganese concentration in order to obtain a high percentage of active CGTase mutants. Therefore, the amplification of the gene encoding the US132 CGTase was performed using a MnCl2 concentration ranging between 0 and 0.5 mM. The finding showed that a manganese concentration of 0.04 mM allowed for 90 % of active mutants. A simple method to rapidly screen the obtained mutants was also developed. After the examination of a small library (of less than 1000 clones), the active mutant named MJ13 was selected for a significant decrease in the cyclization activity, thereby showing a remarkable change in the enzyme specificity towards starch dextrinizing. Sequence analysis showed that MJ13 is a triple mutant with two mutations in the catalytic domain (K47E and S382P) and one substitution in the starch binding domain (N655S).
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Acknowledgments
This research was supported by Grants-in-Aid for Scientific Research from the Ministry of High Education and Scientific Research of Tunisia (Contrat Programme CBS-LMB). We are thankful to Mrs. Salma Karray, an EFL teacher, for her valuable language polishing services.
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Sonia Jemli and Mouna Jaoua have equally contributed to the present study.
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Jemli, S., Jaoua, M. & Bejar, S. US132 Cyclodextrin Glucanotransferase Engineering by Random Mutagenesis for an Anti-Staling Purpose. Mol Biotechnol 58, 551–557 (2016). https://doi.org/10.1007/s12033-016-9952-z
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DOI: https://doi.org/10.1007/s12033-016-9952-z