US132 Cyclodextrin Glucanotransferase Engineering by Random Mutagenesis for an Anti-Staling Purpose.
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).
Laboratoire de Biotechnologie Microbienne et d'Ingénierie des Enzymes, Centre de Biotechnologie de Sfax, Université de Sfax, Route de Sidi Mansour Km 6, BP 1177, 3038, Sfax, Tunisie. soniajemli@yahoo.fr.
Sonia
S
Jemli
Laboratoire de Biotechnologie Microbienne et d'Ingénierie des Enzymes, Centre de Biotechnologie de Sfax, Université de Sfax, Route de Sidi Mansour Km 6, BP 1177, 3038, Sfax, Tunisie.
Mouna
M
Jaoua
Laboratoire de Biotechnologie Microbienne et d'Ingénierie des Enzymes, Centre de Biotechnologie de Sfax, Université de Sfax, Route de Sidi Mansour Km 6, BP 1177, 3038, Sfax, Tunisie.
Samir
S
Bejar
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D003500:Cyclization; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry; D016296:Mutagenesis; D056507:Paenibacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D013213:Starch / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008345:Manganese; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase
Active mutant; Anti-staling; Cyclodextrin glucanotransferase; Error-prone PCR; Library screening