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Serratia marcescens B4A chitinase thermostability enhancement by S390I QuikChange site directed mutagenesis | ||
| Iranian Journal of Fisheries Sciences | ||
| مقاله 217، دوره 18، شماره 4، دی 2019، صفحه 1046-1059 اصل مقاله (586.05 K) | ||
| نوع مقاله: Research Paper | ||
| شناسه دیجیتال (DOI): 10.22092/ijfs.2019.119292. | ||
| نویسندگان | ||
| Zeinab Emruzi tubkanlu؛ Saeed Aminzadeh* ؛ Ali Asghar Karkhane؛ Jahan Alikhajeh؛ Ahmad Ghoroghi | ||
| NIGEB | ||
| چکیده | ||
| Thermostable chitinases are useful for economical, industrial and biotechnological applications. In this paper we attempted to stabilize chitinase from Serratia marcescens B4A by rational mutagenesis and changing of Ser 390 to Ile. This stabilization was performed through entropic stabilization by reduction of the loop length and also by increasing of the beta chain length. On the other hand, with this replacement, polar uncharged residue changed to non-polar one and increased the hydrophobic interactions, furthermore Isoleucine has branched β-carbon that restricted the backbone conformation more than nonbranched residues. Finally all of these factors lead to entropic stabilization and thermal stabilization. The results exhibited that the optimal temperature and pH for enzyme activity of native chitinase weren’t changed by mutagenesis which showed mutation didn’t effect at original characteristics of enzyme, the Km values of native and mutant chitinase was differed very little that showed the affinity of enzyme toward the substrate and natural flexibility of chitinase didn’t changed by mutation, the Vmax value of mutant chitinase was decreased and pH stability of mutant chitinase was increased briefly but its thermal stability was increased remarkably. Mutation made chitinase tolerated high temperatures to 90°C. In addition its activity was increased at 50°C, 60°C for 120 min and up to 2 hours of incubation period and the mutant chitinase showed a high level of activity at 60°C. These results show that entropic stabilization works well for chitinase and this approach may be generally applicable for stabilization of other proteins. | ||
| کلیدواژهها | ||
| Chitinase؛ Thermal stability؛ Serratia marcescens B4A؛ Loop؛ β chain؛ unfolding | ||
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