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کاربرد نوع شرط:
- جایگاه : پژوهشی
- مجله: Iranian Journal of Fisheries Sciences
- نوع مقاله: Journal Article
- کلمات کلیدی: Chitinase,Thermal stability,Serratia marcescens B4A,loop,β chain,unfolding
- چکیده:
- چکیده انگلیسی: 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.
- انتشار مقاله: 16-08-1395
- نویسندگان: Zeinab Emruzi Tubkanlu,Saeed Aminzadeh,Ali Asghar Karkhane,Jahan Alikhajeh,Ahmad Ghoroghi
- مشاهده
- جایگاه : پژوهشی
- مجله: Advanced Research in Microbial Metabolites & Technology
- نوع مقاله: Journal Article
- کلمات کلیدی: Amylopullulanase,Pullulan,Recombinant protein,Zymogram
- چکیده:
- چکیده انگلیسی: Starch debranching enzymes that merely hydrolyze α-(1→6) glycosidic linkages are classified into isoamylases (EC 3.2.1.68) and pullulanases (EC 3.2.1.41). An exception to this definition would be amylopullulanase, a type of pullulanase that is capable of cleaving both α-(1→4) and α-(1→6) linkages. Amylopullulanases are in demand in liquid sugar industries to generate glucose and some other starch derivatives. Pullulanases can be used in conjunction with amylases to improve sugar availability during sugar syrup production. Here, a thermophilic Cohnella sp. A01 amylopullulanase (EC 3.2.1.41) gene, namely Coh4159, was PCR amplified and cloned in pET-26b(+) and transformed into BL21(DE3). Recombinant Coh4159 was heterologously expressed in the presence of 0.5 mM IPTG and purified via affinity chromatography, and further characterized. Enzyme activity was demonstrated via zymogram analysis in the presence of pullulan. The enzyme had a hydrolytic effect on pullulan with Vmax = 2.85 µmol.min-1 and Km = 0.5 mM. Temperature optima and pH were 60 ˚C and 6.0. In which the enzyme kept its activity at wide pH (4-9) and temperature (30-70 ˚C) ranges. The recombinant enzyme kept 50% of its activity for 60 min, 100 min and 120 min when incubated at 80, 70 and 60 ˚C, respectively. Amongst metal ions tested, Mn2+, and Ca2+ have improved the enzyme activity both at 5 and 10 mM. The results promise the capability of producing a commercial industrial enzyme, well-suited to liquid sugar syrup industry specification.
- انتشار مقاله: 02-11-1396
- نویسندگان: Parvin Valiulahi,Saeed Aminzadeh,Mehdi Shamsara,Naser Farrokhi,Jahan Alikhajeh
- مشاهده