Improvement of Creatinase Thermal Stability Through Saturation Mutagenesis

Abstract:

To improve the thermal stability of creatinase（EC 3.5.3.3，CRE)，the "hot-spot" Lys195 in Arthrobacter nicotianae 23710 CRE is identified by sequence alignment，and saturation mutagenesis is conducted at this site. In contrast to wild-type enzyme，the mutants K195V，K195T，K195C，and K195L exhibited 260%，230%，60%，and 20% increase in half-life at 50 ℃，respectively；the specific activity of K195V and K195C are respectively increased by 80.7% and 88.2%. Furthermore，the catalytic efficiency（k_cat/K_m）of mutants K195V，K195T，K195C，and K195L are increased by 131%，218%，83% and 100%，respectively. As indicated by structure analysis，the number of hydrogen bonds of the K195V，K195T，and K195L are increased by 7，12 and 13 in comparison with the wild-type enzyme，respectively. The results indicated mutations at Lys195 could affect the thermal stability and catalytic efficiency of CRE，and the increase in hydrogen bonds may account for the improved thermal stability of CRE.