Domain analysis and site-directed mutagenesis of a thermophilic pullulanase from Thermotoga maritima MSB8

Abstract

Abstract The structure of Thermotoga maritima MSB8 pullulanase (PulA) was modified for checking the relationship of domain and site-directed mutagenesis with enzymatic properties. PulA was roughly linked by five domains as CBM41-X-CBM48-catalytic domain-C domain from N-terminal to C-terminal. The deleted certain domains mutants as PulA1 (deleted CBM41) and PulA2 (deleted CBM41-X) were survived with different properties, whereas the PulA3 (deleted CBM41-X-CBM48) and PulA4 (deleted C domain) lose their enzyme activities. The apparent melting temperatures (Tmapp) measured by differential scanning fluorimetry of PulA, PulA1 and PulA2 were 76.5 oC, 78.8 oC and 66.6 oC, respectively. The thermal stability of PulA1 was increased slightly but PulA2 was reduced significantly, which shows that the existence of X domain may play a positive role for stability of PulA. To investigate the interaction of CBM48 with X domain, a few site-directed mutants as M1 (E172G), M2 (E172G/R190G), M3 (E172G/D198G), M4 (E172G/R190G/D198G) on the surface of X domain were constructed to reduce the hydrogen bond between X domain with CBM48. The optimum temperature of those four mutants and PulA were 90 oC. The Tmapp values of M1, M2, M3, and M4 were 77.4 oC, 71.7 oC, 73.2 oC and 71.9 oC, respectively. The specific activity of M1 and M2 were increased slightly, while the pH stability of M3 and M4 were significantly improved at low pH. This study provides the information of the structure-function relationship of pullulanase.