Improving The Thermal Stability Of The Aciduric Pullulanase By Molecular Modification And Expression

Pullulanase belongs to starch debranching enzyme with high value. It’s hard to apply in industry because of the limited ability for producing pullulanase using the natural wild strain. In fact, gene engineering technology was usually used on improving the enzymatic property and promoting the level of secretory expression of protein. The structure of pullulanase was analyzed by researchers. On this basis, the emzymatic property and the level of secretory expression of pullulanases were improved using computer simulation design.The gene of pullulanase pul from Bacillus naganoensis(ATCC53909) was expressed inductively in E.coli. The optimum temperature and pH of the combinatant pullulanase Pul is 60℃ and 4, respectively. It was very suitable for starch processing with the synergy of other glucoamylases to enhance the utilization efficiency of amylase. But it has poor thermal stability and the enzyme activity was losed after incubating for 10 min at 60℃.Therefore, in this research, it is important to improve the thermal stability of Pul.Four aciduric pullulanase sequences with high similarity from Pullulanibacillus naganoensis were analysed, and the N-terminal and C-terminal deletion sites were determined according to the different structural domains of Pul using homologous modeling method. Only Pul-D1 F had the excellent enzyme characteriatics in the mutants. The optimum temperature and pH was 60 ℃ and 4.5, as some as the wild Pul, respectively. The activity of pullulanase was totally losed after the further truncation of N-terminal and C-terminal. It suggested that these structural domains played an important role for the structure and function of Pul.Site-specific mutagenesis was carried out using Pul-D1 F as the experimental subject, and 9 mutants were constructed for the study of thermostability. While keep warm 0,2,5,10,20,30,40,50,60 min at 60℃,the hermostability of N387 D was the best among 9 single point mutants, followed by D328 H and A414 P. And when keep warm 20 min at 60℃, which residual activity respectively 58.32%,48.37% and 13.06%.The deuce and three-point mutations were constructed to further improve the thermostability of pullulanase. Three single point mutants N387 D,D328H and A414 P were used into combination mutation. The thermostability of A414P/N387D/D328 H was the best and followed by the N387D/D328 H and A414P/N387 D. And when keep warm 60 min at 60℃, which residual activity respectively 57.47%,40.88% and 26.54%.