Efficient Expression And Improvement Of The Thermal Stability Of A Aciduric Pullulanase

Pullulanase?EC 3.2.1.41?plays an important role in the specific hydrolysis of branch points in amylopectin,with high value in starch saccharification.PulB from B.naganoensis?ATCC53909?has excellent enzymatic properties.However,its poor thermostability and low expression level make it hard to apply.In order to improve the thermostability and expression of PulB,in this study,we performed the efficient expression study of it in terms of optimization of translation profile and thermal stability.To improve the expression level of PulB,we optimized the pulb from three respects:extreme rare codon,SD-like sequence and m RNA second structure.Through the optimization of extreme rare codon of Arg,the expression levels of PulB-R573,PulB-R645 and PulB-R573/R645 were 34.37%,16.89%and 47.73%higher than that of PulB-WT,respectively.Through the knockout of the SD-like sequence in pulb,the expression level of SD2 mutant was 34.6%higher than that of PulB-WT.Besides,in the study of improving the thermal stability of PulB,the expression of Pul-N387D1 using GAC codon was decreased sharply.When codon GAC was replaced to synonym codon GAU,the stem-loop structure was removed in mRNA second structure,and the expression level of Pul-N387D2 was improved almost one time of Pul-N387D1 significantly.As the poor thermostability of PulB(its t_1/2 is only 3.5 min at 65oC),a series of site-specific mutations through homology modeling and sequence alignment were constructed previously in our lab.Based on the PulB-N387D with improved expression,in this study,combining three positive one-site mutants?D328H?N387D?A414P?,PulB-D328H/N387D and PulB-D328H/N387D/A414P mutant were constructed.And the PulB-D328H/N387D/A414P mutant had the best thermal stability.The t_1/2 of it was 12.9-fold longer than that of PulB-WT at 65°C and the total increase in T_m of it?4.91°C?was almost 60%greater than the sum of individual increases?3.1°C?.In addition,kinetic studies revealed that the k_cat and the k_cat/K_m of PulB-328/387/414 increased by 38.8%and 12.9%,respectively.Pullulanase activity of PulB-328/387/414 on the pHY300PLK in Bacillus licheniformis in fermentation broth reached 45 U/mL,more than a dozen times of which in Escherichia coli.