Directional Evolution Of Pullulanase From Bacillus Naganoensis

Pullulanase specifically hydrolyzes ?-1,6 glycosidic bonds in amylopectin,and is industrially valuable in bioprocessing and biotransformation of starch.In the previous study,heterogeneous overexpression of Bacillus naganoensis pullulanase(PulA)was obtained,and high-efficiency preparation as well as industrial production of Pul A were achieved.However,during enzymatic production of glucose from starch,it was found that the optimal temperature and pH of PulA did not matched with these of the saccharifying enzyme in the current process,which increased the complexity of the process.In the present study,the amino acid residues that may affect the stability of PulA were screened,PulA mutants were prepared by site-directed mutagenesis,and the enzymatic properties of the mutants were systematically analyzed.The main results are described as follows:(1)Multiple amino acid residues of PulA molecules may be related to its instability under high temperature and low pH.Through analysis of the primary structure of PulA and prediction of the mutation stability,a total of 641 amino acid residues were predicted to contribute to the intolerance of PulA to high temperature and low pH treatment.Fifteen asparagine residues were predicted to be related to the instability of PulA under high temperature and acidic conditions,which were N317,N322,N342,N450,N467,N492,N521,N523,N587,N709 and N734.Further analysis of three-dimensional structure of PulA showed that mutagenesis in N467,N492 or N709 caused more significant structural changes,which indicated their potential role in improving the heat and acid resistance of PulA.(2)Mutations in N467 or N709 significantly affected the enzymatic properties of PulA.The optimal temperature of PulA-1-1(N467G)was increased by 5?,and the optimal pH was reduced by 0.5 pH unit,compared with that of PulA.The optimal temperature of PulA-1-3(N709R)was increased by 5?,and its optimal pH did not change significantly,while its activity at pH 5.0 and pH 6.0 was much higher,compared with that of PulA.After mutagenesis in N492,the optimal temperature and pH of PUlA-1-2(N492A)did not change significantly compared with that of PulA.(3)The combined mutations of N467G,N492A and N709R significantly increased the catalytic activity of PulA at higher temperature and lower pH.PulA-3 was obtained through combined mutagenesis of N467G,N492A and N709R.The optimal pH of PulA-3 was 4.5,the relative enzyme activity was over 90%between pH 4.0 and pH 5.0,and it is more acid-tolerant than PulA.The optimal temperature of PulA-3 was 60?,and the relative enzyme activity was over 90%at the temperature range of 50-60?,while the temperature optimum of PulA was 55?,and the relative enzyme activity was over 80%in the same temperature range.At pH 4.5 and 60?,the catalytic activity of PulA-3 was 20%higher than that of PulA.