Pullulanase, a starch debranching enzyme, is required for the preparation of high glucose/maltose syrup from starch. In order to expand its narrow reaction conditions and improve its application value, Bacillus naganoensis pullulanase (PulA) was mutated by site-directed mutagenesis and the biochemical characteristics of the mutants were studied. The mutant PulA-N3 with mutations at asparagine 467, 492 and 709 residues was obtained. It displayed the activity maximum at 60 °C and pH 4.5 and exceeded 90% activities between 45 and 60 °C and from pH 4.0 to pH 5.5, which was improved greatly compared with wild-type PulA. Its thermostability and acidic pH stability were also remarkably improved. Its catalytic rate (kcat/Vmax) was 2.76 times that of PulA. In the preparation of high glucose syrup, the DX (glucose content, %) values of glucose mediated by PulA-N3 and glucoamylase reached 96.08%, which were 0.82% higher than that of PulA. In conclusion, a new pullulanase mutant PulA-N3 was successfully developed, which has high debranching activity in a wide range of temperature and pH, thereby paving the way for highly efficient starch saccharification.
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