The invention discloses a recombinant
bacillus subtilis of high-yield
pullulanase and a construction method thereof. The construction method of the recombinant
bacillus subtilis of the high-yield comprises the following steps that an artificial
operon BPB used for expressing the
pullulanase is used for constructing a recombinant
plasmid pGE-BPB; the
nucleotide sequence of the artificial
operon BPB is shown in the graph SEQ ID NO.4; the constructed recombinant
plasmid pGE-BPB is converted into a
bacillus subtilis competent cell, and through secondary recombination, a
neutral protease gene nprE in the bacillus subtilis
competent cell is replaced by the artificial
operon BPB in situ. According to the recombinant bacillus subtilis of the high-yield
pullulanase and the construction method thereof, firstly, the acidproof and heatproof pullulanase
gene of an original
bacterial strain is optimized, based on the
synthetic biology method, a plurality of molecular elements capable of improving the
gene transcriptional level are assembled into the artificial operon, and the recombinant bacillus subtilis can be obtained through construction. The recombinant
bacterial strain can ferment to generate the high-yield pullulanase, and the
enzyme activity each unit can reach or exceed 300 U / ml.