Secreted expression of the pullulanase in Bacillus amyloliquefaciens BF7658
According to the sequence of pullulanase gene from Bacillus deramificans (NCBI accession number: BD140672), the gene encoding mature peptide of pullulanase was synthesized and designated BdP. BdP was inserted into the downstream of the DNA fragment encoding signal peptide of pUC980, a secreting expression vector of Bacillus. The constructed plasmid was designated pUC980-BiP. Subsequently, pUC980-BdP was introduced into Bacillus amyloliquefaciens BF7658, an industrial mesophilic amylase producer. Active pullulanase was expressed by recombinant B. amyloliquefaciens. Recombinant pullulanase was secreted into medium, the optimal time of pullulanase production was 48 h and the enzymatic activity was 2.8 U/mL. Its optimum pH was 5.0 and its optimum temperature was 60 ℃. More than 50 % of its initial enzymatic activity was still detectable after incubated at 60 ℃ for 3 h. The enzymatic property showed that the recombinant pullulanase could be used in saccharification process of starch .
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