title: A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens.
abstract: Extracellular α-amylase from Pyrococcus furiosus (PFA) shows great starch-processing potential for industrial application due to its thermostability, long half-life and optimal activity at low pH; however, it is difficult to produce in large quantities. In contrast, α-amylase from Bacillus amyloliquefaciens (BAA) can be produced in larger quantities, but shows lower stability at high temperatures and low pH. Here, we describe a BAA protein expression pattern-mimicking strategy to express PFA in B. amyloliquefaciens using the expression and secretion elements of BAA, including the codon usage bias and mRNA structure of gene, promoter, signal peptide, host and cultivation conditions. This design was assessed to be successful by comparing the various genes (mpfa and opfa), promoters (PamyA and P43), and strains (F30, F31, F32 and F30-∆amyA). The final production of PFA yielded 2714 U/mL, about 3000- and 14-fold that reportedly produced in B. subtilis or E. coli, respectively. The recombinant PFA was optimally active at ~100 °C and pH 5 and did not require Ca(2+) for activity or thermostability, and >80% of the enzyme activity was retained after treatment at 100 °C for 4 h.
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Peili
authors.initials: P
authors.lastname: Wang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Tian
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Yu
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Meihui
authors.initials: M
authors.lastname: Chang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Chu
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Ningfeng
authors.initials: N
authors.lastname: Wu
mesh_terms: D000069977:Bacillus amyloliquefaciens / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins; D014169:Transformation, Bacterial; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000516:alpha-Amylases