Dataset Information

A new strategy to express the extracellular ?-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens.

ABSTRACT: Extracellular ?-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, ?-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.

SUBMITTER: Wang P

PROVIDER: S-EPMC4768087 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens.

Wang Ping P Wang Peili P Tian Jian J Yu Xiaoxia X Chang Meihui M Chu Xiaoyu X Wu Ningfeng N

Scientific reports 20160226

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 ...[more]

PMID: 26916714

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Project description:BackgroundOur laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low.ResultsIn this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SPYojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SPYojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL-1, which is the highest level that has been reported to date.ConclusionsThis is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.

Dataset Information

A new strategy to express the extracellular ?-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens.

Publications

A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens.

Similar Datasets

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