Abstract
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.
Highlights
Our laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties
Heterologous expression of the α-amylases from B. licheniformis, B. amyloliquefaciens and B. stearothermophilus have been reported in Escherichia coli, B. licheniformis, B. subtilis, and other hosts [4,5,6]
Heterologous expression of B. stearothermophilus α‐amylase in B. subtilis WS11 To produce B. stearothermophilus α-amylase (AmyS) in B. subtilis, the expression strain WS11QS was constructed by transforming B. subtilis WS11 with the recombinant vector pHYQamyS, which contained the α-amylase gene amyS
Summary
Our 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. Heterologous expression of the α-amylases from B. licheniformis, B. amyloliquefaciens and B. stearothermophilus have been reported in Escherichia coli, B. licheniformis, B. subtilis, and other hosts [4,5,6]. Its genetic manipulation is relatively simple and the product is directly secreted into the extracellular medium. For these reasons, B. subtilis has been used to produce a variety of heterologous proteins and chemicals. Heterologous expression of α-amylases from different microbial sources in B. subtilis results in very different extracellular production levels. HJ21 expression in B. subtilis are 2012 U mL−1 [7], 5086 U mL−1 [8], 14 μg mL−1 [9], and 19.6 U mL−1 [10], respectively
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