Dataset Information

Enhanced extracellular expression of Bacillus stearothermophilus ?-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and ?-amylase mutant selection.

ABSTRACT:

Background

Our laboratory has constructed a Bacillus stearothermophilus ?-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 ?-amylase in B. subtilis is very low.

Results

In this study, the extracellular production level of B. stearothermophilus ?-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and ?-amylase mutant selection. The ?-amylase optimal signal peptide (SP_YojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular ?-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 ?-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 SP_YojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in ?-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the ?-amylase activity of the culture supernatant was 9201.1 U mL^-1, which is the highest level that has been reported to date.

Conclusions

This is the first report that describes an improvement of B. stearothermophilus ?-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for ?-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of ?-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.

SUBMITTER: Yao D

PROVIDER: S-EPMC6458788 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Publications

Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection.

Yao Dongbang D Su Lingqia L Li Na N Wu Jing J

Microbial cell factories 20190411 1

<h4>Background</h4>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.<h4>Results</h4>In this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide op ...[more]

PMID: 30971250

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