Abstract
A chimeric gene mHG (669 bp) was constructed by substitution of Clostridium thermocellum ZJL4 lichenase (CG) N-terminal fragment (except its signal sequence) for the counterpart of Bacillus sp. A3 lichenase (BG). To acquire high-level secretion of the chimeric lichenase (mHG) in Bacillus subtilis, a series of site-mutated signal peptides were designed. The activity of mHG, which was directed by an artificial hydrophobic signal peptide H1 (MMARKIAGMATSLLVIFSSSAVA) from cytoplasm into growth medium, reached 80.56 U/ml after 22-h incubation, indicating that signal peptide hydrophobicity appears to be critical for early stages in mHG export. By purification of the mHG (∼25.3 kDa) from cultures of B. subtilis (pBSG-H1), enzymatic property assays showed that the common optima for mHG were 70°C and pH 5.0. The residual activity of mHG at 90°C for 10 min was 83.45% of its maximum activity, which was almost similar to that of CG (90°C, 10 min, 84.33%). This constructed shuttle expression vector with a novel signal peptide exhibited its applicability for high-level production of heterologous proteins from B. subtilis. Moreover, the high-level secreted mHG with relatively high thermostability could be a potential candidate for feed industrial applications.
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Acknowledgments
This work was supported by grants from the Zhejiang Provincial Scientific Program of the People’s Republic of China (no. 2004C32031) and the National Natural Science Foundation of the People’s Republic of China (no. 30771579).
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Fu, Ll., Xu, Zr., Shuai, Jb. et al. High-Level Secretion of a Chimeric Thermostable Lichenase from Bacillus subtilis by Screening of Site-Mutated Signal Peptides with Structural Alterations. Curr Microbiol 56, 287–292 (2008). https://doi.org/10.1007/s00284-007-9077-5
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DOI: https://doi.org/10.1007/s00284-007-9077-5