Keratinase is a kind of protease that can specifically degrade the keratin, and has wide application prospects in the fields of waste recycling, leather or textile industry and animal feed addition. However, less extracellular protein is still the main problem of recombinant keratinase. A recombinant Bacillus subtilis WB600-P43NMK-Ker has been obtained with an extracellular keratinase activity of 10.4 kU/mL in the shake flask. SDS-PAGE analysis showed that the color of keratinase band was lighter, indicating that its extracellular expression was low. This study verified the effect of signal peptides and ribosome binding site (RBS) on the extracellular expression of keratinase. 244 endogenous signal peptides from Bacillus subtilis were screened in online database. By predicting the secretory pathway and the subcellular localization, 20 signal peptides that were predicted as Sec signal peptides and lead the pre-protein to extracellular localization. Through the replacing of endogenous signal peptides from the Bacillus subtilis, the secretion capability of the recombinant strain contained SPdacB sequence was significantly increased. Its extracellular keratinase activity reached 84.3 kU/mL, which was approximately 8.1-fold of the original strain. Furthermore, the properties of different efficient signal peptides and their effects on the extracellular keratinase activity were studied. We also analyzed the relationship between the properties and the extracellular expression. It was found that high content of hydrophobic amino acid in H-region and high Gibbs free energy of mRNA folding normally leads to a high extracellular keratinase activity. Conserved sequences in signal peptides generally correspond to high extracellular expression. However, the results of this study shows that M-K-K sequence in the N-region of signal peptides or A-X-A in the C-region may not lead to high extracellular expression. The secretory efficiency of signal peptides has been described as the combined results of many different properties. Furthermore, it is shown that efficient signal peptides for a specific protein are not universally efficient in the expression of other proteins, and signal peptides generally considered to be highly efficient are not universally suitable for the expression of all proteins, such as SPdacB or SPpel. In order to further enhance the extracellular keratinase activity and reduce the number of mutants in the library, the translation initiation rate of different RBS mutation sites was predicted and compared by RBS library calculator. We performed saturation mutations on selected mutation sites. The extracellular keratinase activity of the obtained mutant strain R16D12 showed 59.1 kU/mL, which was 10.5 times that of the original strain. Overall, the strategy of combining signal peptide screening with RBS optimization significantly improved the extracellular expression of keratinase in Bacillus subtilis, laying a foundation for subsequent research and applications.
TAN Muyang
,
CHEN Xiwen
,
PENG Zheng
,
ZHANG Juan
,
ZHANG Guoqiang
. Enhancing the extracellular expression of Bacillus licheniformis keratinase in Bacillus subtilis by combinatorial strategies of signal peptides screening and ribosome binding site optimization[J]. Food and Fermentation Industries, 2023
, 49(4)
: 8
-14
.
DOI: 10.13995/j.cnki.11-1802/ts.031799
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