角蛋白酶(keratinase)是一类能够特异性降解角蛋白的蛋白酶,在废弃物回收、皮革纺织和饲料添加等领域有着广泛的应用前景。然而,较低的胞外蛋白含量仍是重组角蛋白酶开发的主要问题。前期研究中已经获得了1株重组枯草芽孢杆菌Bacillus subtilis WB600-p43NMK-Ker,在摇瓶水平下,其胞外角蛋白酶活力为10.4 kU/mL,在SDS-PAGE分析中发现角蛋白酶所在条带颜色较浅,说明其胞外表达量较低。该研究考察了信号肽和核糖体结合位点(ribosome binding site,RBS)对角蛋白酶胞外表达量的影响。通过枯草芽孢杆菌内源信号肽替换,筛选得到的带有SPdacB的重组菌株的分泌能力显著提升,其胞外角蛋白酶活力达到84.3 kU/mL,约为出发菌株胞外酶活力的8.1倍。在此基础上,通过对RBS进行基于高效突变位点的半理性预测的饱和突变,筛选得到的1株高产菌株R16D12,其胞外酶活力达到109.1 kU/mL,较RBS优化前提高了29%,是出发菌株胞外酶活力的10.5倍。研究结果表明,信号肽筛选和RBS优化的组合策略显著提高了角蛋白酶在枯草芽孢杆菌中的胞外表达量,为后续研究与应用奠定了基础。
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.
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