枯草芽孢杆菌氨肽酶的理性设计及其高效表达

Rational design and efficient expression of aminopeptidase from Bacillus subtilis

摘要:

氨肽酶是食品和医药等领域用于蛋白质深度水解的常用协同酶，但因发酵活力低且热稳定性差，限制了其工业应用范围。本研究通过理性设计提高枯草芽孢杆菌来源氨肽酶168AP的热稳定性，并实现其在工程菌株中的高效异源表达，以拓宽其应用范围。首先，利用在线预测软件预测可能提高168AP热稳定性的突变热点，并对经过初步筛选后热稳定性提升的2个氨肽酶突变体的酶学性质进行测定。随后，运用分子对接技术模拟氨肽酶及其突变体与底物的相互作用，并通过分子动力学模拟解析R39I热稳定性提升的机制。最后，在解淀粉芽孢杆菌中实现热稳定性提高的氨肽酶突变体的高效异源表达。最终成功筛选到2个热稳定性提高的氨肽酶突变体，其中，突变酶R39I在60 ℃处理30 min后残余酶活力是野生型的1.61倍；与此同时，分子对接结果显示R39I与底物的对接结合自由能在所有突变体中最低，为–4.93 kcal/mol；分子动力学模拟结果表明，氨肽酶突变前后引发的疏水作用力改变是其热稳定性提高的主要原因。解淀粉芽孢杆菌Δ6/pLY-3-R39I在7 L发酵罐发酵酶活力达到43 131.57 U/mL，是摇瓶发酵水平的5.11倍。本研究成功筛选得到热稳定性提高的氨肽酶突变体，并实现其在工程菌株中的高效异源表达，拓宽了氨肽酶的工业应用范围。

Abstract:

Aminopeptidases are commonly used co-enzymes for the deep hydrolysis of proteins in food and medicine fields. However, their industrial application is limited due to the low fermentation activity and poor thermal stability. In this study, we employed rational design to enhance the thermal stability of the aminopeptidase 168AP derived from Bacillus subtilis and achieve its efficient heterologous expression in engineered strains, thereby improving and broadening its application potential. First, online prediction software was used to identify mutation hotspots that could potentially improve the thermal stability of 168AP. The enzymatic properties of two aminopeptidase mutants, which exhibited enhanced thermal stability after preliminary screening, were subsequently evaluated. Molecular docking was employed to simulate the interactions between the aminopeptidase mutant R39I and its substrate, and molecular dynamics simulation was conducted to elucidate the mechanism behind the thermal stability enhancement of R39I. Finally, the efficient heterologous expression of the aminopeptidase mutant with enhanced thermal stability was achieved in Bacillus amyloliquefaciens. We successfully identified two aminopeptidase mutants with improved thermal stability. Notably, the residual enzyme activity of R39I after incubation at 60 ℃ for 30 min was 1.61 times that of the wild type. Molecular docking results revealed that R39I had the lowest binding free energy of −4.93 kcal/mol with the substrate among all mutants. Furthermore, molecular dynamics simulation results indicated that the change in hydrophobic interactions before and after mutagenesis was the primary reason for the improved thermal stability. Finally, the enzyme activity of Δ6/pLY-3-R39I in a 7 L fermentation tank reached 43 131.57 U/mL, 5.11 times that of flask fermentation. This study successfully identified aminopeptidase mutants with enhanced thermal stability and achieved their efficient heterologous expression in engineered strains, thus broadening the industrial application range of aminopeptidases.