Characterization, Semirational Design for pH Robustness, and the Application in Bioactive Peptide Production of a X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis MY-3.

PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze ß-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.

GlnR-mediated regulation of KstR controls cholesterol catabolism in Mycobacterium smegmatis.

Tuberculosis, caused by mycobacteria, continues to pose a substantial public health threat. Mycobacteria typically use cholesterol from the membranes of host macrophages as a carbon and energy source. Most genes that control cholesterol degradation are regulated by KstR, which is highly conserved in Mycobacterium tuberculosis and Mycobacterium smegmatis. Through bioinformatic analysis, we found a typical global nitrogen regulator (GlnR)-binding motif (CCGAC-AACAGT-GACAC) in the promoter region of kstR of M. smegmatis, and we determined its binding activity in vitro using electrophoretic mobility shift assays. Using RT-qPCR, we found that nine genes involved in side-chain or sterol-ring oxidation were upregulated in a ΔglnR M. smegmatis strain compared to the WT strain and glnR-complemented strains under nitrogen limitation. ATP assays in macrophages revealed that coordinated GlnR-KstR regulation significantly reduced the viability of M. smegmatis in macrophages. Thus, we found that various genes involved in cholesterol catabolism are regulated by GlnR via KstR in response to environmental nitrogen, and that they further affect the invasive ability of M. smegmatis. These findings revealed a novel regulatory mechanism of cholesterol catabolism, which may be useful in the development of new strategies for controlling tuberculosis.