Phosphorylation residue T175 in RsbR protein is required for efficient induction of sigma B factor and survival of Listeria monocytogenes under acidic stress / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. RsbR, an upstream regulator of the sigma B (SigB) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion (ΔrsbR), complementation (C-ΔrsbR), and phosphorylation site mutations in the rsbR (RsbR-T175A, RsbR-T209A, and RsbR-T175A-T209A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsbR reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175A disabled RsbR complementation, while RsbR-T209A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of RsbR are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.

Phosphorylation residue T175 in RsbR protein is required for efficient induction of sigma B factor and survival of Listeria monocytogenes under acidic stress / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. RsbR, an upstream regulator of the sigma B (SigB) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion (ΔrsbR), complementation (C-ΔrsbR), and phosphorylation site mutations in the rsbR (RsbR-T175A, RsbR-T209A, and RsbR-T175A-T209A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsbR reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175A disabled RsbR complementation, while RsbR-T209A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of RsbR are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.

Improving the thermostability of α-amylase from Rhizopus oryzae by rational design / 生物工程学报

Fungal α-amylases are widely used in the production of maltose syrup, while additional production costs may be required in the syrup production process due to the loss of enzyme activity, because of the poor thermostability exhibited in this type of enzyme. After deeply studying the importance of thermostability of fungal α-amylases applied in industrial production, with attempt to improve the thermostability of Rhizopus oryzae α-amylase (ROAmy), single-point mutations and combined mutations that based on analysis of B-factor values and molecular dynamics simulations were carried out for amino acid residues G128, K269 and G393 of ROAmy by overlapping PCR. The results showed that all the 7 mutants obtained presented better thermostability than the wild-type enzyme, and the best mutant was G128L/K269L/G393P which showed a 5.63-fold increase in half-life at 55 ℃ compared with the wild-type enzyme. Meanwhile, its optimum temperature increased from 50 ℃ to 65 ℃, the maximum reaction rate (Vmax) and catalytic efficiency (kcat/Km) increased by 65.38% and 99.86%. By comparing and analyzing the protein structure and function between the mutants and the wild-type enzyme, it was found that the increase of the number of hydrogen bonds or the introduction of proline in special position may be the main reasons for the improved thermostability that found in the mutants.

Deletion of a dynamic surface loop improves thermostability of (R)-selective amine transaminase from Aspergillus terreus / 生物工程学报

Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T₅₀¹⁰ (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.