The anthrax lethal factor (LF), a Zn-dependent endopeptidase, is considered the dominant virulence factor of anthrax. Because pharmacological inhibition of the catalytic activity of LF is considered a plausible mechanism for preventing the lethality of anthrax, a high-throughput screening experiment based on LF-catalyzed cleavage of a fluorescent substrate was performed to identify novel inhibitors of LF. The RNA-targeting antibiotics, neomycin B and some synthetic dimeric aminoglycosides, were found to be nanomolar active-site inhibitors of LF.

Humans have used high salinity for the production of bean-based fermented foods over thousands of years. Although high salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment, it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people’s health. Therefore, it is imperative to develop novel defined starter cultures for reduced-salt fermentation in a sterile environment. Here, we explored the microbial assembly and function in the fermentation of traditional Chinese broad bean paste with 12% salinity. The results revealed that the salinity and microbial interactions together drove the dynamic of community and pointed out that five dominant genera (Staphylococcus, Bacillus, Weissella, Aspergillus, and Zygosaccharomyces) may play different key roles in different fermentation stages. Then, core species were isolated from broad bean paste, and their salinity tolerance, interactions, and metabolic characteristics were evaluated. The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function. Last, we reconstructed the synthetic microbial community with five strains (Aspergillus oryzae, Bacillus subtilis, Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii) under different salinities and realized efficient fermentation of broad bean paste for 6 weeks in a sterile environment with 6% salinity. In general, this work provided a bottom-up approach for the development of a simplified microbial community model with desired functions to improve the fermentation efficiency of bean-based fermented foods by deconstructing and reconstructing the microbial structure and function. IMPORTANCE Humans have mastered high-salinity fermentation techniques for bean-based fermented product preparation over thousands of years. High salinity was used to select the functional microbiota and conducted food fermentation production with unique flavor. Although a high-salinity environment is beneficial for suppressing harmful microbes in the open fermentation environment, the fermentation efficiency of functional microbes is partially inhibited. Therefore, application of defined starter cultures for reduced-salt fermentation in a sterile environment is an alternative approach to improve the fermentation efficiency of bean-based fermented foods and guide the transformation of traditional industry. However, the assembly and function of self-organized microbiota in an open fermentation environment are still unclear. This study provides a comprehensive understanding of microbial function and the mechanism of community succession in a high-salinity environment during the fermentation of broad bean paste so as to reconstruct the microbial community and realize efficient fermentation of broad bean paste in a sterile environment.

Keratinases are becoming biotechnologically important since they have shown potential in hydrolysis of recalcitrant keratins with highly rigid and strongly cross-linked structures. However, the large-scale application of keratinases has been limited by the inefficient expression level and low enzyme activity. In this work, we employed pro-peptide engineering and saturation mutagenesis to construct excellent keratinase variants with improved activities. It turned out that amino acid substitutions at the pro-peptide cleavage site (P1) could accelerate the release of active mature enzymes, resulting in a 3-fold activity increase. Eighteen sites of the pro-peptide area were targeted for codon mutagenesis, and a multisite saturation mutagenesis library of the six potential sites was generated, achieving a significant improvement of keratinase activity from 179 to 1114 units/mL. Also, the mutants exhibited alterant catalytic properties. Finally, fermentation for keratinase production in a 15 L fermenter was carried out, and the enzyme activity reached up to over 3000 units/mL. Our results demonstrated that propeptide engineering played a crucial role in high expression and engineering of proteases. This study provides a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein.

No abstract

Using indirect competitive time-resolved fluoroimmunoassay (TRFIA), a rapid, highly selective and extremely sensitive method has been established for the determination of ochratoxin A (OA). Tests can be performed in a 96-well microplate using the toxin-specific polyclonal antibodies, obtained from rabbits immunized with ochratoxin A-keyhole limpet's hemocyanin (OA-KLH). In indirect TRFIA format, ochratoxin A-bovine serum albumin conjugate (OA-BSA) is coated onto the microtitre plate and incubated with standard toxin (samples) and anti-OA antibody. A goat anti-rabbit IgG Eu(3+) conjugate is used to enable the detection. The suitability of the assay for quantification of OA is also studied and samples are determined by OA-TRFIA using autoDELFIA1235 system. The results show that the polyclonal antibodies can be used at a dilution exceeding 1:8,000 and the OA detection limit is 0.02 microg/l for indirect competitive TRFIA formats. The 80, 50, and 20% inhibition binding effect dose (ED80, ED50, ED20) of OA were 0.195, 1.018, and 5.314 microg/l, respectively. The assay ranges from 0.02 to 400 microg/l. The cross reactivity with ochratoxin B is 5.6% and antibodies do not react with aflatoxin B1, phenylalanine and BSA. The within-run and between-run CVs of the OA-TRFIA are 2.6 and 5.2%, respectively. The mean recoveries from the OA-free cereals spiked with 1-200 microg of OA/kg of cereals sample were 95.8%. Both OA-TRFIA and OA-ELISA tests are applied for the quantitative measurement of OA in the same cereals, and the coefficient of correlation is 0.912. The results show that the novel TRFIA method can be applied to detect the OA contamination in cereals. It provides very high sensitivity and optimal range and will be useful to screen OA contamination easily, simply, and economically when the number of samples is large.