Co-production of surfactin and fengycin by Bacillus subtilis BBW1542 isolated from marine sediment: a promising biocontrol agent against foodborne pathogens.

Pathogenic bacteria contaminations and related diseases in food industries is an urgent issue to solve. The present study aimed to explore natural food biopreservatives from microorganisms. Using dilution-plate method, a strain BBW1542 with antimicrobial activities against various foodborne pathogenic bacteria was isolated from the seabed silt of Beibu Gulf, which was identified as Bacillus subtilis by the morphological observation and 16S rDNA sequences. The antimicrobial substances of B. subtilis BBW1542 exhibited an excellent stability under cool/heat treatment, UV irradiation, acid/alkali treatment, and protease hydrolysis. The genome sequencing analysis and antiSMASH prediction indicated that B. subtilis BBW1542 contained the gene cluster encoding lipopeptides and bacteriocin subtilosin A. MALDI-TOF-MS analysis showed that the lipopeptides from B. subtilis BBW1542 contained C14 and C15 surfactin homologues, together with fengycin homologues of C18 fengycin A/C16 fengycin B and C19 fengycin A/C17 fengycin B. In silico analysis showed that an eight-gene (sboA-albABCDEFG) operon was involved in the biosynthesis of subtilosin A in B. subtilis BBW1542, and the encoded subtilosin A presented an evident closed-loop structure containing 35 amino acids with a molecular weight of 3425.94 Da. Overall, the antagonistic B. subtilis BBW1542 displayed significant resource value and offered a promising alternative in development of food biopreservation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05864-3.

Structural and enzymatic characterization of a novel metallo-serine keratinase KerJY-23.

KerJY-23 was a novel keratinase from feather-degrading Ectobacillus sp. JY-23, but its enzymatic characterization and structure are still unclear. In this study, the KerJY-23 was obtained by heterologous expression in Escherichia coli BL21(DE3), and enzymatic properties indicated that KerJY-23 was optimal at 60 °C and pH 9.0 and could be promoted by divalent metal ions or reducing agents. Furthermore, KerJY-23 had a broad substrate specificity towards casein, soluble keratin, and expanded feather powder, but its in vitro degradation against chicken feathers required an additional reducing agent. Homology modeling indicated that KerJY-23 contained a highly conserved zinc-binding HELTH motif and a His-Asp-Ser catalytic triad that belonged to the typical characteristics of M4-family metallo-keratinase and serine-keratinase, respectively. Molecular docking revealed that KerJY-23 achieved a reinforced binding on feather keratin via abundant hydrogen bonding interactions. This work not only deepened understanding of the novel and interesting metallo-serine keratinase KerJY-23, but also provided a theoretical basis for realizing the efficient use of waste feather keratin.

Effective biodegradation on chicken feather by the recombinant KerJY-23 Bacillus subtilis WB600: A synergistic process coupled by disulfide reductase and keratinase.

Keratin wastes are abundantly available but rich in hard-degrading fibrous proteins, and the keratinase-producing microorganisms have gained significant attention due to their biodegradation ability against keratinous materials. In order to improve the degradation efficiency of feather keratins, the keratinase gene (kerJY-23) from our previously isolated feather-degrading Ectobacillus sp. JY-23 was overexpressed in Bacillus subtilis WB600 strain. The recombinant KerJY-23 strain degraded chicken feathers rapidly within 48 h, during which the activities of disulfide reductase and keratinase KerJY-23 were sharply increased, and the free amino acids especially the essential phenylalanine and tyrosine were significantly accumulated in feather hydrolysate. The results of structural characterizations including scanning electron microscopy, Fourier transform infrared spectrum, X-ray diffraction, and X-ray photoelectron spectroscopy, demonstrated that the feather microstructure together with the polypeptide bonds and SS bonds in feather keratins were attacked and destroyed by the recombinant KerJY-23 strain. Therefore, the recombinant KerJY-23 strain contributed to feather degradation through the synergistic action of the secreted disulfide reductase to break the SS bonds and keratinase (KerJY-23) to hydrolyze the polypeptide bonds in keratins. This study offers a new insight into the underlying mechanism of keratin degradation, and provides a potential recombinant strain for the valorization of keratin wastes.

Analysis of Osmotic Tolerance, Physiological Characteristics, and Gene Expression of Salmonella enterica subsp. enterica Serotype Derby.

Salmonella is an important foodborne pathogen, and recent epidemiological studies have shown high infection rates of Salmonella enterica subsp. enterica serotype Derby (S.Derby) in poultry in western China and other regions. S.Derby presents increasing concerns with the development of resistance to hypertonic environments; however, there are few reports investigating the mechanism of resistance. Therefore, in this study, we examined hypertonic adaptation in S.Derby at the physiological and molecular levels. The K-B paper method, wiping glass bead method, crystal violet staining, and RT-PCR combined with comparative genomics analysis were employed to characterize virulence, drug resistance, biofilm formation, and changes in gene expression of genes related to hypertonic adaptation in S.Derby. Hypertonic-adapted S.Derby exhibited resistance to OXA, AMP, PEN, and CEP antibiotics, and biofilm-forming ability was 1.25 times that of nonadapted S.Derby. RT-PCR results showed that compared with nonadapted S.Derby, the expression of virulence-related genes in hypertonic-adapted S.Derby increased by 2-3 times, that of biofilm-related genes increased by 2-4 times, and that of OXA, AMP, PEN, and CEP-related drug resistance genes was relatively high. Four hypertonic tolerance-related genes (otsA, proV, proW, omsV) were preliminarily identified in S.Derby. The expression of proW was always relatively high in hypertonic-adapted S.Derby, the expression of otsA gradually became higher than that of proW with increasing time of osmotic stress, and the expression of proV and omsV was only high in non-hypertonic-adapted S.Derby.

A marine lipopeptides-producing Bacillus amyloliquefaciens HY2-1 with a broad-spectrum antifungal and antibacterial activity and its fermentation kinetics study.

The antagonistic Bacillus amyloliquefaciens HY2-1 was a marine microbiology that was isolated previously from the seabed silt of Beibu Gulf in China by dual culture with Penicillium digitatum. As a continuous study, the present work focused on evaluating the antimicrobial activity, identifying the produced active components, and revealing the fermentation characteristics of B. amyloliquefaciens HY2-1, respectively. It was found that B. amyloliquefaciens HY2-1 exhibited a broad-spectrum antimicrobial activity against the tested seven phytopathogenic fungi and five pathogenic bacteria by producing Bacillus lipopeptides such as fengycin A (C14 to C19 homologues) and surfactin (C14 and C15 homologues). Morphological observation of P. digitatum under light microscope, scanning electron microscopy, transmission electron microscopy, and fluorescence microscope inferred that B. amyloliquefaciens exerted the antagonistic activity by damaging the fungal cell membrane, thus inhibiting the mycelium growth and sporification of phytopathogenic fungi. As a marine microbiology, our results showed that B. amyloliquefaciens could survive and metabolize even at the culture condition with 110 g/L of NaCl concentration, and the produced antimicrobial compounds exhibited excellent thermostability and acid-alkali tolerance. The dynamic models were further constructed to theoretically analyze the fermentation process of B. amyloliquefaciens HY2-1, suggesting that the synthesis of antimicrobial compounds was coupled with both cell growth and cell biomass. In conclusion, the marine lipopeptides-producing B. amyloliquefaciens HY2-1 showed a promising prospect to be explored as a biocontrol agent for plant disease control of crops and postharvest preservation of fruits and vegetables, especially due to its outstanding stress resistance and the broad-spectrum and effective antagonist on various phytopathogenic fungi.

Surface Engineering of HEK293 Cell-Derived Extracellular Vesicles for Improved Pharmacokinetic Profile and Targeted Delivery of IL-12 for the Treatment of Hepatocellular Carcinoma.

Background: Extracellular vesicles (EVs) are considered a promising drug delivery platform. Naïve EVs face numerous issues that limit their applications, such as fast clearance, hepatic accumulations, and a lack of target-specific tropism. We aimed to explore a series of surface engineering approaches to: 1) reduce the non-specific adhesion of EVs, and 2) improve their enrichment in the target tissue. As a proof-of-concept, we investigated the therapeutic potentials of a multi-modal EVs system carrying a tumor-specific nanobody and the immuno-stimulant interleukin-12 (IL12) using in vivo models of hepatocellular carcinoma. Methods: The major cell adhesion molecule on the HEK293-derived EVs, integrin ß1 (ITGB1), was knocked out (KO) by CRISPR/Cas9-mediated gene editing, followed by deglycosylation to generate ITGB1-Deg EVs for the subsequent pharmacokinetic and biodistribution analyses. ITGB1-Deg EVs were further loaded with glypican-3 (GPC3)-specific nanobody (HN3) and mouse single-chain IL12 (mscIL12) to generate ITGB1-mscIL12+HN3+Deg EVs, for evaluation of tumor tropism and therapeutic potential in a mice model of hepatocellular carcinoma. Results: Removal of ITGB1 led to the broad suppression of integrins on the EVs surface, resulting in a decrease in cellular uptake. Deglycosylation of ITGB1- EVs gave rise to inhibition of the EVs uptake by activated RAW264.7 cells. ITGB1 removal did not significantly alter the pharmacokinetic behaviors of HEK293-EVs, whereas the ITGB1-Deg EVs exhibited enhanced systemic exposure with reduced hepatic accumulation. Loading of HN3 conferred the ITGB1-Deg EVs with tumor-specific tropism for both subcutaneous and metastasized tumors in mice. The ITGB1-mscIL12+HN3+Deg EVs activated mouse splenocytes with high potency. Systemic administration of the EVs with the equivalent dose of 1.5µg/kg of exosomal IL12 achieved satisfactory tumor growth inhibition and good tolerability. Conclusion: The combinatorial approach of EVs surface engineering conferred HEK293-EVs with reduced non-specific clearance and enhanced tumor targeting efficacy, which constituted an efficient delivery platform for critical cancer therapeutics like IL12.

Nitrogen Removal Characteristics of a Marine Denitrifying Pseudomonas stutzeri BBW831 and a Simplified Strategy for Improving the Denitrification Performance Under Stressful Conditions.

A marine aerobic denitrifying bacterium was isolated and identified as Pseudomonas stutzeri BBW831 from the seabed silt of Beibu Gulf in China. According to the genome analysis, P. stutzeri BBW831 possessed a total of 14 genes (narG, narH, narI, narJ, napA, napB, nirB, nirD, nirS, norB, norC, norD, norQ, and nosZ) responsible for fully functional enzymes (nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase) involved in the complete aerobic denitrification pathway, suggesting that it had the potential for reducing nitrate to the final N2. Denitrification results showed that P. stutzeri BBW831 exhibited efficient nitrogen removal characteristics. Within 12 h, the NO3--N removal efficiency and rate reached 94.64% and 13.09 mg·L-1·h-1 under 166.10 ± 3.75 mg/L NO3--N as the sole nitrogen source, and removal efficiency of the mixed nitrogen (50.50 ± 0.55, 62.28 ± 0.74, and 64.26 ± 0.90 mg/L of initial NH4+-N, NO3--N, and NO2--N, respectively) was nearly 100%. Furthermore, a simplified strategy, by augmenting the inoculation biomass, was developed for promoting the nitrogen removal performance under high levels of NO2--N and salinity. As a result, the removal efficiency of the initial NO2--N up to approximately 130 mg/L reached 99.46% within 8 h, and the NO3--N removal efficiency achieved at 59.46% under the NaCl concentration even up to 50 g/L. The C/N ratio of 10 with organic acid salt such as trisodium citrate and sodium acetate as the carbon source was most conducive for cell growth and nitrogen removal by P. stutzeri BBW831, respectively. In conclusion, the marine P. stutzeri BBW831 contained the functional genes responsible for a complete aerobic denitrification pathway (NO3--N → NO2--N → NO → N2O → N2), and had great potential for the practical treatment of high-salinity nitrogenous mariculture wastewater.

Isolation, characterization, and genome sequencing of a novel chitin deacetylase producing Bacillus aryabhattai TCI-16.

Chitin deacetylase (CDA) is a chitin degradation enzyme that catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues, playing an important role in the high-value utilization of waste chitin. The shells of shrimp and crab are rich in chitin, and mangroves are usually recognized as an active habitat to shrimp and crab. In the present study, a CDA-producing bacterium, strain TCI-16, was isolated and screened from the mangrove soil. Strain TCI-16 was identified and named as Bacillus aryabhattai TCI-16, and the maximum CDA activity in fermentation broth reached 120.35 ± 2.40 U/mL at 36 h of cultivation. Furthermore, the complete genome analysis of B. aryabhattai TCI-16 revealed the chitin-degrading enzyme system at genetic level, in which a total of 13 putative genes were associated with carbohydrate esterase 4 (CE4) family enzymes, including one gene coding CDA, seven genes encoding polysaccharide deacetylases, and five genes encoding peptidoglycan-N-acetyl glucosamine deacetylases. Amino acid sequence analysis showed that the predicted CDA of B. aryabhattai TCI-16 was composed of 236 amino acid residues with a molecular weight of 27.3 kDa, which possessed a conserved CDA active like the known CDAs. However, the CDA of B. aryabhattai TCI-16 showed low homology (approximately 30%) with other microbial CDAs, and its phylogenetic tree belonged to a separate clade in bacteria, suggesting a high probability in structural novelty. In conclusion, the present study indicated that the novel CDA produced by B. aryabhattai TCI-16 might be a promising option for bioconversion of chitin to the value-added chitosan.

HSP60 in cancer: a promising biomarker for diagnosis and a potentially useful target for treatment.

Heat shock proteins (HSPs), most of which are molecular chaperones, are highly conserved proteins produced by cells under physiological stress or pathological conditions. HSP60 (57-69 kDa) can promote or inhibit cell apoptosis through different mechanisms, and its abnormal expression is also related to tumour cell metastasis and drug resistance. In recent years, HSP60 has received increasing attention in the field of cancer research due to its potential as a diagnostic and prognostic biomarker or therapeutic target. However, in different types of cancer, the specific mechanisms of abnormally expressed HSP60 in tumour carcinogenesis and drug resistance are complicated and still require further study. In this article, we comprehensively review the regulative mechanisms of HSP60 on apoptosis, its applications as a cancer diagnostic biomarker and a therapeutic target, evidence of involvement in tumour resistance and the applications of exosomal HSP60 in liquid biopsy. By evaluating the current findings of HSP60 in cancer research, we highlight some core issues that need to be addressed for the use of HSP60 as a diagnostic or prognostic biomarker and therapeutic target in certain types of cancer.