Immobilization of purified enzyme EreB in metalorganic framework (MOF) mesopores for erythromycin degradation.

Erythromycin, a commonly used macrolide antibiotic, plays a crucial role in both human medicine and animal husbandry. However, its abuse has led to residual presence in the environment, with problems such as the emergence of resistant bacteria and enrichment of resistance genes. These issues pose significant risks to human health. Thus far, there are no effective, environmentally friendly methods to manage this problem. Enzymes can specifically degrade erythromycin without causing other problems, but their unrecyclability and environmental vulnerability hinder large-scale application. Enzyme immobilization may help to solve these problems. This study used Cu-BTC, a synthetic metal-organic framework, to immobilize the erythromycin-degrading enzyme EreB. The loading temperature and enzyme quantity were optimized. The Cu-BTC and EreB@Cu-BTC were characterized by various methods to confirm the preparation of Cu-BTC and immobilization of EreB. The maximum enzyme loading capacity was 66.5 mg g-1. In terms of enzymatic properties, immobilized EreB had improved heat (25-45 °C) and alkaline (6.5-10) tolerance, along with greater affinity between the enzyme and its substrate; Km decreased from 438.49 to 372.30 mM. Recycling was also achieved; after 10 cycles, 57.12% of the enzyme activity was maintained. After composite degradation, the antibacterial activity of erythromycin-containing wastewater was examined; the results showed that the novel composite could completely inactivate erythromycin. In summary, Cu-BTC was an ideal carrier for immobilization of the enzyme EreB, and the EreB@Cu-BTC composite has good prospects for the treatment of erythromycin-containing wastewater.

The maize transcription factor EREB58 mediates the jasmonate-induced production of sesquiterpene volatiles.

Over the past two decades, Zea mays (maize) has been established as a model system for the study of indirect plant defense against herbivores. When attacked by lepidopteran larvae, maize leaves emit a complex blend of volatiles, mainly composed of sesquiterpenes, to attract the natural enemies of the herbivores. This is associated with a swift transcriptional induction of terpene synthases such as TPS10; however, the molecular components controlling the complex transcriptional reprogramming in this process are still obscure. Here, by exploiting the finding that the maize TPS10 promoter retained its full responsiveness to herbivory in Arabidopsis, we identified the region from -300 to -200 of the TPS10 promoter as both necessary and sufficient for its herbivore inducibility through 5' deletion mapping. A high-throughput screening of an Arabidopsis transcription factor library using this promoter region as the bait identified seven AP2/ERF family transcription factors. Among their close homologs in maize, EREB58 was the only gene responsive to herbivory, with a spatiotemporal expression pattern highly similar to that of TPS10. Meanwhile, EREB58 was also responsive to Jasmonate. In vivo and in vitro assays indicated that EREB58 promotes TPS10 expression by directly binding to the GCC-box within the region from -300 to -200 of the TPS10 promoter. Transgenic maize plants overexpressing EREB58 constitutively over-accumulate TPS10 transcript, and also (E)-ß-farnesene and (E)-α-bergamotene, two major sesquiterpenes produced by TPS10. In contrast, jasmonate induction of TPS10 and its volatiles was abolished in EREB58-RNAi transgenic lines. In sum, these results demonstrate that EREB58 is a positive regulator of sesquiterpene production by directly promoting TPS10 expression.

Immobilization of EreB on Acid-Modified Palygorskite for Highly Efficient Degradation of Erythromycin.

Erythromycin is one of the most commonly used macrolide antibiotics. However, its pollution of the ecosystem is a significant risk to human health worldwide. Currently, there are no effective and environmentally friendly methods to resolve this issue. Although erythromycin esterase B (EreB) specifically degrades erythromycin, its non-recyclability and fragility limit the large-scale application of this enzyme. In this work, palygorskite was selected as a carrier for enzyme immobilization. The enzyme was attached to palygorskite via a crosslinking reaction to construct an effective erythromycin-degradation material (i.e., EreB@modified palygorskite), which was characterized using FT-IR, SEM, XRD, and Brunauer-Emmett-Teller techniques. The results suggested the successful modification of the material and the loading of the enzyme. The immobilized enzyme had a higher stability over varying temperatures (25-65 °C) and pH values (6.5-10.0) than the free enzyme, and the maximum rate of reaction (Vmax) and the turnover number (kcat) of the enzyme increased to 0.01 mM min-1 and 169 min-1, respectively, according to the enzyme-kinetics measurements. The EreB@modified palygorskite maintained about 45% of its activity after 10 cycles, and degraded erythromycin in polluted water to 20 mg L-1 within 300 min. These results indicate that EreB could serve as an effective immobilizing carrier for erythromycin degradation at the industrial scale.

Prevalence of Multidrug-Resistant Enterococcus faecalis in Hospital-Acquired Surgical Wound Infections and Bacteremia: Concomitant Analysis of Antimicrobial Resistance Genes.

BACKGROUND: The study aimed to assess the prevalence of Enterococcus faecalis infections among patients with hospital-acquired surgical wound sepsis and bacteremia in surgical wards and identify the antimicrobial susceptibility in these pathogens. Genetic role of erythromycin, vancomycin, and cephalosporin resistance in these pathogens was also examined. METHODS: Two hundred samples were collected from surgical wound infections and 100 blood cultures from patients with suggested bacteremia to identify E faecalis by phenotypic and genotypic methods. Antimicrobial susceptibility to 12 antimicrobial agents was tested. The presence of resistance genes was examined by polymerase chain reaction (PCR) assay. RESULTS: E faecalis was isolated with a frequency of 24/200 (12%) from surgical wound samples and 2/100 (2%) from blood cultures. All isolates were completely resistant to cefepime, ampicillin, and tetracycline, 96% of isolates were resistant to erythromycin, 53.8% to vancomycin, and 23.1% to linezolid. Multidrug resistance (MDR) was found in 100% of isolates. ere(B) and erm(B) genes were present in 20/25 (80%) and 17/25 (68%) of erythromycin-resistant isolates, respectively, 15 (60%) isolates carry both ere(B) and erm(B) genes. Van A gene was detected in 71.4% of vancomycin-resistant isolates. All isolates were negative for mef(A/E), blaSHV, and blaTEM genes. CONCLUSION: MDR in all isolates (100%) and high-level resistance to gentamicin, erythromycin, and vancomycin were reported in E Faecalis isolates. In the studied isolates, erythromycin resistance mainly related to the presence of ere(B) and erm(B) genes and vancomycin resistance was mainly related to the presence of vanA gene.