Biodegradation of combined pollutants of polyethylene terephthalate and phthalate esters by esterase-integrated Pseudomonas sp. JY-Q with surface-co-displayed PETase and MHETase.

The waste pollution problem caused by polyethylene terephthalate (PET) plastics poses a huge threat to the environment and human health. As plasticizers, Phthalate esters (PAEs) are widely used in PET production and become combined pollutants with PET. Synthetic biology make it possible to construct engineered cells for microbial degradation of combined pollutants of PET and PAEs. PET hydroxylase (PETase) and monohydroxyethyl terephthalate hydroxylase (MHETase) isolated from Ideonella sakaiensis 201-F6 exhibit the capability to depolymerize PET. However, PET cannot enter cells, thus enzymatic degradation or cell surface displaying technology of PET hydrolase are the potential strategies. In this study, Pseudomonas sp. JY-Q was selected as a chassis strain, which exhibits robust stress tolerance. First, a truncated endogenous outer membrane protein cOmpA and its variant Signal (OprF)-cOmpA were selected as anchor motifs for exogenous protein to display on the cell surface. These anchor motifs were fused at the N-terminal of PET hydrolase and MHETase and transformed into Pseudomonas sp. JY-Q, the mutant strains successfully display the enzymes on cell surface, after verification by green fluorescent protein labeling and indirect immunofluorescence assay. The resultant strains also showed the catalytic activity of co-displaying PETase and MHETase for PET biodegradation. Then, the cell surface displaying PET degradation module was introduced to a JY-Q strain which genome was integrated with PAEs degrading enzymes and exhibited PAEs degradation ability. The resultant strain JY-Q-R1-R4-SFM-TPH have the ability of degradation PET and PAEs simultaneously. This study provided a promising strain resource for PET and PAEs pollution control.

Enhanced Interfacial Modification by Ordered Discotic Liquid Crystals for Thermotolerance Perovskite Solar Cells.

Traditionally used phenylethylamine iodide (PEAI) and its derivatives, such as ortho-fluorine o-F-PEAI, in interfacial modification, are beneficial for perovskite solar cell (PSC) efficiency but vulnerable to heat stability above 85 °C due to ion migration. To address this issue, we propose a composite interface modification layer incorporating the discotic liquid crystal 2,3,6,7,10,11-hexa(pentoxy)triphenylene (HAT5) into o-F-PEAI. The triphenyl core in HAT5 promotes π-π stacking self-assembly and enhances its interaction with o-F-PEAI, forming an oriented columnar phase that improves hole extraction along the one-dimensional direction. HAT5 repairs structural defects in the interfacial layer and retains the layered structure to inhibit ion migration after annealing. Ultimately, our approach increases the efficiency of solar cells from 23.36% to 25.02%. The thermal stability of the devices retains 80.1% of their initial efficiency after aging at 85 °C for 1008 hours without encapsulation. Moreover, the optimized PSCs maintained their initial efficiency of 82.4% after aging under one sunlight exposure for 1008 hours. This study provides a novel strategy using composite materials for interface modification to enhance the thermal and light stability of semiconductor devices.

Phase 1a study of ESG401, a Trop2 antibody-drug conjugate, in patients with locally advanced/metastatic solid tumors.

This phase 1a study assesses ESG401 in patients with heavily pretreated locally advanced or metastatic solid tumors, focusing on metastatic breast cancer. Forty patients are enrolled: three experience dose-limiting toxicities, establishing the maximum tolerated dose at 16 mg/kg on days 1, 8, and 15 of a 28-day cycle. The most common grade ≥3 treatment-related adverse events are neutropenia and leukopenia. Among 38 efficacy-evaluable patients, the objective response rate (ORR) is 34.2%, the disease control rate (DCR) is 65.8%, and the clinical benefit rate (CBR) is 50.0% (including stable disease for at least 6 months). The median progression-free survival is 5.1 months, and the median duration of response is 6.3 months. In patients receiving therapeutically relevant doses, the ORR, DCR, and CBR are 40.6%, 75.0%, and 56.3%, respectively. ESG401 demonstrates a favorable safety profile and promising antitumor activity in this heavily treated population. The trial is registered at ClinicalTrials.gov (NCT04892342).

Antibody-based near-infrared fluorogenic probes for wash-free imaging of cell-surface proteins.

BACKGROUND: Cell-surface proteins, which are closely associated with various physiological and pathological processes, have drawn much attention in drug discovery and disease diagnosis. Thus, wash-free imaging of the target cell-surface protein under its native environment is critical and helpful for early detection and prognostic evaluation of diseases. RESULTS: To minimize the interference from autofluorescence and fit the penetration depth towards tissue samples, we developed a fluorogenic antibody-based probe, Ab-Cy5.5, which will liberate > 5-fold turn-on near-infrared (NIR) emission in the presence of its target antigen within 10 min. SIGNIFICANCE: By taking advantage of the fluorescence-quenched dimeric H-aggregation of Cy5.5, Ab-Cy5.5 with Cy5.5 attached at the N-terminus showed negligible background signal, allowing direct imaging of the target cell-surface protein in both living cells and tissue samples without washing.

A proton channel, Otopetrin 1 (OTOP1) is N-glycosylated at two asparagine residues in third extracellular loop.

A proton (H+) channel, Otopetrin 1 (OTOP1) is an acid sensor in the sour taste receptor cells. Although OTOP1 is known to be activated by extracellular acid, no posttranslational modification of OTOP1 has been reported. As one of the posttranslational modifications, glycosylation is known to modulate many ion channels. In this study, we investigated whether OTOP1 is glycosylated and how the glycosylation affects OTOP1 function. Pharmacological and enzymatic examinations (using an N-glycosylation inhibitor, tunicamycin and peptide: N-glycanase F [PNGase F]) revealed that overexpressed mouse OTOP1 was N-glycosylated. As the N-glycans were Endoglycosidase H (Endo H)-sensitive, they were most likely high-mannose type. A site-directed mutagenesis approach revealed that both two asparagine residues (N238 and N251) in the third extracellular loop between the fifth transmembrane region and the sixth transmembrane region (L5-6) were the glycosylation sites. Prevention of the glycosylations by the mutations of the asparagine residues or by tunicamycin treatment diminished the whole-cell OTOP1 current densities. The results of cell surface biotinylation assay showed that the prevention of the glycosylations reduced the surface expression of OTOP1 at the plasma membrane. These results indicate that mouse OTOP1 is N-glycosylated at N238 and N251, and that the glycosylations are necessary for OTOP1 to show the maximum degree of H+ current densities at the plasma membrane through promoting its targeting to the plasma membrane. These findings on glycosylations of OTOP1 will be a part of a comprehensive understanding on the regulations of OTOP1 function.

Characterization of probiotics isolated from dietary supplements and evaluation of metabiotic-antibiotic combinations as promising therapeutic options against antibiotic-resistant pathogens using time-kill assay.

BACKGROUND: The global probiotics dietary supplements market size is continuously growing. To overcome probiotics' health concerns, metabiotics are recognized as a safer alternative. Aiming to deal with the escalating antimicrobial resistance, the current work demonstrates synergistic metabiotic-antibiotic combinations against antibiotic-resistant pathogens. METHODS: The probiotic properties of lactic acid bacteria (LAB) strains isolated from 3 commercial dietary supplements were characterized in vitro. The combinations of the cell-free supernatants (CFS) of selected probiotic strains and conventional antibiotics against Staphylococcus aureus and Escherichia coli clinical isolates were evaluated using the time-kill assay. To our knowledge, the current literature lacks sufficient time-kill assay studies revealing the kinetics of such metabiotic-antibiotic combinations against S. aureus and E. coli. RESULTS: Four LAB strains isolated from dietary supplements as well as two reference strains were included in this study. The isolated LAB strains were identified by MALDI-TOF mass spectrometry as follows: P2: Lactobacillus acidophilus, P3: Lactiplantibacillus plantarum, P4: Lacticaseibacillus rhamnosus, and P5: Pediococcus acidilactici. The identification matched with that annotated by the manufacturers, except for P3. The tested strains could resist the acidic environment at pH 3. Excluding P2, the examined strains showed less than 1 log reduction in survivors upon the addition of reconstituted skimmed milk to pepsin at pH 2 and displayed an acceptable tolerance to 0.3% ox-bile. All the strains tolerated pancreatin. The hydrophobicity and autoaggregation capacities ranged between 7-92% and 36-66%, respectively. P2 was excluded owing to its inferior probiotic potential. Although the remaining strains showed excellent growth at 0.2% phenol, their growth was reduced at higher concentrations. L. plantarum and P. acidilactici strains possessed bile salt hydrolysis activity. The time-kill assay revealed promising synergistic activities of the combinations of CFS of L. rhamnosus P4 with either ceftazidime or gentamicin against E. coli and with only ceftazidime against S. aureus, as well as CFS of P. acidilactici P5 and ceftazidime against S. aureus. CONCLUSIONS: Strict identification and evaluation of the probiotic strains incorporated in dietary supplements is crucial to ensure their safety and efficacy. The CFS of probiotics could be utilized to formulate novel biotherapeutics targeting problematic pathogens. However, future in vivo studies are required to evaluate the appropriate treatment regimen.

Insights into the recognition of hypermucoviscous Klebsiella pneumoniae clinical isolates by innate immune lectins of the Siglec and galectin families.

Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA.

GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.

Evaluation of the Effect of Menopausal Status and BMI on Polymorphisms in Fas/Fas L and the Risk of Breast Cancer.

BACKGROUND: FAS and FAS ligand play an essential role in cell apoptosis. An identifying feature of malignant cells is the loss of FAS and increased FASL expression. A study analyzing the effects of menopausal status and body mass index (BMI) on functional polymorphisms of FAS-(1377G/A; rs2234767 & 670 A/G; rs1800682) and FASL (-844T/C; rs763110 & Ivs-2nt; rs5030772) in breast cancer evaluated these effects. PATIENTS AND METHODS: 316 blood samples were collected from breast cancer patients and healthy controls in this case/control study. RFLP-PCR was used after DNA extraction to determine genotypes. Age, BMI, menopausal status, smoking, and family history were also analyzed with genotypes. It was analyzed using SPSS software, X2 statistical tests, logistic regression, and Pearson's correlation. The study evaluated the role of indices and polymorphisms in breast cancer risk. RESULTS: While BMI and family history were significantly different, age, menopause status, and smoking were not. Examining the average BMI between menopausal and nonmenopausal people in the 2 groups showed a statistically significant difference between menopausal people (P <0.0001). As a result of 1377AA, 670GG, 844TT, and IVS-2ntGG, the risk of breast cancer increased by 1.83 times, 2.35 times, and 2.38 times respectively. In addition, mutant alleles increased disease risk significantly. The risk of disease increased considerably for postmenopausal females with certain genotypes (except 1377GA and 844CT genotypes) and high BMI. CONCLUSION: Having a high BMI during postmenopause increases your risk of breast cancer. In addition to menopause, BMI also influences disease progression. Different genotypes are needed to clarify this issue.

On-Demand Vaccine Production via Dock-and-Display of Biotinylated Antigens on Bacterial Extracellular Vesicles.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.

Targeting overexpressed surface proteins: A new strategy to manage the recalcitrant triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous cancer that lacks all three molecular markers, Estrogen, Progesterone, and Human Epidermal Growth Factor Receptor 2 (HER2). This unique characteristic of TNBC makes it more resistant to hormonal therapy; hence, chemotherapy and surgery are preferred. Active targeting with nanoparticles is more effective in managing TNBC than a passive approach. The surface of TNBC cells overexpresses several cell-specific proteins, which can be explored for diagnostic and therapeutic purposes. Immunohistochemical analysis has revealed that TNBC cells overexpress αVß3 integrin, Intercellular Adhesion Molecule 1 (ICAM-1), Glucose Transporter 5 (GLUT5), Transmembrane Glycoprotein Mucin 1 (MUC-1), and Epidermal Growth Factor Receptor (EGFR). These surface proteins can be targeted using ligands, such as aptamers, antibodies, and sugar molecules. Targeting the surface proteins of TNBC with ligands helps harmonize treatment and improve patient compliance. In this review, we discuss the proteins expressed, which are limited to αVß3 integrin proteins, ICAM-1, GLUT-5, MUC1, and EGFR, on the surface of TNBC, the challenges associated with the preclinical setup of breast cancer for targeted nanoformulations, internalization techniques and their challenges, suggestions to overcome the limitations of successful translation of nanoparticles, and the possibility of ligand-conjugated nanoparticles targeting these surface receptors for a better therapeutic outcome.

Nanosensor-Enabled Detection and Identification of Intracellular Bacterial Infections in Macrophages.

Opportunistic bacterial pathogens can evade the immune response by residing and reproducing within host immune cells, including macrophages. These intracellular infections provide reservoirs for pathogens that enhance the progression of infections and inhibit therapeutic strategies. Current sensing strategies for intracellular infections generally use immunosensing of specific biomarkers on the cell surface or polymerase chain reaction (PCR) of the corresponding nucleic acids, making detection difficult, time-consuming, and challenging to generalize. Intracellular infections can induce changes in macrophage glycosylation, providing a potential strategy for signature-based detection of intracellular infections. We report here the detection of bacterial infection in macrophages using a boronic acid (BA)-based pH-responsive polymer sensor array engineered to distinguish mammalian cell phenotypes by their cell surface glycosylation signatures. The sensor was able to discriminate between different infecting bacteria in minutes, providing a promising tool for diagnostic and screening applications.

Cell Surface Engineering by Phase-Separated Coacervates for Antibody Display and Targeted Cancer Cell Therapy.

Cell therapies such as CAR-T have demonstrated significant clinical successes, driving the investigation of immune cell surface engineering using natural and synthetic materials to enhance their therapeutic performance. However, many of these materials do not fully replicate the dynamic nature of the extracellular matrix (ECM). This study presents a cell surface engineering strategy that utilizes phase-separated peptide coacervates to decorate the surface of immune cells. We meticulously designed a tripeptide, Fmoc-Lys-Gly-Dopa-OH (KGdelta; Fmoc = fluorenylmethyloxycarbonyl; delta = Dopa, dihydroxyphenylalanine), that forms coacervates in aqueous solution via phase separation. These coacervates, mirroring the phase separation properties of ECM proteins, coat the natural killer (NK) cell surface with the assistance of Fe3+ ions and create an outer layer capable of encapsulating monoclonal antibodies (mAb), such as Trastuzumab. The antibody-embedded coacervate layer equips the NK cells with the ability to recognize cancer cells and eliminate them through enhanced antibody-dependent cellular cytotoxicity (ADCC). This work thus presents a unique strategy of cell surface functionalization and demonstrates its use in displaying cancer-targeting mAb for cancer therapies, highlighting its potential application in the field of cancer therapy.

Sorting of GPI-anchored proteins at the trypanosome surface is independent of GPI insertion signals.

The segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) to distinct domains on the plasma membrane of eukaryotic cells is important for their correct cellular function, but the mechanisms by which GPI-APs are sorted are yet to be fully resolved. An extreme example of this is in African trypanosomes, where the major surface glycoprotein floods the whole cell surface while most GPI-APs are retained in a specialised domain at the base of the flagellum. One possibility is that anchor attachment signals direct differential sorting of proteins. To investigate this, we fused a monomeric reporter to the GPI-anchor insertion signals of trypanosome proteins that are differentially sorted on the plasma membrane. Fusions were correctly anchored by GPI, post-translationally modified, and routed to the plasma membrane, but this delivery was independent of retained signals upstream of the ω site. Instead, ω-minus signal strength appears key to efficacy of GPI addition and to GPI-AP cellular level. Thus, at least in this system, sorting is not encoded at the time of GPI anchor addition or in the insertion sequence retained in processed proteins. We discuss these findings in the context of previously proposed models for sorting mechanisms in trypanosomes.

Small Molecules Targeting GRP78 Mitigate Anti- GRP78 Autoantibody-Mediated Tissue Factor Procoagulant Activity in Cultured Endothelial Cells.

BACKGROUND: The 78-kDa glucose-regulated protein (GRP78) expressed on the cell surface (csGRP78) has been reported to regulate tissue factor procoagulant activity (TF PCA) in lesion-resident endothelial cells (ECs), which is further enhanced by circulating anti-GRP78 autoantibodies that bind to the Leu98-Leu115 epitope in GRP78. OBJECTIVES: Determine the effects of the engagement of the anti-GRP78 autoantibody to cell surface GRP78 on endothelial cells and the underlying mechanisms that impact TF PCA. METHODS: Immunofluorescent staining was used to determine the presence of csGRP78 in TNFα-treated endothelial cells. An established TF PCA assay was used to evaluate human ECs following treatment with anti-GRP78 autoantibodies. The Fura 2-AM assay was used to quantify changes in intracellular Ca2+ levels. Small molecules predicted to bind csGRP78 were identified using artificial intelligence. ELISAs were used to assess the ability of these GRP78 binders to mitigate TF activity and interfere with the autoantibody/csGRP78 complex. RESULTS: In TNFα-treated ECs, anti-GRP78 autoantibodies increased TF PCA. This observation was further enhanced by ER-stress-induced elevation of csGRP78 levels. Anti-GRP78 autoantibody treatment increased intracellular Ca2+ levels. Sequestering the anti-GRP78 autoantibody with a conformational peptide or blocking with heparin molecules attenuated anti-GRP78 autoantibody-induced TF PCA. We identified B07*, a GRP78 binder that diminished anti-GRP78 autoantibody-induced TF PCA on ECs. CONCLUSIONS: These findings show how anti-GRP78 autoantibodies enhance TF PCA that contributes to thrombosis and identify novel GRP78 binders that represent a potential novel therapeutic strategy for treating and managing atherothrombotic disease.

Germ cell tumors, cell surface markers, and the early search for human pluripotent stem cells.

Many strands of research by different groups, starting from teratocarcinomas in the laboratory mouse, later moving the corresponding human tumors, contributed to the isolation and description of human pluripotent stem cells (PSCs). In this review, I highlight the contributions from my own research, particularly at the Wistar Institute during the 1980s, when with my colleagues we characterized one of the first clonal lines of pluripotent human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, and identified key features including cell surface antigen markers that have since found a place in the study and exploitation of human PSC. Much of this research depended upon close teamwork with colleagues, many in other laboratories, who contributed different expertise and experience. It was also often driven by circumstance and chance rather than pursuit of a grand design.

Extracellular vesicle-liposome hybrids via membrane fusion using cell-penetrating peptide-conjugated lipids.

Extracellular vesicles (EVs) are natural carriers for intercellular communication within the human body. Mimicking and utilizing EVs by combining them with artificial nanocarriers such as liposomes for drug delivery has garnered considerable attention. However, current technologies for manipulating EVs to facilitate their fusion with liposomes are limited; the existing technique of polyethylene glycol (PEG)-induced fusion is highly inefficient for fusion. In our previous study, we demonstrated that membrane fusion could be induced by Tat peptide (YGRKKRRQRRR)-conjugated poly(ethylene glycol)-phospholipids (Tat-PEG-lipids), in which the Tat peptide and lipid domain facilitate membrane attachment and subsequent fusion between cells and liposomes. This approach is promising for forming EV and liposomal hybrids. In this study, we aim to fuse EVs and liposomes using Tat-PEG-lipids. We isolated and characterized EVs derived from HEK293T cell culture medium and treated a mixture of EVs and liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol (1:1, molar ratio), with Tat-PEG-lipids with different lipid chain lengths. Here, we used nonanoyl (C9), dodecanoyl (C12), and myristoyl (C14) groups as lipid anchors with 5 kDa PEG chains. Dynamic light scattering analysis revealed a large increase in the apparent size of mixture of EVs and liposomes by adding Tat-PEG-lipids (especially C14, C12, followed by C9). Fluorescence resonance energy transfer, confocal laser scanning microscopy, and transmission electron microscopy, used to analyze the reaction process, revealed that the membrane fusion occurred between EVs and liposomes but not their aggregates. The short lipid domain of Tat-PEG-lipids effectively induced membrane fusion and the formation of hybrid EVs and liposomes. Thus, Tat-PEG-lipids (C9 and C12) could be promising candidates for inducing membrane fusion to fabricate EV-liposome hybrids.

Reducing residual chlortetracycline in wastewater using a whole-cell biocatalyst.

Antibiotic contamination has become an increasingly important environmental problem as a potentially hazardous emergent and recalcitrant pollutant that poses threats to human health. In this study, manganese peroxidase displayed on the outer membrane of Escherichia coli as a whole-cell biocatalyst (E. coli MnP) was expected to degrade antibiotics. The manganese peroxidase activity of the whole-cell biocatalyst was 13.88 ± 0.25 U/L. The typical tetracycline antibiotic chlortetracycline was used to analyze the degradation process. Chlortetracycline at 50 mg/L was effectively transformed via the whole-cell biocatalyst within 18 h. After six repeated batch reactions, the whole-cell biocatalyst retained 87.2 % of the initial activity and retained over 87.46 % of the initial enzyme activity after storage at 25°C for 40 days. Chlortetracycline could be effectively removed from pharmaceutical and livestock wastewater by the whole-cell biocatalyst. Thus, efficient whole-cell biocatalysts are effective alternatives for degrading recalcitrant antibiotics and have potential applications in treating environmental antibiotic contamination.

Surface display provides an efficient expression system for production of recombinant proteins and bacterial whole cell biosensor in E. coli.

A novel bacterial display vector based on Escherichia coli has been engineered for recombinant protein production and purification. Accordingly, a construct harboring the enhanced green fluorescent protein (EGFP) and the ice nucleation protein (INP) was designed to produce EGFP via the surface display in E. coli cells. The fusion EGFP-expressed cells were then investigated using fluorescence measurement, SDS- and native-PAGE before and after TEV protease digestion. The displayed EGFP was obtained with a recovery of 57.7 % as a single band on SDS-PAGE. Next, the efficiency of the cell surface display for mutant EGFP (EGFP S202H/Q204H) was examined in sensing copper ions. Under optimal conditions, a satisfactorily linear range for copper ions concentrations up to 10 nM with a detection limit of 0.073 nM was obtained for cell-displayed mutant EGFP (mEGFP). In the presence of bacterial cell lysates and purified mEGFP, response to copper was linear in the 2-10 nM and 0.1-2 µM concentration range, respectively, with a 1.3 nM and 0.14 µM limit of detection. The sensitivity of bacterial cell lysates and surface-displayed mEGFP in the detection of copper ions is higher than the purified mEGFP.

Construction of a bioelectrocatalytic system with bacterial surface displayed enzyme-nanomaterial hybrids.

To take advantage of the high specificity of enzymatic catalysis along with the high efficiency of electrochemical cofactor regeneration, a bacterial surface displayed enzyme-nanomaterial hybrid bioelectrocatalytic system is herein developed. A cofactor-dependent xylose reductase, capable of reducing xylose to xylitol, is displayed on the surface of Bacillus subtilis, followed by the attachment of copper nanomaterials via the binding of His-tagged enzyme with the nickel ion. This hybrid system can regenerate NADPH with a highest efficiency of 71.6% in 4 h without the usage of extra electron mediators, and 2.35 mM of xylitol can be synthesized after a series of optimization processes. This work opens up new possibilities for the construction and application of bioelectrocatalytic systems with enzyme-nanomaterial hybrids.