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Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

ABSTRACT: Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.

SUBMITTER: Ahsan A

PROVIDER: S-EPMC10898825 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

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Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

Ahsan Afrin A Wagner Dominique D Varaljay Vanessa A VA Roman Victor V Kelley-Loughnane Nancy N Reuel Nigel F NF

Synthetic biology (Oxford, England) 20240213 1

Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hyd ...[more]

PMID: 38414826

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Project description:Polyester urethanes (PUR) are widely used in industries and have led to a worldwide plastic waste problem. Thus, novel solutions for PUR degradation are required to reduce environmental pollution. This work investigates the PUR biodegradation efficiency of 33 fungal species using a polyester-polyurethane colloid branded Impranil DLN (Impranil) compared to Aspergillus niger, which served as the positive control. The biodegradation is evaluated based on its ability to clear Impranil in media. Eleven fungi can clear Impranil in both solid- and liquid-medium assays. The highest degradation was attributed to Embarria clematidis cultured with Impranil as a carbon source. The degradation was confirmed by the Sturm test, Fourier-transform infrared (FTIR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). From the Sturm test, CO2 at a concentration of 0.85 g/L was found in E. clematidis cultured with 150 mL of Impranil solution after a 2-week incubation period while the CO2 at a concentration of 0.53 g/L was detected from A. niger in the same conditions. The biodegradation was further confirmed by evaluating the clearance percentage of supernatant of E. clematidis and A. niger culturing with Impranil from the Sturm test. The clearance percentage of E. clematidis and A. niger supernatant was 88.84 and 48.97%, respectively. Moreover, the degradation of soft segment and breakdown of ester linkages were observed, as evidenced by the decrease of the carbonyl (1,715 cm-1) and N-H stretching (1,340 cm-1 and 1,020 cm-1) FTIR spectral peaks, respectively. GC-MS detected 3Z-heptenol, 5Z-octenol, 2E,4E-hexadienol acetate, and 3E,6Z-nonadienol as degradation products from the E. clematidis culture supernatant. This fungus was screened for its ability to produce extracellular esterase, protease, and urease enzymes. Extracellular esterase, very low urease, and no protease activities were detected in the culture supernatant of E. clematidis in the presence of Impranil. E. clematidis can degrade Impranil partially via hydrolysis of ester linkages by cell-bound esterases at a considerable rate without any prior treatment. This fungus not only degraded Impranil but also mineralized them into CO2 and H2O. E. clematidis can be applied in the process of biochemical depolymerization of PUR for the pure monomers recycling.

Project description:BackgroundComputational methods for mining of biomedical literature can be useful in augmenting manual searches of the literature using keywords for disease-specific biomarker discovery from biofluids. In this work, we develop and apply a semi-automated literature mining method to mine abstracts obtained from PubMed to discover putative biomarkers of breast and lung cancers in specific biofluids.MethodologyA positive set of abstracts was defined by the terms 'breast cancer' and 'lung cancer' in conjunction with 14 separate 'biofluids' (bile, blood, breastmilk, cerebrospinal fluid, mucus, plasma, saliva, semen, serum, synovial fluid, stool, sweat, tears, and urine), while a negative set of abstracts was defined by the terms '(biofluid) NOT breast cancer' or '(biofluid) NOT lung cancer.' More than 5.3 million total abstracts were obtained from PubMed and examined for biomarker-disease-biofluid associations (34,296 positive and 2,653,396 negative for breast cancer; 28,355 positive and 2,595,034 negative for lung cancer). Biological entities such as genes and proteins were tagged using ABNER, and processed using Python scripts to produce a list of putative biomarkers. Z-scores were calculated, ranked, and used to determine significance of putative biomarkers found. Manual verification of relevant abstracts was performed to assess our method's performance.ResultsBiofluid-specific markers were identified from the literature, assigned relevance scores based on frequency of occurrence, and validated using known biomarker lists and/or databases for lung and breast cancer [NCBI's On-line Mendelian Inheritance in Man (OMIM), Cancer Gene annotation server for cancer genomics (CAGE), NCBI's Genes & Disease, NCI's Early Detection Research Network (EDRN), and others]. The specificity of each marker for a given biofluid was calculated, and the performance of our semi-automated literature mining method assessed for breast and lung cancer.ConclusionsWe developed a semi-automated process for determining a list of putative biomarkers for breast and lung cancer. New knowledge is presented in the form of biomarker lists; ranked, newly discovered biomarker-disease-biofluid relationships; and biomarker specificity across biofluids.

Project description:UnlabelledPolyurethane (PU) is widely used in many aspects of modern life because of its versatility and resistance. However, PU waste disposal generates large problems, since it is slowly degraded, there are limited recycling processes, and its destruction may generate toxic compounds. In this work, we isolated fungal strains able to grow in mineral medium with a polyester PU (PS-PU; Impranil DLN) or a polyether PU (PE-PU; Poly Lack) varnish as the only carbon source. Of the eight best Impranil-degrading strains, the six best degraders belonged to the Cladosporium cladosporioides complex, including the species C. pseudocladosporioides, C. tenuissimum, C. asperulatum, and C. montecillanum, and the two others were identified as Aspergillus fumigatus and Penicillium chrysogenum The best Impranil degrader, C. pseudocladosporioides strain T1.PL.1, degraded up to 87% after 14 days of incubation. Fourier transform infrared (FTIR) spectroscopy analysis of Impranil degradation by this strain showed a loss of carbonyl groups (1,729 cm(-1)) and N-H bonds (1,540 and 1,261 cm(-1)), and gas chromatography-mass spectrometry (GC-MS) analysis showed a decrease in ester compounds and increase in alcohols and hexane diisocyanate, indicating the hydrolysis of ester and urethane bonds. Extracellular esterase and low urease, but not protease activities were detected at 7 and 14 days of culture in Impranil. The best eight Impranil-degrading fungi were also able to degrade solid foams of the highly recalcitrant PE-PU type to different extents, with the highest levels generating up to 65% of dry-weight losses not previously reported. Scanning electron microscopy (SEM) analysis of fungus-treated foams showed melted and thinner cell wall structures than the non-fungus-treated ones, demonstrating fungal biodegradative action on PE-PU.ImportancePolyurethane waste disposal has become a serious problem. In this work, fungal strains able to efficiently degrade different types of polyurethanes are reported, and their biodegradative activity was studied by different experimental approaches. Varnish biodegradation analyses showed that fungi were able to break down the polymer in some of their precursors, offering the possibility that they may be recovered and used for new polyurethane synthesis. Also, the levels of degradation of solid polyether polyurethane foams reported in this work have never been observed previously. Isolation of efficient polyurethane-degrading microorganisms and delving into the mechanisms they used to degrade the polymer provide the basis for the development of biotechnological processes for polyurethane biodegradation and recycling.

Dataset Information

Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

Publications

Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

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