Biology subjects

yang, l.

Publications and source records attributed to yang, l..

3 recordsLinked to original sources

bioRxiv2025

A coupled fluorescence assay for high-throughput screening of polyurethane-degrading enzymes

The global accumulation of plastic waste, particularly persistent polymers like polyurethane (PU), demands urgent solutions. Enzymatic depolymerization offers a viable strategy for PU waste valorization. However, progress has been hindered by the lack of reliable high-throughput screening (HTS) assays capable of precise and quantitative evaluation of enzymatic activity. Current enzyme screening assays face significant limitations due to poor substrate relevance and inadequate quantification methods. Here, we present a novel HTS assay featuring a chemically defined, synthetic poly (ethylene adipate)-based PU substrate that enables unambiguous structure-activity analysis and direct quantification of degradation products (adipic acid and ethylene glycol). Coupled with this substrate is a highly sensitive fluorescence-based detection cascade, in which released ethylene glycol is stoichiometrically converted to resorufin via a two-enzyme system (glycerol dehydrogenase and diaphorase). This assay overcomes key limitations of commercial substrates (e.g., Impranil DLN) by providing quantitative, real-time monitoring of PU hydrolysis with molecular precision. Validation with known PU-degrading enzymes demonstrated that the assay can sensitively distinguish differences in enzymatic activity with high reproducibility and quantitative accuracy. Our platform enables rapid, cost-effective screening of enzyme libraries and engineered variants, significantly advancing enzyme discovery and optimization efforts. By bridging the gap between laboratory research and industrial application, this HTS assay accelerates the development of sustainable PU recycling solutions, offering a critical tool against plastic pollution. Graphical Abstract Legend O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/663332v4_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@17ba5c0org.highwire.dtl.DTLVardef@bef89aorg.highwire.dtl.DTLVardef@1b3770eorg.highwire.dtl.DTLVardef@14d5ef5_HPS_FORMAT_FIGEXP M_FIG (A) Workflow of the enzymatic degradation assay. Enzymes are incubated with chemically defined PEA-PU films. The resulting hydrolysate is collected and analysed using a fluorescence-based assay in microplate format (Ex/Em = 535/588 nm). (B) Fluorescence-based detection mechanism. Assay principle based on ethylene glycol (EG), one of the major degradation products. EG is converted through a two-step enzymatic cascade involving glycerol dehydrogenase (GldA) and diaphorase, leading to the generation of fluorescent resorufin from resazurin. C_FIG

synthetic biology↗

bioRxiv2024

Cas9AEY (Cas9-facilitated Homologous Recombination Assembly of non-specific Escherichia coli yeast vector) method of constructing large-sized DNA.

Saccharomyces cerevisiae is widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector can replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure plasmid. Saccharomyces cerevisiae cells are widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector has the ability to replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure knocked out plasmid.

synthetic biology↗

bioRxiv2019

Temperature dependence of allelopathy duality and its influence on boreal forest succession-A case analysis of Picea schrenkiana

Global warming in conjunction with various biotic or abiotic interferences has been jeopardizing the ecosystem of boreal forests. By integrating field inspection with experimental simulation, this work comprehensively investigated the allelopathic effects of a key allelochemical 3,4-dihydroxyacetophenone (DHAP) in the exudates of P. schrenkiana needles on its seed and seedling growth, endogenous hormone metabolism and antioxidant enzyme activity, identified the existence of DHAP allelopathy duality at a certain temperature with an inflection concentration point (e.g. about 0.25 mM at dark/light temperature of 4/12 {degrees}C) as the boundary between promotional and inhibitory effect, and verified that the inflection point of DHAP concentration would inevitably shift to a lower level as temperature increased. Consequently, this paper gives a scientific explanation into the intrinsic mechanism of P. schrenkiana degradation due to allelopathy, but also presents a new approach to explore the relationship between forest evolution and global warming.\n\nHighlightA quantitative description on the duality of 3, 4-dihydroxyacetophenone (DHAP) as a promoter or an inhibitor to affect the seed germination, seedling growth and root development of P. schrenkiana, as well as the antioxidant enzyme activities and hormone contents.\n\nThe new findings of DHAP inflection concentration as boundary to divide the promotional and inhibitory effect of allelopathy which would decrease as environment temperatures rise.\n\nAn explanation into the intrinsic mechanism of P. schrenkiana degradation due to allelopathy, and a new approach to explore the relationship between forest evolution and global warming.

plant biology↗