Immobilization of D-amino acid dehydrogenase(DAADH)by the assembly of peptide linker was studied for the biosynthesis of Dphenylalanine.Hybrid material of zeolitic imidazolate framework-8(ZIF-8)combined with reduced g...Immobilization of D-amino acid dehydrogenase(DAADH)by the assembly of peptide linker was studied for the biosynthesis of Dphenylalanine.Hybrid material of zeolitic imidazolate framework-8(ZIF-8)combined with reduced graphene oxide(RGO)was applied for the immobilization of DAADH from Ureibacillus thermosphaericus.The recovery rate of DAADH/ZIF-8/RGO was 165.6%.DAADH/ZIF-8/RGO remained 53.4%of its initial activity at 50°C for 10 h while the free enzyme was inactivated.DAADH/ZIF-8/RGO maintained 70.5%activity in hyperalkaline solution with pH 12.Kinetic parameters indicated that DAADH/ZIF-8/RGO had greater affinity of phenylpyruvate as V_(max)/K_(m)of DAADH/ZIF-8/RGO was 1.27-fold than free enzyme.After seven recycles,the activity of DAADH/ZIF-8/RGO remained 64.3%.Furthermore,one-step separation and in situ immobilization of DAADH by ZIF-8/RGO/Ni was carried out with 1.5-fold activity enhancement.Combining peptide linker and metal-organic framework(MOF)immobilization,thermostability and activity of the immobilized DAADH were significantly improved.展开更多
A new method has been proposed to realize the visual detection of D-amino acids （DAAs） via the antiaggregation of 4-mercaptobenzoic acid modified gold nanoparticles （AuNPs） in the presence of D-amino acid oxidase ...A new method has been proposed to realize the visual detection of D-amino acids （DAAs） via the antiaggregation of 4-mercaptobenzoic acid modified gold nanoparticles （AuNPs） in the presence of D-amino acid oxidase （DAAO）. The negatively charged AuNPs were prepared using sodium citrate as a reducer and stabilizer. The presence of 4-mercaptobenzoic acid （4-MBA） and Cu2＋ induces the aggregation of AuNPs, resulting in a color change from ruby red to royal purple. However, DAAO could oxidize DAAs to generate H2O2. In the presence of H2O2, the mercapto （-SH） group in 4-mercaptobenzoic acid can be oxidized to form a disulfide （-S-S-） bond. Based on these facts, the pre-incubation of DAAs and 4-mercaptobenzoic acid with DAAO would significantly reduce the concentration of free 4-mercaptobenzoic acid molecules, thus the aggregation of AuNPs was interrupted since due to the lack of inducer. As the concentration of DAAs increases, the color of the AuNPs solution would progress from royal purple to ruby red. Consequently, DAAs could be monitored by the colorimetric response of AuNPs using a UV-vis spectrophotometer or even naked eyes. This DAAO mediated visual detection method could determine D- alanine （D-Ala） as a representative DAA with concentrations ranging from 1.5 × 10^-7mol L 1 to 3.0 × 10^-5 mol L^-1, and the detection limit was as low as 7.5 × 10^-8 mol L^-1. The proposed method is convenient, low-cost and free of complex equipment, making it feasible to analyze the concentration of D-AIa in real samples of β-amyloid peptide （Aβ1-42）.展开更多
The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants.However,screening edited alleles,particularly those with multiplex editing,from herbicide-or antibiotic-resistant ...The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants.However,screening edited alleles,particularly those with multiplex editing,from herbicide-or antibiotic-resistant transgenic plants and segregating out the Cas9 transgene represent two laborious processes.Current solutions to facilitate these processes rely on different selection markers.Here,by taking advantage of the opposite functions of a D-amino acid oxidase(DAO)in detoxifying D-serine and in metabolizing non-toxic D-valine to a cytotoxic product,we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of Cas9-containing progeny in Arabidopsis thaliana.Among five DAOs tested in Escherichia coli,the one encoded by Trigonopsis variabilis(TvDAO)could confer slightly stronger D-serine resistance than other homologs.Transgenic expression of TvDAO in Arabidopsis allowed a clear distinction between transgenic and nontransgenic plants in both D-serine-conditioned positive selection and D-valine-conditioned negative selection.As a proof of concept,we combined CRISPR-induced single-strand annealing repair of a dead TvDAO with D-serine-based positive selection to help identify transgenic plants with multiplex editing,where D-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin.Subsequently,D-valine-based negative selection successfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inherited mutations.Collectively,this work provides a novel strategy to ease CRISPR mutant identification and Cas9 transgene elimination using a single selection marker,which promises more efficient and simplified multiplex CRISPR editing in plants.展开更多
基金supported by the National Natural Science Foundation of China(Nos.21776233 and 22078273)National Key Research and Development Program(No.2022YFA0912000).
文摘Immobilization of D-amino acid dehydrogenase(DAADH)by the assembly of peptide linker was studied for the biosynthesis of Dphenylalanine.Hybrid material of zeolitic imidazolate framework-8(ZIF-8)combined with reduced graphene oxide(RGO)was applied for the immobilization of DAADH from Ureibacillus thermosphaericus.The recovery rate of DAADH/ZIF-8/RGO was 165.6%.DAADH/ZIF-8/RGO remained 53.4%of its initial activity at 50°C for 10 h while the free enzyme was inactivated.DAADH/ZIF-8/RGO maintained 70.5%activity in hyperalkaline solution with pH 12.Kinetic parameters indicated that DAADH/ZIF-8/RGO had greater affinity of phenylpyruvate as V_(max)/K_(m)of DAADH/ZIF-8/RGO was 1.27-fold than free enzyme.After seven recycles,the activity of DAADH/ZIF-8/RGO remained 64.3%.Furthermore,one-step separation and in situ immobilization of DAADH by ZIF-8/RGO/Ni was carried out with 1.5-fold activity enhancement.Combining peptide linker and metal-organic framework(MOF)immobilization,thermostability and activity of the immobilized DAADH were significantly improved.
基金supported by the National Natural Science Foundation of China(No.21272263)the State Key Laboratory of Natural and Biomimetic Drugs(No.K20130206)+1 种基金the University of Chinese Academy of Sciences(No.08JT011J01)the Twelfth Five-Year Plan for National Sciences&Technology Support Project(No.2012BAI37B03)
文摘A new method has been proposed to realize the visual detection of D-amino acids （DAAs） via the antiaggregation of 4-mercaptobenzoic acid modified gold nanoparticles （AuNPs） in the presence of D-amino acid oxidase （DAAO）. The negatively charged AuNPs were prepared using sodium citrate as a reducer and stabilizer. The presence of 4-mercaptobenzoic acid （4-MBA） and Cu2＋ induces the aggregation of AuNPs, resulting in a color change from ruby red to royal purple. However, DAAO could oxidize DAAs to generate H2O2. In the presence of H2O2, the mercapto （-SH） group in 4-mercaptobenzoic acid can be oxidized to form a disulfide （-S-S-） bond. Based on these facts, the pre-incubation of DAAs and 4-mercaptobenzoic acid with DAAO would significantly reduce the concentration of free 4-mercaptobenzoic acid molecules, thus the aggregation of AuNPs was interrupted since due to the lack of inducer. As the concentration of DAAs increases, the color of the AuNPs solution would progress from royal purple to ruby red. Consequently, DAAs could be monitored by the colorimetric response of AuNPs using a UV-vis spectrophotometer or even naked eyes. This DAAO mediated visual detection method could determine D- alanine （D-Ala） as a representative DAA with concentrations ranging from 1.5 × 10^-7mol L 1 to 3.0 × 10^-5 mol L^-1, and the detection limit was as low as 7.5 × 10^-8 mol L^-1. The proposed method is convenient, low-cost and free of complex equipment, making it feasible to analyze the concentration of D-AIa in real samples of β-amyloid peptide （Aβ1-42）.
基金supported by the National Key Research and Development Program of China(grant 2019YFA0906202)J.-F.L.,the National Natural Science Foundation of China(grants 31900305 and 32370294)the Natural Science Foundation of Guangdong Province(grant 2020A1515010465)to F.-Z.W.
文摘The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants.However,screening edited alleles,particularly those with multiplex editing,from herbicide-or antibiotic-resistant transgenic plants and segregating out the Cas9 transgene represent two laborious processes.Current solutions to facilitate these processes rely on different selection markers.Here,by taking advantage of the opposite functions of a D-amino acid oxidase(DAO)in detoxifying D-serine and in metabolizing non-toxic D-valine to a cytotoxic product,we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of Cas9-containing progeny in Arabidopsis thaliana.Among five DAOs tested in Escherichia coli,the one encoded by Trigonopsis variabilis(TvDAO)could confer slightly stronger D-serine resistance than other homologs.Transgenic expression of TvDAO in Arabidopsis allowed a clear distinction between transgenic and nontransgenic plants in both D-serine-conditioned positive selection and D-valine-conditioned negative selection.As a proof of concept,we combined CRISPR-induced single-strand annealing repair of a dead TvDAO with D-serine-based positive selection to help identify transgenic plants with multiplex editing,where D-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin.Subsequently,D-valine-based negative selection successfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inherited mutations.Collectively,this work provides a novel strategy to ease CRISPR mutant identification and Cas9 transgene elimination using a single selection marker,which promises more efficient and simplified multiplex CRISPR editing in plants.
