RESUMO
c-Myc (Myc) is an important transcriptional regulator in embryonic stem (ES) cells, somatic cell reprogramming, and cancer. Here, we identify a Myc-centered regulatory network in ES cells by combining protein-protein and protein-DNA interaction studies and show that Myc interacts with the NuA4 complex, a regulator of ES cell identity. In combination with regulatory network information, we define three ES cell modules (Core, Polycomb, and Myc) and show that the modules are functionally separable, illustrating that the overall ES cell transcription program is composed of distinct units. With these modules as an analytical tool, we have reassessed the hypothesis linking an ES cell signature with cancer or cancer stem cells. We find that the Myc module, independent of the Core module, is active in various cancers and predicts cancer outcome. The apparent similarity of cancer and ES cell signatures reflects, in large part, the pervasive nature of Myc regulatory networks.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Acetilação , Animais , Linhagem Celular , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Much attention has focused on a small set of transcription factors that maintain human or mouse embryonic stem (ES) cells in a pluripotent state. To gain a more complete understanding of the regulatory network that maintains this state, we identified target promoters of nine transcription factors, including somatic cell reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) and others (Nanog, Dax1, Rex1, Zpf281, and Nac1), on a global scale in mouse ES cells. We found that target genes fall into two classes: promoters bound by few factors tend to be inactive or repressed, whereas promoters bound by more than four factors are largely active in the pluripotent state and become repressed upon differentiation. Furthermore, we propose a transcriptional hierarchy for reprogramming factors and broadly distinguish targets of c-Myc versus other factors. Our data provide a resource for exploration of the complex network maintaining pluripotency.
Assuntos
Células-Tronco Embrionárias/metabolismo , Redes Reguladoras de Genes , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Imunoprecipitação da Cromatina , Código das Histonas , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
Lipase immobilization with hydrophobic interaction is of interesting exploration, and some functionalized groups on supports are special for activity increasing. To achieved a good performance of cost-effective immobilization on macro-supports for feasible usage and recycle, eco-friendly PLA-based 3D printing macro-scaffolds with fabrication was designed, and phenyl groups with different length of linkers and combined two kinds of groups were anchored for lipase YCJ01 binding with improving payload, the highest enzyme expression of 2227.5 U/g, activity recovery of 137.3%, and increasing specific activity of 815.9 U/mg were attained by using PLA@AMTS-C7-Ph/PLA@AMTS-C9-Ph scaffolds as carries. The immobilized lipase YCJ01 on bifunctionalized 3D printing scaffolds was further applied to the efficient resolution of racemic 1-indanol (267 mM) with high stereoselectivity using a binary solvent system. The immobilized lipase YCJ01 could control the over transesterification of (S)-1-indanol and exhibit good operational stability of repetitive usage for 9 cycles. This is beneficial to obtain the high enantiomerical pure product by feasible separation of immobilized biocatalyst without rigorous operation.
Assuntos
Enzimas Imobilizadas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Indanos/química , Indanos/isolamento & purificação , Lipase/metabolismo , Impressão Tridimensional , Burkholderia/enzimologia , Reutilização de Equipamento , Solventes/química , EstereoisomerismoRESUMO
Paper devices have attracted great attention for their rapid development in multiple fields, such as life sciences, biochemistry, and materials science. When manufacturing paper chips, flexible materials, such as cellulose paper or other porous flexible membranes, can offer several advantages in terms of their flexibility, lightweight, low cost, safety and wearability. However, traditional cellulose paper sheets with chaotic cellulose fiber constitutions do not have special structures and optical characteristics, leading to poor repeatability and low sensitivity during biochemical sensing, limiting their wide application. Recent evidence showed that the addition of ordered structure provides a promising method for manufacturing intelligent flexible devices, making traditional flexible devices with multiple functions (microfluidics, motion detection and optical display). There is an urgent need for an overall summary of the evolution of paper devices so that readers can fully understand the field. Hence, in this review, we summarized the latest developments in intelligent paper devices, starting with the fabrication of paper and smart flexible paper devices, in the fields of biology, chemistry, electronics, etc. First, we outlined the manufacturing methods and applications of both traditional cellulose paper devices and modern smart devices based on pseudopaper (order paper). Then, considering different materials, such as cellulose, nitrocellulose, nature sourced photonic crystals (photonic crystals sourced from nature directly) and artificial photonic crystals, we summarized a new type of smart flexible device containing an ordered structure. Next, the applications of paper devices in biochemical sensing, wearable sensing, and cross-scale sensing were discussed. Finally, we summarized the development direction of this field. The aim of this review is to take an integral cognition approach to the development of smart flexible paper devices in multiple fields and promote communications between materials science, biology, chemistry and electrical science.
Assuntos
Dispositivos Eletrônicos Vestíveis , Celulose , Eletricidade , Eletrônica , PorosidadeRESUMO
A penicillin G acylase (PGA) from Achromobacter xylosoxidans PX02 was newly isolated, and site-directed mutagenesis at three important positions αR141, αF142, ßF24 was carried out for improving the enzymatic synthesis of ß-lactam antibiotics. The efficient mutant ßF24A was selected, and the (Ps/Ph)ini (ratio between the initial rate of synthesis and hydrolysis of the activated acyl donor) dramatically increased from 1.42-1.50 to 23.8-24.1 by means of the optimization of reaction conditions. Interestingly, the efficient enzymatic synthesis of ampicillin (99.1% conversion) and amoxicillin (98.7% conversion) from a high concentration (600 mM) of substrate 6-APA in the low acyl donor/nucleus ratio (1.1:1) resulted in a large amount of products precipitation from aqueous reaction solution. Meanwhile, the by-product D-phenylglycine was hardly precipitated, and 93.5% yield of precipitated ampicillin (561 mM) and 94.6% yield of precipitated amoxicillin (568 mM) were achieved with high purity (99%), which significantly simplified the downstream purification. This was the first study to achieve efficient ß-lactam antibiotics synthesis process with in situ product removal, with barely any by-product formation. The effect enzymatic synthesis of antibiotics in aqueous reaction solution with in situ product removal provides a promising model for the industrial semi-synthesis of ß-lactam antibiotics.
Assuntos
Achromobacter denitrificans/enzimologia , Antibacterianos/biossíntese , Penicilina Amidase/metabolismo , beta-Lactamas/metabolismo , Antibacterianos/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Penicilina Amidase/genética , Penicilina Amidase/isolamento & purificação , Solubilidade , beta-Lactamas/químicaRESUMO
Paper has unique advantages over other materials, including low cost, flexibility, porosity, and self-driven liquid pumping, thus making it widely used in various fields in biology, chemistry, physics and materials science. Recently, many multifunctional and highly integrated membrane-based devices have been achieved with the rapid development of membrane-building materials such as paper and pseudo-paper. Therefore, the rigid boundary between paper and other membranes has become blurred; paper can be considered a flexible membrane, and membranes with appropriately flexible or porous structures can also be defined as paper. Paper can manipulate liquids and respond photoelectrically to external objects to be measured, making it suitable for (bio)chemical sensing (chromatographic analysis, electrochemical analysis and wearable sensing). This review focuses on the development of microfluidic devices built with both traditional paper and other flexible membranes, including fabrication, (bio)chemical sensing, microfluidics manipulation and multiple applications.
RESUMO
Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0-9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived from Brevibacillus borstelensis LK01 and AmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP: AmPNP (2:20 µg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.
Assuntos
Bacillales/enzimologia , Nucleosídeos de Purina/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Bacillales/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Clonagem Molecular , Estabilidade Enzimática , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/genética , Pirimidina Fosforilases/metabolismo , TermodinâmicaRESUMO
The 62nd residue of glycosyltransferase UGTBL1 was identified as a "hotspot" for glycosylation at 3-OH of resveratrol. Via semi-rational design including structure-guided alanine scanning and saturation mutations, the mutation I62G significantly switched the regioselectivity from 4'-OH to 3-OH of resveratrol and mainly produced polydatin (87.7%), a therapeutic natural product.
Assuntos
Proteínas de Bactérias/química , Glucosídeos/síntese química , Glucosiltransferases/química , Engenharia de Proteínas/métodos , Estilbenos/síntese química , Bacillus licheniformis/enzimologia , Proteínas de Bactérias/genética , Glucosiltransferases/genética , Glicosilação , Cinética , Mutação , Resveratrol/químicaRESUMO
High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting steps. In this study, three PGAs isolated from Providencia rettgeri (PrPGA), Alcaligenes faecalis (AfPGA), and Achromobacter xylosoxidans (AxPGA) were efficiently expressed in E. coli by replacing with applicable signal peptide. Different bottlenecks of the expression process were analyzed for PrPGA, AfPGA, and AxPGA. Subsequently, five efficient signal peptides, including OmpA, pelB, Lpp, PhoA, and MalE, were used to replace the original signal peptides of the PGAs. With respect to AfPGA and AxPGA, translocation was the primary limitation, and the use of pelB signal peptide effectively overcame this barrier. For PrPGA, which was almost not expressed in wild type, the translation initiation efficiency was optimized by replacing with MalE signal peptide. In addition, low temperature (20 °C) slowed down the transcription and translation, thereby facilitating the posttranslational process and preventing the formation of inclusion bodies. Furthermore, combined induction with IPTG and arabinose not only enhanced the cell density but also remarkably improved the expression of PGAs. Final specific activities of the three PGAs reached 2100 (PrPGA), 9200 (AfPGA), and 1400 (AxPGA) U/L/OD600, respectively. This simple and robust strategy by fitting replacement of signal peptide might dramatically improve the expression of PGAs from various bacteria, which was significant in the production of many valuable ß-lactam antibiotics.
Assuntos
Regulação Enzimológica da Expressão Gênica , Microbiologia Industrial/métodos , Penicilina Amidase/genética , Sinais Direcionadores de Proteínas/genética , Achromobacter denitrificans/enzimologia , Achromobacter denitrificans/genética , Alcaligenes faecalis/enzimologia , Alcaligenes faecalis/genética , Escherichia coli/genética , Providencia/enzimologia , Providencia/genéticaRESUMO
OBJECTIVE: To improve the stability of ß-galactosidase from Bacillus megaterium YZ08 (BMG) in aqueous hydrophilic solvents and promote its application in the galactosylation of natural products. RESULTS: The addition of 5 mM Mg2+ significantly enhanced the stability of BMG in aqueous hydrophilic solvents, and the half-lives of BMG in these solutions reached 56 min to 208 h, while they were only 7 min to 5.9 h without addition of Mg2+. Studies on the kinetic parameters in buffer solution and 30% dimethyl sulfoxide (DMSO) indicated that the affinity of BMG to 2-nitrophenyl-ß-D-galactopyranoside and its catalytic efficiency (κ cat/K m) increased with the addition of Mg2+. Furthermore, the addition of Mg2+ facilitated galactosylation reactions in 30% DMSO and increased product conversions by 24-41% due to the reversal of the thermodynamic equilibrium of hydrolysis. CONCLUSION: A convenient approach was established to improve the stability of BMG in aqueous hydrophilic solvents.
Assuntos
Proteínas de Bactérias/química , Produtos Biológicos/metabolismo , Magnésio/química , beta-Galactosidase/química , Bacillus megaterium , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Dimetil Sulfóxido/química , Estabilidade Enzimática , Temperatura Alta , Fenômenos de Química Orgânica , beta-Galactosidase/metabolismoRESUMO
OBJECTIVE: To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining. RESULTS: BbPyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. BbPyNP had a highest 60% identity with that of reported PyNPs. BbPyNP could catalyze the phosphorolysis of thymidine, 2'-deoxyuridine, uridine and 5-methyuridine. BbPyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. BbPyNP had the highest activity at an optimum alkaline pH of 8.5. BbPyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by BbPyNP reached the yield of 61-84%. CONCLUSION: An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.
Assuntos
Brevibacillus/genética , Floxuridina/metabolismo , Engenharia Metabólica/métodos , Nucleosídeos/metabolismo , Pirimidina Fosforilases/metabolismo , Brevibacillus/enzimologia , Estabilidade Enzimática , Escherichia coli/genética , Floxuridina/análise , Temperatura Alta , Concentração de Íons de Hidrogênio , Nucleosídeos/química , Pirimidina Fosforilases/química , Pirimidina Fosforilases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
To achieve efficient production of succinyldaidzin and succinylgenistin, resting cells of a solvent-stable strain Bacillus licheniformis ZSP01 were used to react with pure isoflavones or soybean flour extract in a reaction medium with 10% dimethyl sulfoxide. Strikingly, 0.8 mM daidzein, 0.8 mM genistein, 2.0 mM daidzin, and 2.0 mM genistin were transformed to succinyl isoflavone glycosides in 27 H (yield >90%). The soybean flour extract (6.1%, w/v) contained 0.32 mM daidzein, 0.84 mM daidzin, 0.38 mM genistein, and 1.04 mM genistin. Over 95% of total isoflavones (daidzein, daidzin, genistein, and genistin) in the soybean flour extract were converted to succinyl isoflavone glycosides after 27 H. Strain ZSP01 shows both high glycosylation and succinylation activities. These results suggest that B. licheniformis ZSP01 could be useful for the efficient production of succinyl soybean isoflavone glycosides.
Assuntos
Bacillus/metabolismo , Reatores Biológicos/microbiologia , Glycine max/microbiologia , Glicosídeos/biossíntese , Isoflavonas/biossíntese , Bacillus/classificação , Técnicas de Cultura Celular por Lotes/métodos , Meios de Cultura/química , Meios de Cultura/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Especificidade da Espécie , Água/químicaRESUMO
OBJECTIVE: We knocked out the genes related to lipopolysaccharide in outer membrane of Escherichia coli BL21 (DE3) to study the effects on extracellular secretion of recombinant proteins. METHODS: We generated waaF or msbB knockout mutants [E. coli BL21 (ΔwaaF) or E. coli BL21 (ΔmsbB) ] of E. coli BL21 (DE3) by using lambda-Red recombination system. Then, we transformed recombinant plasmids pET-ffase or pET-pga into E. coli BL21 (AmsbB) , E. coli BL21 (ΔwaaF) and E. coli BL21 (DE3) respectively, to generate the engineering strains E. coli BL21 (ΔmsbB)/ pET-ffase, E. coli BL21 (ΔwaaF)/pET-ffase, E. coli BL21 (DE3)/pET-ffase, E. coli BL21 (ΔmsbB)/pET-pga, E. coli BL21 (ΔwaaF)/pET-pga and E. coli BL21 (DE3)/pET-pga. Finally, we studied the effects of mutants on extracellular secretion of beta-fructofuranosidase (EC 3. 2. 1. 26, beta-FFase) and penicillin G acylase (EC 3. 5. 1. 11) in shaking flask fermentation. RESULTS: After induced expression for 4 hours, up to 19.7% of the beta-FFase activity was found in the culture medium with the msbB deletion mutant, and 50.9% with the waaF deletion mutant, compared to the original 2.6%. Besides, after induced expression for 24 hours, up to 1708 U/L extracellular activity of penicillin G acylase was found in the culture medium with the waaF deletion mutant, which was 4.1 times of the original. CONCLUSION: Knockout mutants (ΔmsbB and ΔwaaF) had significantly higher excretion of beta-FFase and the waaF deletion mutant had higher excretion of penicillin G acylase.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Espaço Extracelular/metabolismo , Proteínas Recombinantes/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Sistemas de Secreção Bacterianos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espaço Extracelular/genética , Técnicas de Inativação de Genes , Transporte Proteico , Proteínas Recombinantes/genética , Transferases/genética , Transferases/metabolismoRESUMO
Nucleoside phosphorylases (NPs) are the key enzymes in the nucleoside metabolism pathway and are widely employed for the synthesis of nucleoside analogs, which are difficult to access via conventional synthetic methods. NPs are generally classified as purine nucleoside phosphorylase (PNP) and pyrimidine or uridine nucleoside phosphorylase (PyNP/UP), based on their substrate preference. Here, based on the evolutionary information on the NP-I family, we adopted an insertions-deletions (InDels) strategy to engineer the substrate promiscuity of nucleoside phosphorylase AmPNPΔS2V102 K, which exhibits both PNP and UP activities from a trimeric PNP (AmPNP) of Aneurinibacillus migulanus. Furthermore, the AmPNPΔS2V102 K exerted phosphorylation activities toward arabinose nucleoside, fluorosyl nucleoside, and dideoxyribose, thereby broadening the unnatural-ribose nucleoside substrate spectrum of AmPNP. Finally, six purine nucleoside analogues were successfully synthesized, using the engineered AmPNPΔS2V102 K instead of the traditional "two-enzymes PNP/UP" approach. These results provide deep insights into the catalytic mechanisms of the PNP and demonstrate the benefits of using the InDels strategy to achieve substrate promiscuity in an enzyme, as well as broadening the substrate spectrum of the enzyme.
RESUMO
Glycosyltransferase is a popular and promising enzyme to produce high-value-added natural products. Rare ginsenoside Rh1 and unnatural ginsenoside 3ß-O-Glc-PPT are promising candidates for drugs. Herein, the microbial glycosyltransferase UGTBL1 was able to catalyze the 20(S)-protopanaxatriol (PPT) 3-O/6-O-glycosylation with poor 6-O-regiospecificity. A structure-guided strategy of mutations involving loop engineering, PSPG motif evolution, and access tunnel engineering was proposed to engineer the enzyme UGTBL1. The variant I62R/M320H/P321Y/N170A from protein engineering achieved a great improvement in 6-O regioselectivity which increased from 10.98 % (WT) to 96.26 % and a booming conversion of 95.57 % for ginsenoside Rh1. A single mutant M320W showed an improved 3-O regioselectivity of 84.83 % and an increased conversion of 98.13 % for the 3ß-O-glc-PPT product. Molecular docking and molecular dynamics (MD) simulations were performed to elucidate the possible molecular basis of the regiospecificity and catalytic activity. The unprecedented high titer of ginsenoside Rh1 (20.48 g/L) and 3ß-O-Glc-PPT (18.04 g/L) was attained with high regioselectivity and yields using fed-batch cascade reactions from UDPG recycle, which was the highest yield reported to date. This work could provide an efficient and cost-effective approach to the valuable ginsenosides.
Assuntos
Ginsenosídeos , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Simulação de Acoplamento Molecular , GlicosilaçãoRESUMO
Site-specific integration (SSI) via recombinase mediated cassette exchange (RMCE) has shown advantages over random integration methods for expression of biotherapeutics. As an extension of our previous work developing SSI host cells, we developed a dual-site SSI system having two independent integration sites at different genomic loci, each containing a unique landing pad (LP). This system was leveraged to generate and compare two RMCE hosts, one (dFRT) compatible with the Flp recombinase, the other (dBxb1) compatible with the Bxb1 recombinase. Our comparison demonstrated that the dBxb1 host was able to generate stable transfectant pools in a shorter time frame, and cells within the dBxb1 transfectant pools were more phenotypically and genotypically stable. We further improved process performance of the dBxb1 host, resulting in desired fed batch performance attributes. Clones derived from this improved host (referred as 41L-11) maintained stable expression profiles over extended generations. While the data represents a significant improvement in the efficiency of our cell line development process, the dual LP architecture also affords a high degree of flexibility for development of complex protein modalities.
Assuntos
Genômica , Recombinases , Cricetinae , Animais , Células CHO , Cricetulus , Recombinases/genética , Células Clonais/metabolismo , Genômica/métodos , TransgenesRESUMO
Embryonic stem (ES) cells are pluripotent and of therapeutic potential in regenerative medicine. Understanding pluripotency at the molecular level should illuminate fundamental properties of stem cells and the process of cellular reprogramming. Through cell fusion the embryonic cell phenotype can be imposed on somatic cells, a process promoted by the homeodomain protein Nanog, which is central to the maintenance of ES cell pluripotency. Nanog is thought to function in concert with other factors such as Oct4 (ref. 8) and Sox2 (ref. 9) to establish ES cell identity. Here we explore the protein network in which Nanog operates in mouse ES cells. Using affinity purification of Nanog under native conditions followed by mass spectrometry, we have identified physically associated proteins. In an iterative fashion we also identified partners of several Nanog-associated proteins (including Oct4), validated the functional relevance of selected newly identified components and constructed a protein interaction network. The network is highly enriched for nuclear factors that are individually critical for maintenance of the ES cell state and co-regulated on differentiation. The network is linked to multiple co-repressor pathways and is composed of numerous proteins whose encoding genes are putative direct transcriptional targets of its members. This tight protein network seems to function as a cellular module dedicated to pluripotency.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Humanos , Imunoprecipitação , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteína Homeobox Nanog , Ligação Proteica , Interferência de RNA , Reprodutibilidade dos TestesRESUMO
Levan-type fructooligosaccharides (FOS) exhibit enhanced health-promoting prebiotic effects on gut microbiota. The wild type (WT) of ß-fructofuranosidase Fru6 could mainly yield 6-ketose. Semirational design and mutagenesis of Fru6 were exploited to promote the transfructosylating capacity for FOS. The promising variants not only improved the formation of 6-kestose but also newly produced tetrasaccharides of 6,6-nystose and 1,6-nystose (a new type of FOS), and combinatorial mutation boosted the production of 6-kestose and tetrasaccharides (39.9 g/L 6,6-nystose and 4.6 g/L 1,6-nystose). Molecular docking and molecular dynamics (MD) simulation confirmed that the mutated positions reshaped the pocket of Fru6 to accommodate bulky 6-kestose in a reactive conformation with better accessibility for tetrasaccharides formation. Using favored conditions, the variant S165A/H357A could yield 6-kestose up to 335 g/L, and tetrasaccharides (6,6-nystose and 1,6-nystose) reached a high level of 121.1 g/L (134.5 times of the mutant S423A). The ß-(2,6)-linked FOS may show the potential application for the prebiotic ingredients.
Assuntos
Oligossacarídeos , beta-Frutofuranosidase , Simulação de Acoplamento Molecular , beta-Frutofuranosidase/genéticaRESUMO
Microneedle (MN) dressings, with the ability of transdermal drug delivery, have played an essential role in the field of wound healing. However, patients may still feel uncomfortable when sensitive unhealing wounds are pieced by strong needles. Here, inspired by the structure of mosquito mouthparts, which possess a fixation part and a liquid-transferring part, we present a novel MN wound dressing with superfine needle tips, personalized pattern design, programmable needle length, and multiple mechanical strengths for intelligent and painless drug delivery. By simply stretching the silicone rubber (Ecoflex) molds before engraving, superfine MNs can be formed in the restored molds. Meanwhile, by utilizing intelligent image recognition, precise treatment for irregular wounds is achieved. Notably, combined with temperature-responsive N-isopropylacrylamide (NIPAM) hydrogel and inverse opal (IO) photonic crystals (PCs), a controllable drug release system has been achieved on MN dressings. Moreover, the performance of the MN dressing in facilitating wound recovery has been demonstrated by full-thickness skin wounds of a mouse model. These results indicate that novel personalized and programmable MN wound dressings are of considerable value in the field of wound management.
Assuntos
Bandagens , Agulhas , Administração Cutânea , Animais , Humanos , Hidrogéis , Camundongos , CicatrizaçãoRESUMO
Polydatin, resveratrol-3-O-ß-glucoside, possesses various biological activities. However, the regioselective glucosylation of resveratrol by UDP-glycosyltransferases (UGTs) constitutes a persistent problem. In this study, semi-rational design and iterative combinatorial mutagenesis were carried out to screen the mutants of UGTBL1 and the high specificity with the glycosylation of the 3-OH group of resveratrol was explored. The triple mutant I62G/M112D/K143G exhibited near-perfect control of polydatin synthesis (regioselectivity â¼ 99%), and the ratio of polydatin to resveratrol-4'-O-ß-glucoside was finally enhanced by 786-fold. Molecular docking revealed that the mutant could form three H-bonds between 3-, 5-, and 4'-OH groups of resveratrol and the residues around the active center, resulting in the oriented-binding of resveratrol. Furthermore, UGTBL1 mutant coupling sucrose synthase AtSuSy can synthesize polydatin at an unprecedented high titer of 10.33 g/L, together with efficient UDPG regeneration (RCmax = 54). This study provides an efficient approach for the regioselective biosynthesis of polydatin.