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1.
ACS Appl Mater Interfaces ; 16(3): 3187-3201, 2024 Jan 24.
Article in English | MEDLINE | ID: mdl-38206677

ABSTRACT

Cancer pain seriously reduces the quality of life of cancer patients. However, most research about cancer focuses solely on inhibiting tumor growth, neglecting the issue of cancer pain. Therefore, the development of therapeutic agents with both tumor suppression and cancer pain relief is crucial to achieve human-centered treatment. Here, the work reports curcumin (CUR) and ropivacaine (Ropi) coincorporating CaCO3/PDA nanoparticles (CaPNMCUR+Ropi) that realized efficient tumor immunotherapy and cancer pain suppression. The therapeutic efficiency and mechanism are revealed in vitro and in vivo. The results indicate that CaPNMCUR+Ropi underwent tumor microenvironment-responsive degradation and realized rapid release of calcium ions, Ropi, and CUR. The excessive intracellular calcium triggered the apoptosis of tumor cells, and the transient pain caused by the tumor injection was relieved by Ropi. Simultaneously, CUR reduced the levels of immunosuppressive factor (TGF-ß) and inflammatory factor (IL-6, IL-1ß, and TNF-α) in the tumor microenvironment, thereby continuously augmenting the immune response and alleviating inflammatory pain of cancer animals. Meanwhile, the decrease of TGF-ß leads to the reduction of transient receptor potential vanilloid 1 (TRPV1) expression, thereby alleviating hyperalgesia and achieving long-lasting analgesic effects. The design of the nanosystem provides a novel idea for human-centered tumor treatment in the future.


Subject(s)
Cancer Pain , Curcumin , Indoles , Neoplasms , Polymers , Animals , Humans , Transforming Growth Factor beta , Calcium Carbonate , Cancer Pain/drug therapy , Calcium , Quality of Life , Ropivacaine/therapeutic use , Neoplasms/drug therapy , Curcumin/therapeutic use , Immunotherapy , Tumor Microenvironment
2.
ACS Appl Bio Mater ; 6(11): 4998-5008, 2023 11 20.
Article in English | MEDLINE | ID: mdl-37880964

ABSTRACT

Sonodynamic therapy (SDT) and chemotherapy have received great attention as effective methods for tumor treatment. However, the inherent hypoxia of the tumor greatly hinders its therapeutic efficacy. In this work, a tumor microenvironment-responsive biodegradable nanoplatform SiO2-MnO2-PEG-Ce6&DOX (designated as SMPC&D) is fabricated by encapsulating manganese oxide (MnO2) into silica nanoparticles and anchoring poly(ethylene glycol) (PEG) onto the surface for tumor hypoxia relief and delivery, then loaded with sonosensitizer Chlorin e6 (Ce6) and chemotherapeutic drug doxorubicin (DOX) for hypoxic tumor treatment. We evaluated the physicochemical properties of SMPC&D nanoparticles and the tumor therapeutic effects of chemotherapy and SDT under ultrasound stimulation in vitro and in vivo. After endocytosis by tumor cells, highly expressed glutathione (GSH) triggers biodegradation of the nanoplatform and MnO2 catalyzes hydrogen peroxide (H2O2) to generate oxygen (O2), thereby alleviating tumor hypoxia. Depleting GSH and self-supplying O2 effectively improve the SDT efficiency both in vitro and in vivo. Ultrasonic stimulation promoted the release and cellular uptake of chemotherapy drugs. In addition, the relieved hypoxia reduced the efflux of chemotherapy drugs by downregulating the expression of the P-gp protein, which jointly improved the effect of chemotherapy. This study demonstrates that the degradable SMPC&D as a therapeutic agent can achieve efficient chemotherapy and SDT synergistic therapy for hypoxic tumors.


Subject(s)
Manganese Compounds , Oxygen , Humans , Hydrogen Peroxide , Silicon Dioxide , Oxides , Hypoxia , Doxorubicin/therapeutic use , Glutathione
3.
Discov Nano ; 18(1): 69, 2023 Apr 21.
Article in English | MEDLINE | ID: mdl-37382740

ABSTRACT

The relatively low output performance of triboelectric nanogenerator (TENG), which faces a challenge in performance improvement, limits its practical applications. Here, a high-performance TENG consisting of a silicon carbide@silicon dioxide nanowhiskers/polydimethylsiloxane (SiC@SiO2/PDMS) nanocomposite film and a superhydrophobic aluminum (Al) plate as triboelectric layers is demonstrated. The 7 wt% SiC@SiO2/PDMS TENG presents a peak voltage of 200 V and a peak current of 30 µA, which are ~ 300 and ~ 500% over that of the PDMS TENG, owing to an increase in dielectric constant and a decrease in dielectric loss of the PDMS film because of electric insulated SiC@SiO2 nanowhiskers. Furthermore, a 10 µF capacitor can be charged up to 3 V within ~ 87 s, which can be continuously operated on the electronic watch for 14 s. The work provides an effective strategy for improving output performance of TENG by adding core-shell nanowhiskers to modulate the dielectric properties of organic materials.

4.
Biomed Res Int ; 2022: 3768880, 2022.
Article in English | MEDLINE | ID: mdl-36033571

ABSTRACT

Dihuang Yinzi, as a classical Chinese medicine prescription, plays an important role for the treatment of ischemic stroke. Gut microbiota play a functional role for the expression of proinflammatory cytokines and anti-inflammatory cytokines, which further affect central nervous system and change brain function. Our research confirmed that Dihuang Yinzi can exert brain protection by inhibiting inflammatory reaction. Dihuang Yinzi can significantly decrease the contents of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-17 (IL-17) in brain, serum, and colon tissues and increase the contents of transforming growth factor-ß (TGF-ß) and interleukin-10 (IL-10) in cerebral ischemia-reperfusion model rats. The results of 16s rRNA high-throughput sequencing showed that Dihuang Yinzi had a significant effect on microbiome in rats. The firmicutes, bacteroidetes, and proteobacteria were dominant in Dihuang Yinzi group. The content of firmicutes increased with the increase of dosage of Dihuang Yinzi. Especially, the content of actinomycetes in the high-dose group was higher than other groups. At the genus level, the number of bacteroides in the antibiotic groups was significantly higher than that in the other treatment groups. The results suggest that Dihuang Yinzi may play important roles in treatment of ischemic stroke by regulating the gut microbiota and the inflammatory reaction in the colon tissues, serum, and brain of the model rats, to verify the scientific nature of this prescription in relieving brain inflammatory reaction and brain injury by this way and to reveal the brain-gut related mechanism of Dihuang Yinzi in treating ischemic stroke.


Subject(s)
Brain Ischemia , Gastrointestinal Microbiome , Ischemic Stroke , Animals , Cerebral Infarction , Cytokines , Inflammation , RNA, Ribosomal, 16S , Rats , Rats, Sprague-Dawley , Reperfusion
5.
PLoS One ; 17(6): e0269153, 2022.
Article in English | MEDLINE | ID: mdl-35687548

ABSTRACT

Nontypeable Haemophilus influenzae (NTHi) is a common opportunistic pathogen that colonizes the nasopharynx. NTHi infections result in enormous global morbidity in two clinical settings: otitis media in children and acute exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Thus, there is an urgent need to design and develop effective vaccines to prevent morbidity and reduce antibiotic use. The NTHi outer membrane protein P6, a potential vaccine candidate, is highly conserved and effectively induces protective immunity. Here, to enhance mucosal immune responses, P6-loaded mannose-modified chitosan (MC) microspheres (P6-MCMs) were developed for mucosal delivery. MC (18.75%) was synthesized by the reductive amination reaction method using sodium cyanoborohydride (NaBH3CN), and P6-MCMs with an average size of 590.4±16.2 nm were successfully prepared via the tripolyphosphate (TPP) ionotropic gelation process. After intranasal immunization with P6-MCMs, evaluation of humoral immune responses indicated that P6-MCMs enhance both systemic and mucosal immune responses. Evaluation of cellular immune responses indicated that P6-MCMs enhance cellular immunity and trigger a mixed Th1/Th2-type immune response. Importantly, P6-MCMs also trigger a Th17-type immune response. They are effective in promoting lymphocyte proliferation and differentiation without toxicity in vitro. The results also demonstrate that P6-MCMs can effectively induce MHC class I- and II-restricted cross-presentation, promoting CD4+-mediated Th immune responses and CD8+-mediated cytotoxic T lymphocyte (CTL) immune responses. Evaluation of protective immunity indicated that immunization with P6-MCMs can reduce inflammation in the nasal mucosa and the lung and prevent NTHi infection. In conclusion, MCMs are a promising adjuvant-delivery system for vaccines against NTHi.


Subject(s)
Chitosan , Haemophilus Infections , Haemophilus Vaccines , Animals , Antibodies, Bacterial , Bacterial Outer Membrane Proteins , Haemophilus Infections/prevention & control , Haemophilus influenzae , Immunity, Mucosal , Mannose , Mice , Mice, Inbred BALB C , Microspheres , Nasal Mucosa
6.
J Biomed Nanotechnol ; 18(3): 763-777, 2022 Mar 01.
Article in English | MEDLINE | ID: mdl-35715902

ABSTRACT

Although the development of safe and efficient cancer therapeutic agents is essential, this process remains challenging. In this study, a mitochondria-targeted degradable nanoplatform (PDA-MnO2-IR780) for synergistic photothermal, photodynamic, and sonodynamic tumor treatment was investigated. PDA-MnO2-IR780 exhibits superior photothermal properties owing to the integration of polydopamine, MnO2, and IR780. IR780, a photosensitizer and sonosensitizer, was used for photodynamic therapy and sonodynamic therapy. When PDA-MnO2-IR780 was delivered to the tumor site, MnO2 was decomposed by hydrogen peroxide, producing Mn2+ and oxygen. Meanwhile, alleviating tumor hypoxia promoted the production of reactive oxygen species during photodynamic therapy and sonodynamic therapy. Moreover, large amounts of reactive oxygen species could reduce the expression of heat shock proteins and increase the heat sensitivity of tumor cells, thereby improving the photothermal treatment effect. In turn, hyperthermia caused by photothermal therapy accelerated the production of reactive oxygen species in photodynamic therapy. IR780 selectively accumulation in mitochondria also promoted tumor apoptosis. In this system, the mutual promotion of photothermal therapy and photodynamic therapy/sonodynamic therapy had an enhanced therapeutic effect. Moreover, the responsive degradable characteristic of PDA-MnO2-IR780 in the tumor microenvironment ensured excellent biological safety. These results reveal a great potential of PDA-MnO2-IR780 for safe and highly-efficiency synergistic therapy for cancer.


Subject(s)
Nanocomposites , Nanoparticles , Photochemotherapy , Cell Line, Tumor , Lasers , Manganese Compounds/metabolism , Mitochondria/metabolism , Nanocomposites/therapeutic use , Nanoparticles/therapeutic use , Oxides , Photochemotherapy/methods , Reactive Oxygen Species
7.
J Biomed Nanotechnol ; 18(2): 352-368, 2022 Feb 01.
Article in English | MEDLINE | ID: mdl-35484752

ABSTRACT

The construction of high-efficiency tumor theranostic platform will be of great interest in the treatment of cancer patients; however, significant challenges are associated with developing such a platform. In this study, we developed high-efficiency nanotheranostic agent based on ferroferric oxide, manganese dioxide, hyaluronic acid and doxorubicin (FMDH-D NPs) for dual targeting and imaging guided synergetic photothermal-enhanced chemodynamic/chemotherapy for cancer, which improved the specific uptake of drugs at tumor site by the dual action of CD44 ligand hyaluronic acid and magnetic nanoparticles guided by magnetic force. Under the acidic microenvironment of cancer cells, FMDH-D could be decomposed into Mn2+ and Fe2+ to generate •OH radicals by triggering a Fenton-like reaction and responsively releasing doxorubicin to kill cancer cells. Meanwhile, alleviating tumor hypoxia improved the efficacy of chemotherapy in tumors. The photothermal properties of FMDH generated high temperatures, which further accelerated the generation of reactive oxygen species, and enhanced effects of chemodynamic therapy. Furthermore, FMDH-D NPs proved to be excellent T1/T2-weighted magnetic resonance imaging contrast agents for monitoring the tumor location. These results confirmed the considerable potential of FMDH-D NPs in a highly efficient synergistic therapy platform for cancer treatment.


Subject(s)
Manganese Compounds , Neoplasms , Doxorubicin/pharmacology , Humans , Hyaluronic Acid , Magnetic Resonance Imaging , Manganese Compounds/pharmacology , Neoplasms/diagnostic imaging , Neoplasms/drug therapy , Oxides , Tumor Microenvironment
8.
Biomater Sci ; 9(19): 6528-6541, 2021 Sep 28.
Article in English | MEDLINE | ID: mdl-34582541

ABSTRACT

Lung cancer is the second most common tumor and has the highest mortality rate. Both novel therapeutic targets and approaches are needed to improve the overall survival of patients with lung cancer. MicroRNA-320a-3p belongs to the miR-320a family and has been reported as a tumor suppressor in multiple cancers. However, its definitive role and precise mechanism in the progression of lung cancer remain unclear. In this study, we developed a new type of gold nanorod modified with polyethyleneimine that targets cancer-specific nanoparticles by RGD peptide, which could condense miRNA to self-assemble supramolecular nanoparticles. The designed nanoparticles can achieve integrin αvß3-targeted cancer therapy, realize photosensitive therapy by laser irradiation and attain gene-targeted therapy by miRNAs. These nanoparticles could deliver miR-320a into lung cancer cells specifically and efficiently. Moreover, we demonstrated that Au-RGD-miR-320a nanoparticles combined with laser irradiation dramatically inhibited the proliferation and metastasis, and enhanced the apoptosis of lung cancer, both in vitro and in vivo. In terms of the mechanism, miR-320a inhibits Sp1 expression by directly binding to the 3'UTR of Sp1, and it eventually enhanced the expression of PTEN and inhibited the expression of matrix metallopeptidase 9 (MMP9). These findings provide a new and promising anticancer strategy via the use of Au-RGD-miR-320a nanoparticles, and identify miR-320a/Sp1 as a potential target for future systemic therapy against lung cancer.


Subject(s)
Lung Neoplasms , Metal Nanoparticles , MicroRNAs , Cell Line, Tumor , Cell Proliferation , Gold , Humans , Lung Neoplasms/genetics , Lung Neoplasms/therapy , MicroRNAs/genetics , Photothermal Therapy , Sp1 Transcription Factor/genetics
9.
Biomater Sci ; 9(10): 3662-3674, 2021 May 18.
Article in English | MEDLINE | ID: mdl-33617619

ABSTRACT

Photodynamic therapy (PDT) has been regarded as a promising strategy for tumor therapy. However, heterogeneous tumor microenvironments severely limit the efficacy of photodynamic therapy. In this work, a multifunctional theranostic platform (MnO2-SiO2-APTES&Ce6 (MSA&C)) was fabricated based on MnO2 nanoflowers, which afforded MRI-guided synergistic therapy incorporating PDT and second near-infrared window (NIR-II) photothermal therapy (PTT). Herein, MnO2 nanoflowers are first proposed as a NIR-II photothermal agent. In the MSA&C system, MnO2 nanoflowers were employed for effective photosensitizer loading, relieving tumor hypoxia, and NIR-II PTT and tumor-specific imaging. The large amount of photosensitizer, reduced tumor hypoxia, and hyperthermia all contributed to the improvement of PDT. The quantity of reactive oxygen species (ROS) generated during PDT in turn down-regulated the expression of heat shock proteins (HSP 70), thereby improving photothermal performance. Positively charged (3-aminopropyl)triethoxysilane (APTES) was used to promote cellular uptake, further improving treatment efficiency. In this system, the MSA&C nanoflowers can not only alleviate tumor hypoxia, but they also obviously induce cell apoptosis under laser irradiation through a ROS- and hyperthermia-mediated mechanism, thereby leading to remarkable tumor growth inhibition. Furthermore, the Mn2+ ions generated during treatment can be explored for MR imaging, and this could be used to finally realize MRI-guided enhanced PDT/PTT.


Subject(s)
Hyperthermia, Induced , Photochemotherapy , Magnetic Resonance Imaging , Manganese Compounds , Oxides , Photosensitizing Agents/therapeutic use , Silicon Dioxide
10.
Sci Total Environ ; 715: 136852, 2020 May 01.
Article in English | MEDLINE | ID: mdl-32041041

ABSTRACT

As one of the important nitrogen (N) and phosphorus (P) pollution sources of waters, the paddy water N and P runoff losses are still poorly understood in the double rice cropping system under the interaction of chemical fertilizer and pesticide. In the subtropical hilly region of China, we conducted a 1.5-year continuous and high-frequency monitoring of paddy water N and P concentrations, runoff N and P losses, and grain yield in a double rice-cropping system with different chemical fertilizer and pesticide application rates. The results showed that the high-risk periods for N loss were in the first 5 days after the base fertilizer (BF) application and the first 10 days after the topdressing fertilizer application in both early and late rice seasons, while the high-risk periods for P loss were in the first 5 days after BF application in the early rice season and the first 15 days after BF application in the late rice season. The N and P runoff losses in the early rice season were greater than those in the late rice season, due to that the N and P fertilizers use efficiencies were lower, and thus paddy water N and P concentrations were higher in the early rice season. The paddy N and P concentrations and N and P runoff losses increased significantly with increased fertilizer application rates, while the pesticide application rate did not significantly affect N and P losses. Therefore, special effects (e.g., avoiding high irrigation, fertilizer deep application) should be taken during the high-risk periods of N and P losses to reduce the N and P runoff losses in the double rice cropping system, especially in the early rice season. There are also potentials to reduce fertilizer and pesticide input without reducing rice grain yield for the double rice cropping system in the subtropical hilly region of China.


Subject(s)
Oryza , Agriculture , China , Fertilizers , Nitrogen , Pesticides , Phosphorus
11.
J Biosci Bioeng ; 119(6): 648-51, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25488497

ABSTRACT

A novel 2-aminoacetophenone reductase was purified to homogeneity from Arthrobacter sulfureus BW1010. The enzyme is a monomer with a molecular weight of approximately 60 kDa. Using NADPH as coenzyme, it catalyzes the reduction of ketones, especially amine phenyl ketones, and stereospecifically reduces 2-aminoacetophenone to (S)-2-amino-1-phenylethanol (e.e > 99.8%) with the optimal pH at 7.5.


Subject(s)
Acetophenones/metabolism , Arthrobacter/enzymology , NADP/metabolism , Oxidoreductases/isolation & purification , Oxidoreductases/metabolism , Benzyl Alcohols/metabolism , Hydrogen-Ion Concentration , Ketones/chemistry , Ketones/metabolism , Molecular Weight , Oxidation-Reduction , Oxidoreductases/chemistry , Substrate Specificity
12.
Protein Expr Purif ; 53(2): 239-46, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17317214

ABSTRACT

A novel epoxide hydrolase from Aspergillus niger SQ-6 has now been cloned by inverse PCR. Its gene shows eight exons including a non-coding exon at its 5'-terminal (GenBank Accession No. AY966486). Phylogenetic analysis using deduced amino acid sequence (395 aa) confirms it as an epoxide hydrolase and shares 58.3% identity with that of A. niger LCP521 (GenBank Accession No. AF238460). The predicted catalytic triad is composed of Asp(191), His(369) and Glu(343). Active recombinant epoxide hydrolase has been successfully expressed in Escherichia coli as protein fusions with a poly-His tail. Scale-up fermentation can yield 2.5g/L of recombinant protein. The electrophoretic pure recombinant protein, which shows similar characterization as natural enzyme purified from A. niger SQ-6, can be easily purified by Ni(2+)-chelated affinity and gel-filtration chromatography. Optimal pH and temperature for purified enzyme are pH 7.5 and 37 degrees C, respectively. The K(m), k(cat) and maximal velocity (V(max)) for p-nitrostyrene oxide are determined to be 1.02mM, 172s(-1) and 231micromol min(-1)mg(-1), respectively. The enzyme can be inhibited by oxidant (H(2)O(2)), solvent (Tetrahydrofuran) and several metal ions including Hg(2+), Fe(2+) and Co(2+). This (R)-stereospecific epoxide hydrolase exhibits high enantioselectivity (enantiomeric excess value, 99%) for the less hindered carbon atom of epoxide. It may be an industrial biocatalyst for the preparation of enantiopure epoxides or vicinal diols.


Subject(s)
Aspergillus niger/enzymology , Aspergillus niger/genetics , Epoxide Hydrolases/genetics , Epoxide Hydrolases/isolation & purification , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Fungal/genetics , Epoxide Hydrolases/metabolism , Escherichia coli/genetics , Exons , Gene Expression , Genes, Fungal , Hydrogen-Ion Concentration , Kinetics , Metals/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Phylogeny , Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Solvents , Substrate Specificity , Temperature
13.
J Chromatogr B Analyt Technol Biomed Life Sci ; 850(1-2): 560-3, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17257909

ABSTRACT

A capillary electrophoresis (CE) method for the enantioseparation of phenylglycidates has been developed. Successful enantioseparation was achieved using sulfated beta-cyclodextrin as chiral selector in a phosphate buffer. The effects of varying pH, sulfated beta-cyclodextrin concentration and electrophoresis voltage were systematically investigated and the optimized separation conditions were thus obtained. When the migration time was set at the threshold value, it was found that the best enantioseparation was obtained at 10 kV with 3% (w/v) sulfated beta-cyclodextrin at pH 6.5. A range of substituted phenylglycidates were successfully separated using the method and the results shown to be superior to those obtained using gas chromatography (GC).


Subject(s)
Electrophoresis, Capillary/methods , Glycine/isolation & purification , beta-Cyclodextrins/chemistry , Glycine/analogs & derivatives , Spectrophotometry, Ultraviolet , Stereoisomerism
14.
Wei Sheng Wu Xue Bao ; 46(4): 565-70, 2006 Aug.
Article in Chinese | MEDLINE | ID: mdl-17037056

ABSTRACT

A N-carbamoyl-D-amino acid amidohydrolase gene (hyuC) from Sinorhizobium morelens S-5 was cloned by LA PCR, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of D-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the recombinant enzyme was purified 16.1-fold to homogeneity with a yield of 21.2% by heat treatment and three steps of column chromatography. The results of gel filtration on Superdex 200 HR and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 38-kDa subunits. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and it was strictly D-specific. The enzyme showed broad substrate specificity, and exhibited high activity in the hydrolysis of N-carbamoyl-D-p-hydroxyphenylglycine as substrate. The enzyme did not hydrolyze N-carbamoyl-beta-alanine. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. Enzyme activity was slightly improved by Ca2+ and Fe2+, and nearly not affected by metal chelators and sulfhydryl reagents. The enzyme showed high thermal and oxidative stability. These results show that the enzyme has great potential for industrial application.


Subject(s)
Amidohydrolases/genetics , Amidohydrolases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Sinorhizobium/enzymology , Sinorhizobium/genetics , Cloning, Molecular , Enzyme Stability , Hydrogen-Ion Concentration , Molecular Weight , Temperature
15.
Biochimie ; 88(3-4): 237-44, 2006.
Article in English | MEDLINE | ID: mdl-16546310

ABSTRACT

A d-carbamoylase from Sinorhizobium morelens S-5 was purified and characterized. The enzyme was purified 189-fold to homogeneity with a yield of 19.1% by aqueous two-phase extraction and two steps of column chromatography. The enzyme is a homotetramer with a native molecular mass of 150 kDa and a subunit relative molecular mass of 38 kDa. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. The enzyme showed high thermal and oxidative stability. It was found to have a K(m) of 3.76 mM and a V(max) of 383 U/mg for N-carbamoyl-d-p-hydroxyphenylglycine. The hyuC gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of d-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity from the recombinant. Our results show that the enzyme has great potential for industrial application.


Subject(s)
Amidohydrolases/genetics , Amidohydrolases/isolation & purification , Escherichia coli/genetics , Sinorhizobium/enzymology , Amidohydrolases/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Enzyme Stability , Escherichia coli/metabolism , Gene Expression , Hydrolysis , Kinetics , Molecular Sequence Data , Molecular Weight , Protein Subunits/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sinorhizobium/metabolism , Substrate Specificity , Temperature
16.
J Ind Microbiol Biotechnol ; 33(4): 274-82, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16320035

ABSTRACT

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35 degrees C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30 degrees C for 44 h, glycerol (20 g L(-1)) and corn steep liquor (CSL) (30 g L(-1)) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L(-1) h(-1)) and continuous feeding of CSL (1 g L(-1) h(-1)) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5+/-0.8 g L(-1); maximum EH production was 351.2+/-13.1 U L(-1) with a specific activity of 14.3+/-0.5 U g(-1). Partially purified EH exhibited a temperature optimum at 37 degrees C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.


Subject(s)
Aspergillus niger/metabolism , Epoxide Hydrolases/metabolism , Epoxy Compounds/metabolism , Fermentation , Aspergillus niger/growth & development , Aspergillus niger/isolation & purification , Electrophoresis, Capillary , Epoxide Hydrolases/isolation & purification , Epoxy Compounds/chemistry , Epoxy Compounds/isolation & purification , Ethylene Glycols/chemistry , Ethylene Glycols/isolation & purification , Ethylene Glycols/metabolism , Hydrogen-Ion Concentration , Stereoisomerism , Temperature
17.
Biotechnol Lett ; 26(16): 1255-9, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15483382

ABSTRACT

A new isolate of Arthrobacter sulfureus , when incubated at 50 g resting cells (dry cell wt) l(-1) with 50 g glucose l(-1) and 1 g 2-aminoacetophenone l(-1) in 50 mm potassium buffer (pH 7, 4 ml) at 30 degrees C, produced ( S )-2-amino-1-phenylethanol (e.e. >99%) with 75% yield in 6 h.


Subject(s)
Acetophenones/metabolism , Arthrobacter/growth & development , Arthrobacter/metabolism , Cell Culture Techniques/methods , Electrophoresis, Capillary/methods , Phenylethyl Alcohol/metabolism , Arthrobacter/classification , Biotransformation , Cell Proliferation , Glucose/metabolism , Phosphates/metabolism , Potassium Compounds/metabolism , Soil Microbiology
18.
Biotechnol Lett ; 25(13): 1041-7, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12889812

ABSTRACT

Substitution of Ser113 for Gly113 in the cap domain of hydroxynitrile lyase from Manihot esculenta (MeHNL) was performed by site-directed mutagenesis to improve its self-generated folding and stability under denaturation conditions. The yield of the recombinant mutant HNL1 (mut-HNL1), which had higher specific activity than the wild type HNL0 (wt-HNL0), was increased by 2 to 3-fold. Thermostability of MeHNL was also enhanced, probably due to an increase in content of the beta-strand secondary structure according to CD analysis. Our data in this report suggest that Ser113 significantly contributes to the in vivo folding and stability of MeHNL and demonstrates an economic advantage of mut-HNL1 over the wt-HNL0.


Subject(s)
Aldehyde-Lyases/chemistry , Aldehyde-Lyases/metabolism , Manihot/enzymology , Manihot/genetics , Mutagenesis, Site-Directed , Protein Engineering/methods , Acetonitriles/metabolism , Aldehyde-Lyases/genetics , Circular Dichroism , Enzyme Activation , Enzyme Stability , Escherichia coli/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression Regulation, Enzymologic , Hydrogen-Ion Concentration , Manihot/chemistry , Molecular Weight , Mutation , Protein Conformation , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Temperature
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