- Single-Cell-Based Screening and Engineering of d -Amino Acid Amidohydrolases Using Artificial Amidophenol Substrates and Microbial Biosensors
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Enantiomerically pure d-amino acids are important intermediates as chiral building blocks for peptidomimetics and semisynthetic antibiotics. Here, a transcriptional factor-based screening strategy was used for the rapid screening of d-stereospecific amino acid amidase via an enzyme-specific amidophenol substrate. We used a d-threonine amidophenyl derivative to produce 2-aminophenol that serves as a putative enzyme indicator in the presence of d-threonine amidases. Comparative analyses of known bacterial species indicated that several Bacillus strains produce amidase and form putative indicators in culture media. The estimated amidase was cloned and subjected to rapid directed evolution through biosensor cells. Consequently, we characterized the F119A mutation that significantly improved the catalytic activity toward d-alanine, d-threonine, and d-glutamate. Its beneficial effects were confirmed by higher conversions and recurrent applications of the mutant enzyme, compared to the wild-type. This study showed that rapid directed evolution with biosensors coupled to designed substrates is useful to develop biocatalytic processes.
- An, Jung-Ung,Kim, Haseong,Kwon, Kil Koang,Lee, Dae-Hee,Lee, Hyewon,Lee, Jin-Young,Lee, Seung-Goo,Park, Sung Hyun,Rha, Eugene,Yeom, Soo-Jin
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p. 1203 - 1211
(2022/01/27)
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- A novel strategy for efficient chemoenzymatic synthesis of D-glutamine using recombinant Escherichia coli cells
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D-glutamine is a D type stereoisomer of glutamine which is involved in many metabolic processes. Seeking lower-cost and industrially scalable approaches for the synthesis of D-glutamine is very valuable both in academic career and potential applications. Herein, we developed a novel efficient chemoenzymatic strategy for producing D-glutamine. Initially, DL-glutamine was chemically prepared with cheap and accessible DL-glutamic acid as raw material. Subsequently, the L-glutamine among the racemic mixture was selectively hydrolyzed to L-glutamic acid by Escherichia coli whole-cell system which expressed L-aminopeptidase D-Ala-esterase/amidase (DmpA) from Ochrobactrum anthropi. The left D-glutamine was obtained by isoelectric point precipitation with 70% of the theoretical yield. Furthermore, we optimized enzymatic resolution conditions to determine the optimum parameters as pH 8, 30 °C, 0.1% (v/v) Triton X-100, and 1 mM Mn2+. These results suggested that our strategy might be potentially usable for the synthesis of D-glutamine in industrial productions.
- Du, Qinglin,Zhang, Xiangyang,Pan, Xinru,Zhang, Hongjuan,Yang, Yu-Shun,Liu, Junzhong,Jiao, Qingcai
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- Light-Driven Kinetic Resolution of α-Functionalized Carboxylic Acids Enabled by an Engineered Fatty Acid Photodecarboxylase
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Chiral α-functionalized carboxylic acids are valuable precursors for a variety of medicines and natural products. Herein, we described an engineered fatty acid photodecarboxylase (CvFAP)-catalyzed kinetic resolution of α-amino acids and α-hydroxy acids, which provides the unreacted R-configured substrates with high yields and excellent stereoselectivity (ee up to 99 %). This efficient light-driven process requires neither NADPH recycling nor prior preparation of esters, which were required in previous biocatalytic approaches. The structure-guided engineering strategy is based on the scanning of large amino acids at hotspots to narrow the substrate binding tunnel. To the best of our knowledge, this is the first example of asymmetric catalysis by an engineered CvFAP.
- Xu, Jian,Hu, Yujing,Fan, Jiajie,Arkin, Mamatjan,Li, Danyang,Peng, Yongzhen,Xu, Weihua,Lin, Xianfu,Wu, Qi
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supporting information
p. 8474 - 8478
(2019/05/24)
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- Chromatographic Resolution of α-Amino Acids by (R)-(3,3'-Halogen Substituted-1,1'-binaphthyl)-20-crown-6 Stationary Phase in HPLC
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Three new chiral stationary phases (CSPs) for high-performance liquid chromatography were prepared from R-(3,3'-halogen substituted-1,1'-binaphthyl)-20-crown-6 (halogen = Cl, Br and I). The experimental results showed that R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 (CSP-1) possesses more prominent enantioselectivity than the two other halogen-substituted crown ether derivatives. All twenty-one α-amino acids have different degrees of separation on R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6-based CSP-1 at room temperature. The enantioselectivity of CSP-1 is also better than those of some commercial R-(1,1'-binaphthyl)-20-crown-6 derivatives. Both the separation factors (α) and the resolution (Rs) are better than those of commercial crown ether-based CSPs [CROWNPAK CR(+) from Daicel] under the same conditions for asparagine, threonine, proline, arginine, serine, histidine and valine, which cannot be separated by commercial CR(+). This study proves the commercial usefulness of the R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 chiral stationary phase.
- Wu, Peng,Wu, Yuping,Zhang, Junhui,Lu, Zhenyu,Zhang, Mei,Chen, Xuexian,Yuan, Liming
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supporting information
p. 1037 - 1042
(2017/07/25)
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- SEPARATING AGENT AND MANUFACTURING METHOD THEREOF
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An embodiment of the present invention is a separating agent wherein a group represented by a chemical formula of: or a group represented by a chemical formula of: is introduced on a surface thereof.
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Paragraph 0067; 0068; 0069; 0070; 0071; 0072; 0089; 0090
(2015/01/07)
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- SEPARATING AGENT FOR CHROMATOGRAPHY
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A separating agent for chromatography is provided that is useful for the separation of specific compounds, e.g., for the optical resolution of amino acids. This separating agent for chromatography provides a higher productivity and contains a crown ether-like cyclic structure and optically active binaphthyl. This separating agent for chromatography containing a crown ether-like cyclic structure and optically active binaphthyl is provided by introducing a substitution group for binding to carrier into a specific commercially available 1,1′-binaphthyl derivative that has substituents at the 2, 2′, 3, and 3′ positions, then introducing a crown ether-like cyclic structure, and subsequently chemically bonding the binaphthyl derivative to the carrier through the substitution group for binding to carrier.
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Paragraph 0074; 0075
(2013/08/15)
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- Imaging mass spectrometry and genome mining reveal highly antifungal virulence factor of mushroom soft rot pathogen
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Soft rot diseases caused by a variety of bacteria account for severe losses in agriculture, devastating fruits, vegetables, and cultivated mushrooms. After bacterial infection, often owing to direct contact or transmission by insects, virulence factors and lytic enzymes cause degradation of plant and mushroom tissues, thereby turning crop into mush. In many cases, the chemical mediators of soft rot diseases have remained elusive, as in the long-known mushroom pathogen Janthinobacterium agaricidamnosum. This motile Gram-negative bacterium has been found to be the causative agent of soft rot disease of the cultured button mushroom, Agaricus bisporus. Typical symptoms of the infection are lesions turning into sticky blotches on the cap surface and a complete dissolution of the mushroom within only a few days (Figure 1A, B). We reasoned that knowledge on the causative agent of the soft rot would have a double benefit. Foremost, it could aid in understanding the pathobiology of the mushroom pathogen, which may be a starting point for protective measures. Second, there is an increasing need for novel antifungals, since the incidents of severe and even lethal fungal infections and resistance towards antifungals are on the rise. We hypothesized that mushroom soft rot bacteria could excrete antifungal agents as virulence factors, which might also be active against human pathogens. Herein we report the discovery and full characterization of a highly antifungal virulence factor from the soft rot pathogen Janthinobacterium agaricidamnosum guided by imaging mass spectrometry and genome mining.
- Graupner, Katharina,Scherlach, Kirstin,Bretschneider, Tom,Lackner, Gerald,Roth, Martin,Gross, Harald,Hertweck, Christian
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supporting information
p. 13173 - 13177
(2013/03/14)
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- Preparation of D-amino acids by enzymatic kinetic resolution using a mutant of penicillin-G acylase from E. coli
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We have demonstrated for the first time that d-glutamine (d-Gln) and d-glutamic acid (d-Glu) can be efficiently obtained in high ee (97% and 90%, respectively) by enzymatic kinetic resolution of d,l-Gln and d,l-Glu. This was achieved by enantioselective conversion of the l-enantiomers to their N-phenylacetyl derivatives in aqueous solution, using a mutant of penicillin-G acylase (PGA) from E. coli and phenylacetic acid methylester as the acyl donor. Kinetic modeling studies suggest that the high ee values obtained are both due to a strong enantiopreference for the l-amino acid in the deacylation step of the covalent enzyme intermediate, as well as to completeness of conversion that is transiently obtained as a result of the distinct preference of the mutant PGA for phenylacetic acid methylester over the N-phenylacetyl-l-amino acid product. For the other amino acids tested (Asn, Asp, and Ser), the highest ee values that were obtained for the remaining d-enantiomer are moderate (50-80%) because of lower enantioselectivity in the enzyme deacylation step and due to less complete conversion of the l-amino acid caused by competition for the active site between the acyl donor and the N-phenylacetyl-l-amino acid that is produced. The results demonstrate that the mutated PGA has great potential for the production of optically active D-amino acids by kinetic resolution.
- Carboni, Chiara,Kierkels, Hans G. T.,Gardossi, Lucia,Tamiola, Kamil,Janssen, Dick B.,Quaedflieg, Peter J. L. M.
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p. 245 - 251
(2007/10/03)
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- Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization-mass spectrometry
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Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-μm-i.d., 150-μm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.
- Schultz, Casey L.,Moini, Mehdi
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p. 1508 - 1513
(2007/10/03)
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- Compounds for and methods of inhibiting matrix metalloproteinases
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The present invention relates to compounds of Formula I that inhibit matrix metalloproteinases and to a method of inhibiting matrix metalloproteinases using the compounds More particularly, the present invention relates to a method of treating diseases in which matrix metalloproteinases are involved such as multiple sclerosis, atherosclerotic plaque rupture, restenosis, aortic aneurysm, heart failure, periodontal disease, corneal ulceration, burns, decubital ulcers, chronic ulcers or wounds, cancer metastasis, tumor angiogenesis, osteoporosis, rheumatoid or osteoarthritis, renal disease, left ventricular dilatation, or other autoimmune or inflammatory diseases dependent upon tissue invasion by leukocytes.
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- Method of making a diastereomeric mixture containing two diastereomeric N-acyl-amino acid esters
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In the process of hydrocarboxylating an α-enamide with CO and an organic hydroxyl compound to produce a N-acyl-α-amino acid ester, the improvement comprising using as the organic hydroxyl compound reactant, an organic hydroxyl compound which has a chiral center that is essentially all L or D, thereby producing a reaction mixture having essentially no enantiomeric pairs and containing diastereomeric N-acyl-α-amino acid esters having two chiral centers.
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- Method of making a diastereomeric mixture containing two diastereomeric N-acyl-amino acid esters
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In the process of hydrocarboxylating an α-enamide with CO and an organic hydroxyl compound to produce a N-acyl-α-amino acid ester, the improvement comprising using as the organic hydroxyl compound reactant, an organic hydroxyl compound which has a chiral center that is essentially all L or D, thereby producing a reaction mixture having essentially no enantiomeric pairs and containing diastereomeric N-acyl-α-amino acid esters having two chiral centers.
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- Optimal Conditions for the Enzymatic Production of D-Amino Acids from the Corresponding 5-Substituted Hydantoins
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The reaction conditions for the production of D-p-hydroxyphenylglycine (D-HPG) from DL-5-(p-hydroxyphenyl)hydantoin (DL-HPH) by cells of Pseudomonas sp.AJ-11220 and the cultural conditions for this bacterium for the formation of the D-HPG-producing enzyme involved by this bacterium were investigated.The optimal pH of this reaction was about 8.0 and the optimal temperature about 43 deg C.The D-HPG producing enzyme was inducibly produced in Pseudomonas sp.AJ-11220 in proportion to the cell growth.Cells containing high activity were obtained when Pseudomonas sp.AJ-11220 was grown in a medium containing 20 g of glucose 5 g of (NH4)2SO4, 1g of KH2PO4, 3 g of K2HPO4, 0.5 g of MgSO4.7H2O, 0.01 g of FeSO4.7H2O, 0.01 g of MnSO4.4H2O, 10 g of yeast extract 5 g of DL-5-cyanoethylhydantoin and 20 g of CaCO3 in a total volume of 1 liter (pH 7.0).Under the optimal conditions, 25 mg/ml of D-HPG was asymmetrically and directly produced from 30 mg/ml of DL-HPH with a molar yield of 92percent.Various D-amino acids could also be effectively produced from the corresponding 5-substituted hydantoins.
- Yokozeki, Kenzo,Nakamori, Shigeru,Yamanaka, Shigeru,Eguchi, Chikahiko,Mitsugi, Koji,Yoshinaga, Fumihiro
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p. 715 - 720
(2007/10/02)
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- Chiral Recognition of Natural Products on Optically Active Polysiloxanes
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Polysiloxanes with chiral peptide side chains have been synthesized and used as stationary phases in gas chromatography.With these phases amino acid enantiomers can be separated as N-acyl esters.Due to temperature stability up to 230 deg C all protein amino acids can be separated and the racemization of synthetic biologically active peptides controlled.The phases can be used to assign the absolute configuration of amino acids.A variety of other classes of compounds can be separated into their enantiomers using the chiral polysiloxanes, like hydroxy acids, sulfoxides, phosphorous compounds, glycols, atrop isomers, amines and alcohols.Various applications of these phases are described including investigations of enantiomer selectivity in nature. - Key words: Polysiloxanes, Enantiomers, Racemization
- Bayer, Ernst
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p. 1281 - 1291
(2007/10/02)
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- Process for obtaining amino acids from the raw juices of sugar manufacture
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To recover amino acids from raw sugar juice, as obtained in beet extraction, the juice is treated with acid or with lime to coagulate impurities which are separated off. The juice may be treated before or after coagulation with a pectin-cleaving enzyme. The juice so pretreated can be directly passed through a strong cation exchanger and a weak anion exchanger whereby the amino acids are adsorbed. Switchover to a new cation exchanger is initiated when betaine flows out the column. Elution is effected, preferably with an ammonium compound, and eluate is collected, preferably each fraction being enriched in a different amino acid. The sugar juice may be concentrated and crystallized in known manner and is of comparatively high purity.
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