Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling.

Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2-9. Here we report photobiocatalytic asymmetric sp3-sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5'-phosphate-dependent enzymes, threonine aldolases10-12, for the α-C-H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13-15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox-pyridoxal biocatalysis provides a platform for sp3-sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.

Biosynthesis of Strained Amino Acids by a PLP-Dependent Enzyme through Cryptic Halogenation.

Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an SN2-like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Mechanisms of anammox granular sludge reactor effluent as biostimulant: Shaping microenvironment for anammox metabolism.

Effluent from anammox granular sludge (AnGS) bioreactor contains microbes and microbial products. This study explored mechanisms of utilizing AnGS-effluent as biostimulant for anammox process enhancement. Compared with no AnGS-effluent supplemented control reactor, 5.0 and 1.3 times higher ammonium nitrogen and total inorganic nitrogen removal rates, respectively were obtained with continuous AnGS-effluent supplementation after 98 days' operation. Anammox bacteria from Candidatus Brocadia accounted for 0.1 % (DNA level) and 1.3 %-1.5 % (RNA level) in control reactor, and 2.9 % (DNA level) and 54.5 %-55.4 % (RNA level) in the AnGS-effluent-fed reactor. Influent microbial immigration evaluation showed that bacterial immigration via AnGS-effluent supplementation was not the main contributor to active anammox community development. Amino acids biosynthesis, B-vitamins and coenzymes metabolism related pathways were facilitated by AnGS-effluent supplementation. AnGS-effluent supplementation aided anammox metabolic activity by shaping microenvironment and microbial interactions. This study provides insights into enhancing anammox bacterial metabolism with AnGS-effluent microbial products as biostimulant.

[Engineering of microorganisms for high production of amino acids].

Amino acids as the building blocks of proteins are widely applied in food, medicine, feed, and chemical industries. Amino acid production by microbial cell factories from renewable resources is praised for the environmental friendliness, mild reaction conditions, and high product purity, which helps to achieve the goal of carbon neutrality. Researchers have employed the methods of metabolic engineering and synthetic biology to engineer Escherichia coli and Corynebacterium glutamicum and optimized the culture conditions to construct the microbial cell factories with high performance for producing branched chain amino acids, amino acids of the aspartic acid and glutamic acid families, and aromatic amino acids. We review the engineering process of microbial cell factories for high production of amino acids, in the hope of providing a reference for the creation of high-performance microbial cell factories.

Biosynthesis of macrolactam antibiotics with ß-amino acid polyketide starter units.

Macrolactam antibiotics incorporating ß-amino acid polyketide starter units, isolated primarily from Actinomycetes species, show significant biological activities. This review provides a detailed analysis into the biosynthetic studies of vicenistatin, a macrolactam antibiotic with a 3-aminoisobutyrate starter unit, as well as biosynthetic research on related macrolactam compounds. Firstly, the elucidation of a common mechanism for the incorporation of ß-amino acid starter units into the polyketide synthase (PKS) is described. Secondly, the unique biosynthetic mechanisms of the ß-amino acids that are used to supply the main macrolactam biosynthetic pathways with starter units are discussed. Thirdly, some distinctive post-PKS modification mechanisms that complete macrolactam antibiotic biosynthesis are summarized. Finally, future directions for creating new macrolactam compounds through engineered biosynthesis pathways are described.

Protein metabolism and physical training: any need for amino acid supplementation?

Muscle mass is the major deposit of protein molecules with dynamic turnover between net protein synthesis and degradation. In human subjects, invasive and non-invasive techniques have been applied to determine their skeletal muscle catabolism of amino acids at rest, during and after different forms of physical exercise and training. The aim of this review is to analyse the turnover flux and the relative oxidation rate of different types of muscle proteins after one bout of exercise as well as after resistance and endurance condition of training. Protein feeding in athletes appears to be a crucial nutrition necessity to promote the maintenance of muscle mass and its adaptation to the need imposed by the imposed technical requirements. In resting human individuals, there commended protein daily allowance is about 0.8 g (dry weight) kg−body weight per 24 h knowing that humans are unable to accumulate protein stores in muscle tissues. Nevertheless, practical feeding recommendations related to regular exercise practice are proposed to athletes by different bodies in order to foster their skills and performance. This review will examine the results obtained under endurance and resistance type of exercise while consuming single or repeated doses of various ingestions of protein products (full meat, essential amino acids, specific amino acids and derivatives, vegetarian food). From the scientific literature, it appears that healthy athletes(and heavy workers) should have a common diet of 1.25 g kg−24 h to compensate the exercise training muscle protein degradation and their resyn thesis within the following hours. A nitrogen-balance assay would berecommended to avoid any excessive intake of protein. Eventually, a daily equilibrated food intake would beof primer importance versus inadequate absorption of some specific by-products.

Avaliação de alguns tratamentos na intoxicação por amônia em bovinos / Evaluation of some treatments for ammonia poisoning in cattle

Para avaliar tratamentos na intoxicação por amônia, 15 novilhos, infundidos com solução de cloreto de amônio, foram distribuídos em três grupos de cinco animais cada e tratados, como: 1 - grupo-controle (C), infundido com solução salina fisiológica (H); 2 - grupo O+H, medicado com aminoácidos do ciclo da ureia (O) e "H"; 3 - grupo O+F+H, o mesmo protocolo do grupo 2 acrescido de furosemida (F). Os animais foram monitorados, colhendo-se amostras sanguíneas e todo volume urinário. Os tratamentos O+F+H e O+H promoveram melhora clínica pronunciada, em relação ao grupo H, isto é, permaneceram em estação, retornaram o movimento de rúmen e o apetite e recuperaram-se do edema pulmonar mais rapidamente. Observaram-se, nos grupos O+F+H e O+H, teores plasmáticos mais baixos de amônia e lactato-L, urina mais abundante, excreção pela urina de maior quantidade de amônio e ureia, depuração mais intensa de amônia do sangue e pH urinário mais baixo. Concluiu-se que os tratamentos propostos apresentaram ação efetiva principalmente por diminuírem a hiperamonemia, por meio da maior excreção renal de amônio e maior transformação hepática de amônia em ureia.

Fifteen yearling steers were used to evaluate treatments for ammonia intoxication. The animals subjected to ammonium chloride infusion were distributed into three groups of five animals each, and treated as following: 1 - control group (C), physiological saline solution (H) infusion; 2 - O+H group, urea cycle aminoacids (O) and "H" medication; and 3 - O+F+H group, the same protocol of group 2 plus furosemide (F). The animals were monitored by collection of blood samples and the whole urinary volume. O+F+H and O+H treatments promoted pronounced clinical improvement, in attention to H group, i.e., they remained in standing position, returned ruminal movement and appetite, and recovered more quickly from pulmonary edema. In O+F+H and O+H groups, minor plasmatic levels of ammonia and L-lactate, more abundant urine, major ammonium and urea amount excretion by urine, more intensive ammonia depuration from blood, and lower urinary pH were observed. It was concluded that the proposed treatments had effective action due to hyperammonemia decrease, by major renal ammonium excretion, and hepatic transformation of ammonia in urea.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinantspiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l-1 Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3 percent higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5 percent. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85 percent and 72.1 percent of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41 percent. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9 percent while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l-1 leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l-1 leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1 percent to 46.9 percent.

Cambios en aminoácidos luego de administración intraperitoneal de veneno del escorpión Tityus zulianus en ratones: Estudio con microdiálisis subcutánea y electroforesis capilar

Resumen. El envenenamiento por escorpiones es un problema de salud pública en la zona suroccidental de Venezuela. El Tityus zulianus es uno de los escorpiones que causa, especialmente en niños, edema pulmonar e insuficiencia cardíaca que pueden ser fatales y que se han atribuido en parte a una gran descarga simpática. La administración intraperitoneal de (20 µg/g peso) de veneno del T. zulianus a ratones anestesiados durante microdiálisis subcutánea provocó aumento de secreciones, dificultad respiratoria, convulsiones y muerte entre 30 min a 2 h. En los dializados recolectados antes y después de recibir el veneno, se analizaron 7 aminoácidos por electroforesis capilar con detección mediante fluorescencia inducida por láser (EC-DFIL). Se encontró un aumento en arginina (39%), fenilalanina (40%) y glutamato (94%), sin cambios en valina, serina y aspartato, en los animales que recibieron veneno con respecto a sus valores pretratamiento. Estos cambios porcentuales fueron significativos al comparar veneno vs. vehículo después de la inyección; y antes vs. después de recibir el veneno. Para este momento no hay una explicación clara del significado de estos aumentos de aminoácidos específicos. Se requieren nuevos estudios para conocer si estos cambios bioquímicos tienen o no una relación con los mecanismos de acción molecular del veneno o algunos de sus componentes y/o con las manifestaciones clínicas. De acuerdo con la literatura revisada, este es el primer reporte de la combinación de microdiálisis subcutánea y EC-DFIL en el estudio in vivo del emponzoñamiento por escorpiones en ratones.

Abstract. Scorpion human envenoming is a public health hazard in the southwest of Venezuela. Tityus zulianus is one of the scorpion species whose venom causes lung edema and cardiac failure in children. These occasionally deadly manifestations have been attributed to a massive sympathetic discharge. The intraperitoneal administration of T. zulianus venom (20 µg/g mouse) to anesthetized mice during subcutaneous microdialysis caused increased secretions, dyspnea, seizures and death between 30 min to 2 h. Seven amino acids were analyzed by capillary electrophoresis with laser induced fluorescence detection (CE-LIFD) in the collected samples before and after the venom administration. We found an increase of arginine (39%), phenylalanine (40%) and glutamate (94%), with no changes in valine, serine and aspartate, changes were significant when the injection of venom and vehicle were compared and before vs after venom injection. Further investigation is needed to know if the observed changes could be related to the molecular mechanisms of the venom or some of its components and therefore with the envenoming symptoms. To our knowledge, this is the first report with subcutaneous microdialysis and CE-LIFD coupling in scorpion envenomation studies in vivo, in mice.

Amino acid composition of single and mixed fungal cultures grown in sugar cane vinasse

Foi avaliada a composiçäo de aminoácidos de culturas puras e mista de Cryptococus laurentii e Aspergillus niger desenvolvidas em meio de vinhaça. Os perfis de aminoácidos, determinados por cromatografia de troca iônica, mostraram que a biomassa de A.niger apresentou valores superiores aqueles apresentados por Cr. laurentii para cada aminoácido. No entanto, a cultura mista destes microrganismos apresentou teores ainda mais elevados de aminoácidos. A biomassa da cultura mista foi deficiente em cistina em comparaçäo com o padräo de proteína recomendado pela FAO; ácido glutâmico e prolina em comparaçäo com a caseína; e metionina em comparaçäo com as proteínas da carne, trigo, leite e ovo. Os resultados mostraram ser a cultura mista de A.niger and Cr.laurenti uma boa fonte de proteína

Single cell protein quality produced from lignocellulosic materials by the ascomycete Chrysonilia sitophila (TFB-27441 strain)

Se apresentam dados do conteúdo de aminoácidos de 15 materiais lignocelulósicos, incluindo águas residuais de oliva (alpechin). Os substratos lignocelulósicos selecionados foram: glicose, celobiose, sacarose, celulose microcristalina, tanino, flobafeno, cortiça, resíduos lignocelulósicos resíduos de açúcares derivados do processo organo-solv, casca de arroz, casca de arroz pré-irradiada, bagaço de laranja, alpechin (5 por cento), alpechin (10 por cento), e alpechin pré-irradiado (10 por cento). Säo apresentadas taxas de alguns aminoacidos de significado nutricional em fontes convencionais de proteínas e aqueles induzidos por diferentes materiais lignocelulósicos. Também säo comparadas as taxas de aminoácidos individuais com aminoácidos essenciais (E/A) e aminoácidos essenciais totais recuperados como nitrogênio total (razäo E/T), juntamente com a escore químico dos aminoácidos essenciais, o que permitiu avaliar a qualidade de cada fonte de carbono utilizada. Essas análises mostram que o bagaço de laranja, alpechin (10 por cento) e cortiça foram os materiais mais econômicos e que resultaram em proteína de maior qualidade

Liquid-liquid extraction of pharmaceuticals by aqueous two-phase systems

Aqueous two-phase systems are generally composed of a water solution of two structurally distinct hydrophilic polymers or of one polymer and certain salts. From analytical to commercial scale, aqueous two-phase systems have their application in the purification, characterization and study of biomaterials. In this review, we present some applications of aqueous two-phase systems in the separation and study of various pharmaceuticals, incluing recombinant proteins, antibodies and antigens, antibiotics, amino acids and oligopeptides, lactic acid, enzymes etc. Some new developments of the aqueous two-phase systems are reviewed and the prospects of this excellent technology are discussed...

Como a genética microbiana pode auxiliar a indústria de fermentaçäo / How the microbial genetic can aid the fermentation industry

A fermentaçäo industrial tem recebido, ultimamente, grande impulso, graças às novas técnicas genéticas disponíveis que permitem modificar o patrimônio genético de um microrganismo para que possa ser utilizado mais eficientemente, ou ainda, de modo mais inédito, para fabricar produtos novos que näo podiam ser obtidos por fermentaçäo. Diversos exemplos säo mencionados (ênzimos, antibióticos, proteínas, aminoácidos, entre outros), para ilustrar as enormes potencialidades do melhoramento genético e da engenharia genética como ferramentas valiosíssimas para a indústria de fermentaçöes e säo discutidos os principais problemas oriundos da utilizaçäo desses microrganismos modificados no processo fermentativo

Aspectos fisiológicos y fisiopatológicos del oxido nítrico en los tejidos mamíferos / Physiological and pathophysiological aspects of nitric oxide in mammalian tissues

En los últimos años el óxido nítrico (ON), una molécula sencilla pero altamente reactiva, ha sido el centro de una gran cantidad de investigaciones. Es sintetizado en los tejidos mamíferos a partir del aminoácido L-arginina por una reacción enzimática catalizada por la sintasa de óxido nítrico (SON), produciendo L-citrulina y, ON. El ON tiene una media vida muy corta, es liposoluble, reacciona fácilmente con un gran número de sistemas enzimáticos, y es producido por una amplia variedad de células. La enzima SON existe en, al menos, tres formas: dos isoformas son calcio dependientes y están continuamente presentes en células específicas, llamadas SON constitutivas (CSON). De éstas, una isoforma está presente en el citosol de las células neuronales (nSON), mientras la otra isoforma está presente en forma de proteína ligada a las membranas en las células endoteliales (eSON). Las cSON producen pequeñas cantidades de ON, después de que ocurre una estimulación por agonistas específicos. El ON producido por las cSON frecuentemente media señales celulares y comunicación celulas. Una tercera isofroma de SON es calcio-independientemente, no está presente en células no estimuladas y produce grandes cantidades de ON siguiendo a la estimulación de células apropiadas con citoquinas o lipopolisacáridos. Esta isoforma es llamada SON inducible (iSON). El ON es un mediador tanto de procesos fisiológicos como patológicos. Actúa directamente en sus blancos celulares, de los cuales el más importante es la guanilatociclasa, y produce una gran variedad de efectos biológicos, que van desde citoprotección a citotoxicidad. Es motivo de la presente revisión un análisis de la bio-química y fisiología del ON, así como de sus acciones biológicas y posibles implicaciones terapéuticas