Reisolamento di una rara specie di Lievito: Trigonopsis variabilis Schachner

Zusammenfassung Trigonopsis variabilis, bisher erst einmal isoliert (1929), wurde in einem Traubenmost aus dem Weinbaugebiet des Staates S. Paulo, Brasilien, wieder aufgefunden. Es wird sein morphologisches und physiologisches Verhalten unter verschiedenen Kulturbedingungen beschrieben.     

Simple and rapid determination of the activity of recombinant D-amino acid oxidase in cephalosporin C bioconversion with use of a micro pO2 probe

Summary D-Amino acid oxidase (DAAO) genes were amplified from Trigonopsis variabilis and Rhodotorula gracilis by polymerase chain reactions. T. variabilis DAAO gene was cloned into a pUC19 vector. The expression of the T. variabilis DAAO was directly determined in permeabilized E. coli cells using a micro pO₂ probe. The micro pO₂ probe was sensitive enough to be suitable for a simple and rapid estimation of DAAO activity toward cephalosporin C.     

The stabilizing effects of immobilization in D-amino acid oxidase from <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis>

Background  

Immobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications.     

D-amino acid oxidase,aromatic L-amino aminotransferase,and aromatic lactate dehydrogenase from several yeast species: Comparison of enzyme activities and enzyme specificities

Production of D-amino acid oxidase, L-aromatic aminotransferase and aromatic lactate dehydrogenase by several yeast species was examined. Of 16 strains tested, Trigonopsis variabilis and Rhodosporidium toruloides were found to be most suitable for D-amino acid oxidase production, T. variabilis and Brettanomyces anomalus for L-aromatic aminotransferase production, and Hansenula polymorpha, Cryptococcus terreus, and Candida maltosa for aromatic lactate dehydrogenase production. This selection is based on a high amount of enzyme activity as well as a broad enzyme specificity. The data will be reported here.     

Enantioselective reduction of acetyldimethylphenylsilane by Trigonopsis variabilis (DSM 70714)

Summary Growing and resting cells of the yeast Trigonopsis variabilis (DSM 70714) can be used for the enantioselective reduction of the organosilicon compound acetyldimethylphenylsilane (1) to give optically active (R)-(1-hydroxyethyl)dimethylphenylsilane [(R)-2] in good yields. The enantiomeric purity of the isolated product was determined to be 62–86% ee depending on the substrate concentration used. Both substrate and product caused an inhibition of the reaction at concentrations higher than 0.35 and 0.5 g/l, respectively. Besides, higher substrate and product concentrations led to increased formation of the by-product 1,1,3,3-tetramethyl-1,3-diphenyldisiloxane. Considering the limiting substrate and product concentrations, it was possible to use the same biomass at least 5 times without significant loss of enzyme activity. 3-Methyl-3-phenyl-2-butanone (5) and acetyldimethylphenylgermane (7), which represent carbon and germanium analogues of 1, were also found to be accepted as substrates by Trigonopsis variabilis (DSM 70714). The reduction rates of the silicon (1) and germanium compound (7) were much higher than the transformation rate of the corresponding carbon analogue 5.     

New anamorphic yeast species: Candida infanticola sp. nov., Candida polysorbophila sp. nov., Candida transvaalensis sp. nov. and Trigonopsis californica sp. nov

Three new species of Candida and a new species of Trigonopsis are described based on their recognition from phylogenetic analysis of gene sequences from large subunit ribosomal RNA, ITS1/ITS2 rRNA, mitochondrial small subunit rRNA and cytochrome oxidase II. Candida infanticola sp. nov. (type strain NRRL Y-17858, CBS 7922) was isolated from the ear of an infant in Germany and is closely related to Candida sorbophila. Candida polysorbophila sp. nov. (type strain NRRL Y-27161, CBS 7317) is a member of the Zygoascus clade and was isolated in South Africa as a contaminant from an emulsion of white oil and polysorbate. Candida transvaalensis sp. nov. (type strain NRRL Y-27140, CBS 6663) was obtained from forest litter, the Transvaal, South Africa, and forms an isolated clade with Candida santjacobensis. Trigonopsis californica sp. nov. (type strain NRRL Y-27307, CBS 10351) represents a contaminant from wine in California, and forms a well-supported clade with Trigonopsis cantarellii, Trigonopsis variabilis and Trigonopsis vinaria.     

High yielding culture conditions for the biosynthesis of D-amino acid oxidase byTrigonopsis variabilis

Summary A process was developed for the production ofTrigonopsis variabilis and D-amino acid oxidase. Yields for the yeast were in excess of 220 g/l wet weight and 62 g/l dry weight. Using cephalosporin C as a substrate the enzyme concentration was 7000 units per liter.     

D-Amino-acid oxidase—an improved production of the enzyme by the yeastTrigonopsis variabilis in a laboratory fermentor

The cultivation of the yeastTrigonopsis variabilis producingd-amino-acid oxidase (an enzyme participating in the transformation of cephalosporin C into 7-aminocephalosporanic acid for the production of β-lactam antibiotics) was controlled by changes of dissolved oxygen tension and extended fermentation times. The production technology was optimized on a laboratory scale and scale-up parameters were identified.     

Expression of modified oxidase of D-aminoacids of <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis> in methylotrophic yeasts <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Effective recombinant strains Pichia pastoris that produce functionally active hybrid of Trigonopsis variabilis D-aminoacids bond with chitin-connecting domain of chitinase A1 of Bacillus circulans (DAOcbd) were obtained. The dependence of DAOcbd production levels from production of the number of copies of “expression cassette” integrated in the AOX1 locus of recombinant strains was studied. It was indicated that synthesized DAOcbd may be easily purified and immobilized on chitin sorbents and possessed high specific activity. Produced strains and methods of their cultivation and DAOcbd extraction may be used for development of technologies of obtaining of biocatalyzers in technological processes of obtaining of 7-aminocepha-losporane acid.     

Stepwise engineering of a <Emphasis Type="Italic">Pichia pastoris</Emphasis> D-amino acid oxidase whole cell catalyst

Background  

Trigonopsis variabilis D -amino acid oxidase (Tv DAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of Tv DAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris Tv DAO whole-cell biocatalyst.     

D-amino acid oxidase: structure,catalytic mechanism,and practical application

D-Amino acid oxidase (DAAO) is a FAD-dependent enzyme that plays an important role in microbial metabolism, utilization of endogenous D-amino acids, regulation of the nervous system, and aging in mammals. DAAO from yeasts Rhodotorula gracilis and Trigonopsis variabilis are used to convert cephalosporin C into 7-aminocephalosporanic acid, the precursor of other semi-synthetic cephalosporins. This review summarizes the recent data on the enzyme localization, physiological role, gene cloning and expression, and the studies on the enzyme structure, stability, catalytic mechanism, and practical applications.Translated from Biokhimiya, Vol. 70, No. 1, 2005, pp. 51–67.Original Russian Text Copyright © 2005 by Tishkov, Khoronenkova.     

Efficient and selective catalysts for olefin polymerizations-insights and challenges

Recent developments, present state of art and future potentials of polyolefin production by means of metallocene and other organometallic catalysts are outlined with a view toward practical applicability.

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Riassunto   Catalizzatori efficienti e selettivi per la produzione di poliolefine: approfondimenti e sfide aperte. Vengono passati in rassegna gli sviluppi recenti, lo stato presente dell'arte e le future prospettive della produzione delle poliolefine mediante metalloceni ed altri catalizzatori organometallici, con particolare riguardo alla possibilità di applicazioni pratiche

     

A proposito della imificazione dei generi delle „Torulopsidaceae-Mycotoruleae”

Riassunto L'A., in relazione ad una Nota di Diddens e Lodder relativa alla unificazione dei generi di Mycotoruloideae, ritiene che, nell'ambito di questa sottofamiglia di Torulopsidaceae, debbano essere mantenuti i generi Mycotorula e Candida pur convenendo di eliminare gli altri.

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Summary In answer to the question of unification of the genera of the asporigenous yeast with pseudomycelium, prospected by Diddbns and Lodder, the view of the author is to mantain both Mycotorula and Candida.

     

Properties and applications of microbial D-amino acid oxidases: current state and perspectives

D-Amino acid oxidase (DAAO) is a biotechnologically relevant enzyme that is used in a variety of applications. DAAO is a flavine adenine dinucleotide-containing flavoenzyme that catalyzes the oxidative deamination of D-isomer of uncharged aliphatic, aromatic, and polar amino acids yielding the corresponding imino acid (which hydrolyzes spontaneously to the α-keto acid and ammonia) and hydrogen peroxide. This enzymatic activity is produced by few bacteria and by most eukaryotic organisms. In the past few years, DAAO from mammals has been the subject of a large number of investigations, becoming a model for the dehydrogenase-oxidase class of flavoproteins. However, DAAO from microorganisms show properties that render them more suitable for the biotechnological applications, such as a high level of protein expression (as native and recombinant protein), a high turnover number, and a tight binding of the coenzyme. Some important DAAO-producing microorganisms include Trigonopsis variabilis, Rhodotorula gracilis, and Fusarium solani. The aim of this paper is to provide an overview of the main biotechnological applications of DAAO (ranging from biocatalysis to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment) and to illustrate the advantages of using the microbial DAAOs, employing both the native and the improved DAAO variants obtained by enzyme engineering.      

A rapid method for the purification of D-amino acid oxidase of Trigonopsis variabilis by hydrophobic chromatography

Summary A relatively simple method has been described for the rapid purification of D-amino acid oxidase from Trigonopsis variabilis by hydrophobic chromatography on Phenyl-Sepharose CL-4B and negative adsorption on DEAE-cellulose. The purified enzyme had a specific activity of 22–24 units at 25°C and exhibited three bands on enzymatic staining.     

Un copepode nouveau d'Indonesie

A new species of Cyclopidae (Crustacea, Copepoda) is described from Indonesia. Its morphological features require the creation of a new subgenus in the genus Microcyclops, Microcyclops (Mediocyclops) variabilis n. subgen., n. sp.

     

Effects of cassava varietal mixtures on the whiteflies Aleurotrachelus socialis and Trialeurodes variabilis in Colombia

The effects of cassava varietal mixtures on the population dynamics of the whiteflies Aleurotrachelus socialis Bondar and Trialeurodes variabilis (Quaintance) were investigated under outbreak conditions in Colombia. A regional variety of cassava (MCOL 2257) was grown in pure stand and mixed with a variety, CMC 40, believed to have partial resistance to T. variabilis. The dominant species in the systems, A. socialis, showed no varietal preference and population levels on the regional cultivar were similar between treatments. Of the two cultivars, CMC 40 was less preferred by T. variabilis and its presence in mixed variety plots lowered populations on the regional cassava. Yield losses, primarily due to A. socialis, were equal for regional cassava mixed with CMC 40 and in pure stand. Nevertheless, the use of CMC 40 in varietal mixtures provides a potential strategy for lowering herbivore load and increasing yields in zones with heavy T. variabilis attack.

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Résumé L'influence du mélange de différentes variétés de manioc sur A. socialis Bondar et T. variabilis Quaintance a été examinée dans des conditions de pullulation en Colombie. Une variété régionale (MCOL 2257) a été cultivée seule ou mélangée avec une variété (CMC 40), considérée comme ayant une résistance partielle à T. variabilis. A. socialis, espèce dominante dans la biocénose, n'a pas montré de préférence variétale et son niveau de population sur MCOL 2557 était le même quelles que soient les conditions de culture. T. variabilis a moins préféré CMC 40, dont la présence en culture mixte a réduit le niveau des populations sur manioc local. Les dégâts sur CMC 40, dus d'abord à A. socialis, ont été les mêmes, qu'il ait été seul ou mélangé à MCOL 2257. Les dégats sur MCOL 2257, dus avant tout à A. socialis ont été les mêmes en culture pure ou en mélange avec CMC 40. Quoi qu'il en soit, la culture de CMC 40 en association variétale constitue une méthode qui abaisse potentiellement le fardeau des herbivores en augmentant les récoltes dans les zones à forte contamination par T. variabilis.

     

Reaction of Trigonopsis variabilis d-amino acid oxidase with 2,6-dichloroindophenol: kinetic characterisation and development of an oxygen-independent assay of the enzyme activity

2,6-Dichloroindophenol (DCIP) is shown to be utilised efficiently as electron acceptor replacing dioxygen in the reaction of Trigonopsis variabilis d-amino acid oxidase (TvDAO) with d-methionine as the substrate. The specificity constant for DCIP reduction at 30 °C is one-twelfth that of oxygen conversion into hydrogen peroxide. Time course analysis of simultaneous consumption of DCIP and dioxygen, recorded on-line by absorption and non-invasive fluorescence quenching, respectively, pinpoints the preferential utilisation of dioxygen; and reveals a maximum DCIP conversion rate that is independent of the initial concentration of dioxygen. A robust direct assay of TvDAO activity has been developed that does not require anaerobic reaction conditions. It was down-scaled to microtitre plate format and overcomes practical limitations of other assays due to the low affinity of TvDAO for dioxygen (K_m ≈ 0.7 mmol L⁻¹).     

D-amino acid oxidase fromTrigonopsis variabilis: Comparison of enzyme specificity “in vivo” and “in vitro”

Summary A method is described for permeabilization of intact cells of the yeastTrigonopsis variabilis with respect toin vivo measuring D-amino acid oxidase activity. The kinetic results so obtained differ from those obtained with the purified enzyme, pointing to the advantage of using the purified enzyme or the permeabilized cells in the oxidative deamination of different D-amino acids.