Expression of Brugmansia candida Hyoscyamine 6beta-Hydroxylase gene in Saccharomyces cerevisiae and its potential use as biocatalyst

<p>Abstract</p> <p>Background</p> <p>Tropane alkaloids, mainly hyoscyamine and scopolamine, are widely used in medicine due to their anticholinergic activity. Scopolamine has a higher demand being the more valuable alkaloid due to its fewer side effects and higher physiological activity. Anisodamine (6β-hydroxyhyoscyamine) is the intermediate in the conversion of hyoscyamine into scopolamine. Current studies report that this alkaloid is potentially applicable in medicine. The gene that codifies for Hyoscyamine 6-β hydroxylase, the enzyme responsible for hyoscyamine hydroxylation and epoxidation, leading to scopolamine was isolated from <it>Brugmansia candida</it>.</p> <p>Results</p> <p>The <it>h6h</it>cDNA was cloned into pYES2.1 and pYES2.1/V5-His-TOPO vectors to produce an untagged and a tagged protein, respectively. The H6H enzyme was produced in <it>Saccharomyces cerevisiae </it>in order to obtain a biological catalyst for potential industrial applications. Protein extracts of the induced yeast were analyzed by Western blot. The expression was detected 4 h after induction and no degradation was observed during the period assayed. The tagged and the untagged proteins were able to transform hyoscyamine, showing a functional expression of the <it>h6h</it>cDNA.</p> <p>Conclusion</p> <p>The strains obtained in this work are promising and potentially applicable in biocatalytic processes.</p

Analytical considerations for the successful evaluation of hyoscyamine biotransformation into 6β-hydroxyhyoscyamine and scopolamine

Hyoscyamine, 6β-hydroxyhyoscyamine and scopolamine are anticholinergic agents that belong to the tropane alkaloids, a pharmacological important group of secondary metabolites. Hyoscyamine and scopolamine were historically used in medicine. Additionally, potential medical applications for 6β- hydroxyhyoscyamine were described in the last years. Previous works carried out in our lab allowed us the construction of a Saccharomyces cerevisiae strain harboring the Hyoscyamine-6β-hydroxylase (H6H) enzyme which is responsible of the conversion of hyoscyamine into 6β-hydroxyhyoscyamine and scopolamine. Several factors influenced and complicated the optimization of the hyoscyamine bioconversion process. The aim of this work was to evaluate the analytical factors that critically affect the performance of the alkaloid extraction and the detection and quantification method of the alkaloids implied in the biocatalytical process. The mechanical breakdown of yeast cells by continuous agitation at 4 °C in 2 mL tubes was the method of choice for an efficient recovery of the functional H6H enzyme. In addition, the different pH assayed for the alkaloid extraction caused significant variations in the recovery of the alkaloids, specifically impacting on scopolamine recovery which decreased a 35 % after the increase of the pH of the extraction. The development of robust and sensitive analytical methods was requisite for the correct monitoring and quantification of the alkaloids produced in order to evaluate the technological and economic feasibility of this process.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Pedagogic Content Knowledge (PCK) in university Biotechnology teaching. The microbial specific growth rate (μ) case

En este trabajo se presenta un análisis de las ideas relacionadas al concepto de velocidad específica de crecimiento microbiano (μ) que presentaron estudiantes universitarios que cursaban la orientación en biotecnología del último año de la carrera de Bioquímica (Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina). El estudio se realizó en base a las respuestas que dieron los estudiantes, de manera anónima, frente a la solicitud espontanea de que explicaran qué entendían por μ. El análisis se focalizó en la identificación de los factores que podrían relacionarse con las ideas que mostraron los estudiantes, entre las que se incluye el tratamiento previo del concepto, la tendencia a la reducción funcional, el pragmatismo y la posibilidad de que se trate de concepciones alternativas pero en un campo muy específico y aplicado de las ciencias como es la biotecnología. Se plantean estrategias aplicadas para la reconstrucción del concepto de μ considerando estos factores. Las experiencias y conclusiones que surgen de este trabajo pretenden contribuir al desarrollo del Conocimiento Didáctico del Contenido (CDC) para ciencias aplicadas en general, y para la biotecnología en particular.In this work, a study based on the university student’s conception about microbial specific growth rate (μ) is presented. The study was focused on last year students of the Biochemist career (Buenos Aires University, Argentina). It was developed considering the answers given anonymously by the students when they were spontaneously asked about the meaning of μ. The analysis was focused in the identification of factors which could be related with the students´ ideas about μ, such as the previous work with the subject, the tendency to the functional reduction, the pragmatisms and the possibility of alternative conceptions, but related with a specific field of applied sciences, such as biotechnology. Strategies aiming to the reconstruction of the μ concept were proposed considering these factors. The experiences presented in this work will contribute to the development of the Pedagogical Content Knowledge (PCK) in applied sciences, particularly in biotechnology

Effects of elicitation on solasodine production by Solanum Eleagnifolium Cav. hairy root cultures

El objetivo de este trabajo fue estudiar de distintos elicitores sobre la producción de solasodina por cultivos de raíces transformadas de Solanum eleagnifolium Cav. Se ensayaron: quitosano 10 y 100 mg/l, hemicelulasa 0.1 y 0.2 mg/l, H2O2 17 y 170 mg/l, AgNO3 0,14 mg/l y homogenatos de los hongos Hormonema sp. y Phytium sp., los cuales no produjeron diferencias significativas en la acumulación de solasodina respecto del control. Asimismo cuando se elicitó con un homogenato de Sclerotinia sclerotiorum se produjo una disminución de la concentración del alcaloide de 30% respecto al control, atribuible a la posibilidad de que la elicitación con este hongo haya inducido la producción de sesquiterpenos en vez de alcaloides como la solasodina.The effect of several elicitors on solasodine production by hairy root cultures of Solanum eleagnifolium Cav. was studied. When chitosan 10 and 100 mg/l, hemicelullase 0.10 and 0.2 0 mg/, H2O2 17 and 170 mg/l, Ag- NO3 0,14 mg/l and Hormonema sp. and Phytium sp´s homogenates were used as elicitors no effect on solasodine production was observed. When homogenates of Sclerotinia sclerotiorum were used the solasodine production decreased about 30% respect to the control. This behavior could be attributed to Sclerotinia sclerotiorum elicitation induced the sesquiterpenes biosynthesis instead of alkaloids production.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Effects of elicitation on solasodine production by Solanum Eleagnifolium Cav. hairy root cultures

El objetivo de este trabajo fue estudiar de distintos elicitores sobre la producción de solasodina por cultivos de raíces transformadas de Solanum eleagnifolium Cav. Se ensayaron: quitosano 10 y 100 mg/l, hemicelulasa 0.1 y 0.2 mg/l, H2O2 17 y 170 mg/l, AgNO3 0,14 mg/l y homogenatos de los hongos Hormonema sp. y Phytium sp., los cuales no produjeron diferencias significativas en la acumulación de solasodina respecto del control. Asimismo cuando se elicitó con un homogenato de Sclerotinia sclerotiorum se produjo una disminución de la concentración del alcaloide de 30% respecto al control, atribuible a la posibilidad de que la elicitación con este hongo haya inducido la producción de sesquiterpenos en vez de alcaloides como la solasodina.The effect of several elicitors on solasodine production by hairy root cultures of Solanum eleagnifolium Cav. was studied. When chitosan 10 and 100 mg/l, hemicelullase 0.10 and 0.2 0 mg/, H2O2 17 and 170 mg/l, Ag- NO3 0,14 mg/l and Hormonema sp. and Phytium sp´s homogenates were used as elicitors no effect on solasodine production was observed. When homogenates of Sclerotinia sclerotiorum were used the solasodine production decreased about 30% respect to the control. This behavior could be attributed to Sclerotinia sclerotiorum elicitation induced the sesquiterpenes biosynthesis instead of alkaloids production.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Cultivo de raíces transformadas: Producción de Solasodina por Solanum eleagnifolium Cav. y de tiófenos por Tagetes laxa Cabrera

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Western blot performed with cell lysates of producing the recombinant H6H enzyme from after different post-induction times: 0, 4, 8, 12, 16, 24, 27, 29 h

<p><b>Copyright information:</b></p><p>Taken from "Expression of Hyoscyamine 6beta-Hydroxylase gene in and its potential use as biocatalyst"</p><p>http://www.microbialcellfactories.com/content/7/1/17</p><p>Microbial Cell Factories 2008;7():17-17.</p><p>Published online 27 May 2008</p><p>PMCID:PMC2430943.</p><p></p