RESUMEN
A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications.
Asunto(s)
Aminohidrolasas/aislamiento & purificación , Aminohidrolasas/metabolismo , Betaproteobacteria/enzimología , Clonación Molecular , Escherichia coli/enzimología , Análisis de Secuencia de ADN , Secuencia de Aminoácidos , Aminohidrolasas/química , Aminohidrolasas/genética , Secuencia de Bases , Betaproteobacteria/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Estereoisomerismo , Especificidad por SustratoRESUMEN
A biocatalytic process for the hydration of adiponitrile to 5-cyanovaleramide has been developed which can be run to higher conversion, produces more product per weight of catalyst, and generates significantly less waste products than alternate chemical processes. The biocatalyst consists of Pseudomonas chlororaphis B23 microbial cells immobilized in calcium alginate beads. The cells contain a nitrile hydratase (EC 4.2.1.84) which catalyzes the hydration of adiponitrile to 5-cyanovaleramide with high regioselectivity, and with less than 5% selectivity to byproduct adipamide. Fifty-eight consecutive batch reactions with biocatalyst recycle were run to convert a total of 12.7 metric tons of adiponitrile to 5-cyanovaleramide. At 97% adiponitrile conversion, the yield of 5-cyanovaleramide was 13.6 metric tons (93% yield, 96% selectivity), and the total weight of 5-cyanovaleramide produced per weight of catalyst was 3150 kg/kg (dry cell weight).
Asunto(s)
Amidas/química , Biotecnología/métodos , Nitrilos/química , Pseudomonas/metabolismo , Amidas/metabolismo , Estabilidad de Enzimas , Hidroliasas/química , Hidroliasas/metabolismo , Nitrilos/metabolismoRESUMEN
High levels of active glycolate oxidase from spinach (GO) and active catalase T from Saccharomyces cerevisiae (catT) have been co-produced in the methylotrophic yeast Pichia pastoris (Pp). In sequential rounds of transformation using two selectable markers, multiple copies of the genes encoding GO and catT were integrated into the Pp chromosome under control of the methanol inducible alcohol oxidase I promoter, resulting in a strain designated MSP8.6. MSP8.6 is a second-generation biocatalyst used for the conversion of glycolate to glyoxylate in the presence of a reaction component which inhibits endogenous Pp catalase. This work demonstrates a significant advance in the utility of recombinant Pp for commercial bioprocess development.
Asunto(s)
Oxidorreductasas de Alcohol/biosíntesis , Catalasa/biosíntesis , Proteínas Fúngicas/biosíntesis , Pichia/genética , Proteínas de Plantas/biosíntesis , Oxidorreductasas de Alcohol/genética , Catalasa/genética , Catálisis , Clonación Molecular/métodos , Activación Enzimática , Proteínas Fúngicas/genética , Ingeniería Genética , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Spinacia oleracea , Transformación GenéticaRESUMEN
The methylotrophic yeast Hansenula polymorpha has been developed as an efficient production system for heterologous proteins. The system offers the possibility to cointegrate heterologous genes in anticipated fixed copy numbers into the chromosome. As a consequence co-production of different proteins in stoichiometric ratios can be envisaged. This provides options to design this yeast as an industrial biocatalyst in procedures where several enzymes are required for the efficient conversion of a given inexpensive compound into a valuable product. To this end recombinant strains have been engineered with multiple copies of expression cassettes containing the glycolate oxidase (GO) gene from spinach and the catalase T (CTT1) gene from S. cerevisiae. The newly created strains produce high levels of the peroxisomal glycolate oxidase and the cytosolic catalase T. The strains efficiently convert glycolate into glyoxylic acid, oxidizing the added substrate and decomposing the peroxide formed during this reaction into water and oxygen.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Catalasa/genética , Expresión Génica , Pichia/genética , Proteínas Recombinantes/biosíntesis , Oxidorreductasas de Alcohol/metabolismo , Catalasa/metabolismo , Catálisis , Citosol/enzimología , Fermentación , Glicolatos/metabolismo , Glioxilatos/metabolismo , Microbiología Industrial , Microcuerpos/enzimología , Pichia/enzimología , Saccharomyces cerevisiae/enzimología , Spinacia oleracea/enzimologíaRESUMEN
The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity.
RESUMEN
Glycolate oxidase (GO) is a flavo-enzyme that catalyzes the oxidation of glycolate, and is useful for the biocatalytic production of glyoxylate. We have produced high levels of spinach GO in the methylotrophic yeast Pichia pastoris (Pp), by chromosomal integration of multiple copies of an expression cassette containing the GO coding sequence under control of the methanol-inducible alcohol oxidase I promoter. Under fermentation conditions, greater than 250 units of GO per gram of cells (wet weight) was obtained, corresponding to roughly 20-30% of soluble cell protein. This recombinant Pp strain was used as a whole-cell biocatalyst for conversion of glycolic acid to glyoxylic acid.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Pichia/genética , Secuencia de Bases , Catálisis , Clonación Molecular , Ingeniería Genética , Vectores Genéticos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Recombinantes , Spinacia oleracea/enzimologíaRESUMEN
A variety of methods for the immobilization of glycolate oxidase have been examined for the preparation of a catalyst for the oxidation of glycolic acid to glyoxylic acid. The co-immobilization of glycolate oxidase and catalase on oxirane acrylic beads produced a catalyst which was stable to the reaction conditions used for the oxidation, where glycolic acid and oxygen are reacted in aqueous solution in the presence of the immobilized enzyme catalyst and ethylenediamine. Under optimum reaction conditions, 99% yields of glyoxylic acid were obtained at greater than 99% conversion of glycolic acid, and the recovery and reuse of the co-immobilized enzyme catalyst was demonstrated.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Catalasa/metabolismo , Enzimas Inmovilizadas/metabolismo , Glioxilatos/síntesis química , Aspergillus niger/enzimología , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Cinética , Spinacia oleracea/enzimología , Factores de TiempoRESUMEN
To test the hypothesis that ovarian steroid hormones modulate oxytocin-induced release of prostaglandin F2alpha (PGF2alpha) from uterine endometrium, 2 ovariectomized rabbits were pretreated with progesterone (5 mg/day for 10 days), 2 with estradiol-17 beta (25 microgram/day for 10 days), 2 with both steroids, and one with sesame oil only. On the last day of treatment, endometrial fragments were excised and incubated in vitro with or without oxytocin (100 muU/ml). Although endometrium from rabbits pretreated with combined steroids released more PGF2alpha immediately after excision than did tissue from animals pretreated with either steroid by itself, endometrium from animals pretreated with estradiol-17 beta alone released the most PGF2alpha during sustained incubation in vitro. Moreover, only this tissue exhibited significant oxytocin-dependent release of PGF2alpha. At the dosages used, progesterone completely antagonized both of these effects of estradiol-17 beta. The results support the hypothesis that ovarian steroid hormones regulate oxytocin-dependent release of PGF2alpha from endometrial cells. A posible mechanism of action is suggested.
Asunto(s)
Endometrio/metabolismo , Estradiol/farmacología , Oxitocina/farmacología , Progesterona/farmacología , Prostaglandinas F/metabolismo , Animales , Medios de Cultivo , Femenino , Prostaglandinas F/biosíntesis , Conejos , Tasa de Secreción/efectos de los fármacosRESUMEN
Endometrial and myometrial tissues, obtained from Merino ewes on 5 different days of the estrous cycle, were incubated at 37 C in 30 ml of gassed (95% O2:5% CO2) Krebs-bicarbonate buffer containing, 0, 10, 100 or 1,000 muU/ml oxytocin. Aliquots of the medium were removed at 10 min intervals and examined for prostaglandin F2alpha (PGF2alpha) content by radioimmunoassay. Fresh-frozen (-70 C) samples of endometrial and myometrial tissue were homogenized in Tyrode's solution. Particulate fractions from each tissue, sedimenting between 1,000 X g for 10 min and 165,000 X g for 30 min, were prepared and assayed for [3H]oxytocin-binding activity. Endometrium incubated in vitro released PGF2alpha spontaneously and oxytocin enhanced this release in a dose-dependent manner. The degree of enhancement with low doses of oxytocin appeared to increase as estrus approached, reaching a maximum on the day of estrus. High-affinity binding sites (Kd = 5 to 7 X 10(-10) M) were found in both myometrium and endometrium. The number of high-affinity sites rose to a peak at estrus in both tissues but the binding capacity of endometrium was twice that of the myometrium at this time. Although both tissues released PGF2alpha during incubation, oxytocin enhanced release from endometrial tissue only. The results suggest that (i) the endometrium is a target for oxytocin, (ii) synthesis of PGF2alpha by the uterus may involve interaction between oxytocin and its endometrial receptors and (iii) ovarian steroids may influence uterine PG synthesis by regulating the availability of these receptors.