RESUMO
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.
Assuntos
Aminoidrolases/isolamento & purificação , Aminoidrolases/metabolismo , Betaproteobacteria/enzimologia , Clonagem Molecular , Escherichia coli/enzimologia , Análise de Sequência de DNA , Sequência de Aminoácidos , Aminoidrolases/química , Aminoidrolases/genética , Sequência de Bases , Betaproteobacteria/genética , Escherichia coli/genética , Dados de Sequência Molecular , Estereoisomerismo , Especificidade por SubstratoRESUMO
The literature is surveyed from mid-1998 through to August 2000 for examples of the resolution or desymmetrization of existing/potential pharmaceutical entities and pharmaceutical intermediates, and of chiral building blocks for pharmaceutical manufacture. Examples of commercial interest are included with new developments in the art of resolution.
RESUMO
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).
Assuntos
Amidas/química , Biotecnologia/métodos , Nitrilas/química , Pseudomonas/metabolismo , Amidas/metabolismo , Estabilidade Enzimática , Hidroliases/química , Hidroliases/metabolismo , Nitrilas/metabolismoRESUMO
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.
Assuntos
Oxirredutases do Álcool/biossíntese , Catalase/biossíntese , Proteínas Fúngicas/biossíntese , Pichia/genética , Proteínas de Plantas/biossíntese , Oxirredutases do Álcool/genética , Catalase/genética , Catálise , Clonagem Molecular/métodos , Ativação Enzimática , Proteínas Fúngicas/genética , Engenharia Genética , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Spinacia oleracea , Transformação GenéticaRESUMO
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.
Assuntos
Oxirredutases do Álcool/genética , Catalase/genética , Expressão Gênica , Pichia/genética , Proteínas Recombinantes/biossíntese , Oxirredutases do Álcool/metabolismo , Catalase/metabolismo , Catálise , Citosol/enzimologia , Fermentação , Glicolatos/metabolismo , Glioxilatos/metabolismo , Microbiologia Industrial , Microcorpos/enzimologia , Pichia/enzimologia , Saccharomyces cerevisiae/enzimologia , Spinacia oleracea/enzimologiaRESUMO
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.
RESUMO
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.
