RESUMO
Flavoenzymes are colourful oxidoreductases that catalyze a large variety of different types of reactions. Flavoenzymes have been extensively studied for their structural and mechanistic properties and are gaining momentum in industrial biocatalytic applications. Some of these enzymes catalyze the oxidative modification of protein substrates. New insights in oxidative flavoenzymes and in particular in novel family members point towards their potential application in the pharmaceutical, fine-chemical and food industries.
Assuntos
Enzimas/metabolismo , Flavinas/metabolismo , Flavoproteínas/metabolismo , Oxirredutases/metabolismo , Enzimas/química , Flavinas/química , Flavoproteínas/química , Oxirredutases/químicaRESUMO
The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V(HH) R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mgl(-1) fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V(HH) R9 (R9-ARP) is the preferred orientation.
Assuntos
Anticorpos Monoclonais/biossíntese , Aspergillus , Proteínas Fúngicas/biossíntese , Fungos não Classificados/enzimologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Peroxidase/biossíntese , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Camelídeos Americanos/genética , Camelídeos Americanos/imunologia , Proteínas Fúngicas/genética , Fungos não Classificados/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Peroxidase/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
We report the expression and production of llama variable heavy-chain antibody fragments (V(HH)s) by Aspergillus awamori. Fragments encoding V(HH)s were cloned in a suitable Aspergillus expression vector and transformants secreting V(HH) fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V(HH)s were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted V(HH)s were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.
Assuntos
Aspergillus/genética , Camelídeos Americanos/genética , Camelídeos Americanos/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Transformação GenéticaRESUMO
The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l(-1) in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP.
Assuntos
Aspergillus/enzimologia , Biotecnologia/métodos , Fungos Mitospóricos/enzimologia , Peroxidase/genética , Aspergillus/genética , Aspergillus/crescimento & desenvolvimento , Northern Blotting , Southern Blotting , Fermentação , Heme/metabolismo , Fungos Mitospóricos/genética , Peroxidase/metabolismo , Plasmídeos , Regiões Promotoras GenéticasRESUMO
In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted.
RESUMO
The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D-galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L-arabinose and D-galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding beta-galactosidase and the L-arabinose releasing enzymes were not only expressed in the presence of D-galacturonic acid, but also in the presence of L-arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D-galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L-rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D-galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L-arabinose, L-rhamnose or ferulic acid.
