RESUMO
The coding region of the licB gene from Clostridium thermocellum was truncated at the 3' end. The modified lichenase encoded by the construct (LicBM2) retained the most important properties of the enzyme - its high activity and thermostability. LicBM2 consists of the catalytic domain and part of the Pro-Thr-box. We demonstrated the application of the licBM2 gene as a reporter system for prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells by expressing it either as a transcriptional fusion with selected promoters or as a translational fusion with the E. coli uidA gene. The assays available for LicB activity are sensitive, accurate and simple, and can be used for the analysis of various gene fusion systems or for screening of transformants.
Assuntos
Clostridium/enzimologia , Clostridium/genética , Genes Reporter , Glicosídeo Hidrolases/genética , Animais , Fusão Gênica Artificial , Sequência de Bases , DNA Bacteriano/genética , Estabilidade Enzimática , Escherichia coli/genética , Células Eucarióticas , Expressão Gênica , Genes Bacterianos , Vetores Genéticos , Células PC12 , Células Procarióticas , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , TemperaturaRESUMO
The main properties (pH and temperature range, stability, substrate specificity) of the modified cellulase CelE (endo-beta-1,4-glucanase) from Clostridium thermocellum have been analyzed with the goal of its expression in plants. The modified enzyme is similar to plant cellulases. Deletions in the N-terminus of the enzyme do not affect its biochemical properties. Based on the present investigation, we conclude that the modified beta-1,4-glucanase CelEM1, when expressed in plants, will be a good model to study the role of cellulases in plants.
Assuntos
Carboximetilcelulose Sódica/metabolismo , Celulase/genética , Celulase/metabolismo , Clostridium/enzimologia , Glucanos/metabolismo , Deleção de Sequência/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulase/química , Clostridium/genética , Simulação por Computador , Ativação Enzimática , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Modelos Genéticos , Mutação/genética , Plantas/enzimologia , Plantas/genéticaRESUMO
Major properties (pH and temperature optimum, stability) of lichenase (beta-1,3-1,4-glucanase) deletion variants from Clostridium thermocellum were comparatively studied. The deletion variant LicBM2 was used to create hybrid bifunctional proteins by fusion with sequences of the green fluorescent protein (GFP) from Aequorea victoria. The data show that in hybrid proteins both GFP and lichenase retain their major properties, namely, GFP remains a fluorescent protein and the lichenase retains activity and high thermostability. Based on the results of this investigation and results that have been obtained earlier, the use of the deletion variants of lichenase and the bifunctional hybrid proteins as reporter proteins is suggested.