RESUMO
The nitrile hydratase (NHase, EC 4.2.1.84) genes (alpha and beta subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (alpha subunit, M(r) 23 kDa) and 218 amino acids (beta subunit, M(r) 24 kDa) and the NHase activator of 413 amino acids (M(r) 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5-17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30 degrees C and showed the highest stability at 4 degrees C in thermal inactivation studies between 4 degrees C and 50 degrees C.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Hidroliases/genética , Hidroliases/metabolismo , Rhodococcus equi/enzimologia , Rhodococcus equi/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Genes Bacterianos , Hidroliases/isolamento & purificação , Concentração de Íons de Hidrogênio , Metacrilatos/metabolismo , Nitrilas/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Espectrofotometria Ultravioleta , Estereoisomerismo , Especificidade por Substrato , TemperaturaRESUMO
Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H(2)O/25% NH(3). Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.
Assuntos
Clorofila/biossíntese , Oxigenases/metabolismo , Protoporfirinas/isolamento & purificação , Protoporfirinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Liofilização , Hordeum/enzimologia , Mutação , Oxigenases/genética , Protoclorifilida/metabolismo , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.
Assuntos
DNA/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Deleção de Sequência/genéticaRESUMO
Chlorophyll biosynthesis is a process involving approximately 20 different enzymatic steps. Half of these steps are common to the biosynthesis of other tetrapyrroles, such as heme. One of the least understood enzymatic steps is formation of the isocyclic ring, which is a characteristic feature of all (bacterio)chlorophyll molecules. In chloroplasts, formation of the isocyclic ring is an aerobic reaction catalyzed by Mg-protoporphyrin IX monomethyl ester cyclase. An in vitro assay for the aerobic cyclase reaction required membrane-bound and soluble components from the chloroplasts. Extracts from barley (Hordeum vulgare L.) mutants at the Xantha-l and Viridis-k loci showed no cyclase activity. Fractionation of isolated plastids by Percoll gradient centrifugation showed that xantha-l and viridis-k mutants are defective in components associated with chloroplast membranes. The Xantha-l gene, corresponding to Arabidopsis thaliana CHL27, Rubrivivax gelatinosus acsF, Chlamydomonas reinhardtii CRD1, and CTH1 and situated at the short arm of barley chromosome 3 (3H), was cloned, and the mutations in xantha-l(35), xantha-l(81), and xantha-l(82) were characterized. This finding connected biochemical and genetic data because it demonstrated that Xantha-l encodes a membrane-bound cyclase subunit. The evidence suggests that the aerobic cyclase requires at least one soluble and two membrane-bound components.