RESUMO
A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71 kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg(2+) as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease.
RESUMO
A thermophilic xylan-degrading Actinomadura sp. S14 was isolated from compost in Thailand. Hemicellulase activities such as endo-1,4-ß-xylanase, ß-xylosidase and α-arabinofuranosidase were induced with xylan-containing agriculture wastes and oat spelt xylan. The gene encoding xylanase consisting of 687bp was cloned from Actinomadura sp. S14. The deduced amino acid sequence contained a signal peptide of 41 amino acids and a probable mature xylanase of 188 amino acids. An open reading frame (xynS14) corresponding to a mature xylanase was expressed in Escherichia coli and Pichia pastoris. The specific activity of purified XynS14 (P. pastoris) was 2.4-fold higher than XynS14 (E. coli). Both XynS14s showed the same basic properties such as optimal pH and temperature (pH 6.0 and 80°C) and stability in a broad pH range (pH 5.0-11.0) and at high temperatures up to 80°C. Both XynS14s showed approximately the same substrate specificity and K(m) values toward various xylans, but XynS14 (P. pastoris) showed higher V(max) and K(cat) than XynS14 (E. coli). Higher specific activities of XynS14 (P. pastoris) may be due to protein-folding in the host. Purified XynS14 showed more endo-1,4-ß-xylanase activity on xylan and xylooligosaccharides than on xylotriose.
Assuntos
Actinomycetales/enzimologia , Actinomycetales/genética , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Endo-1,4-beta-Xilanases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade por Substrato , Temperatura , Tailândia , Trissacarídeos/metabolismo , Xilanos/metabolismoRESUMO
Sphingopyxis terrae and the Sphingopyxis macrogoltabida strains 103 and 203 are able to degrade polyethylene glycol (PEG). They possess the peg operon, which is responsible for the conversion of PEG to PEG-carboxylate-coenzyme A (CoA). The upstream (3.0 kb) and downstream (6.5 kb) regions of the operon in strain 103 were cloned and sequenced. The structure was well conserved between S. macrogoltabida strain 203 and S. terrae, except that two sets of transposases are absent in strain 203. The downstream region contains the genes for PEG-carboxylate dehydrogenase (PCDH), glutathione S-transferase (GST), tautomerase, and a hypothetical protein. The genes for pcdh and gst were transcribed constitutively and monocistronically, indicating that their transcription is independent of the operon regulation. PCDH and GST were expressed in Escherichia coli and characterized biochemically. PCDH is a homotetramer of 64-kDa subunits and contains one molecule of flavin adenine dinucleotide per subunit. The enzyme dehydrogenates PEG-carboxylate to yield glyoxylate, suggesting that the enzyme is the third enzyme involved in PEG degradation. GST is a homodimer of 28-kDa subunits. GST activity was noncompetitively inhibited by acyl-CoA and PEG-carboxylate-CoA, suggesting the interaction of GST with them. The proposed role for GST is to buffer the toxicity of PEG-carboxylate-CoA.
Assuntos
Oxirredutases do Álcool/metabolismo , Glutationa Transferase/metabolismo , Polietilenoglicóis/metabolismo , Sphingomonadaceae/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Glutationa Transferase/química , Glutationa Transferase/genética , Dados de Sequência Molecular , Óperon , Sphingomonadaceae/química , Sphingomonadaceae/genética , Sphingomonadaceae/metabolismo , Especificidade por SubstratoRESUMO
Sphingopyxis macrogoltabida strain 103 possesses polyethylene-glycol (PEG)-inducible pegBCDAE operon encoding the genes relevant to PEG degradation. PEG is converted to PEG-carboxylate by PegA (PEG dehydrogenase) and PegC (PEG-aldehyde dehydrogenase). In this study, the recombinant PegE (homologous to acyl-CoA synthetases) was characterized. PegE was an acyl-CoA synthetase active for PEG-carboxylate and fatty acids. Judging from the nature of this kind of protein (located on the cytoplasmic membrane as a translocator), PegE might be responsible for the translocation of PEG-carboxylate from the periplasm into the cytoplasm or for the detoxification of strong acidity of the substrate.
