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Query: EC:4.2.2.10 (PNL)
 341 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonas marginalis N6301 was cloned and expressed in Escherichia coli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2. 1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5 alpha. A plasmid thus obtained was designated as pPNL6301. E. coli DH5 alpha harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pn1 gene in the plasmid pPNL6301 encoding PNL from P. marginalis N6301 was determined. The structural gene of pn1 consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P. marginalis N6301 determined. The nucleotide sequence of the 5'-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichia coli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pn1 gene between P. marginalis N6301 and E. carotovora Er were 60.8% and 57.2%, respectively.
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PMID:Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonas marginalis N6301. 173 76

A pectin lyase (PNL; EC 4.2.2.10) was isolated from culture filtrates of Pseudomonas fluorescens W51 and purified to apparent homogeneity. The enzyme catalyzed a random eliminative cleavage of pectin but not sodium polypectate, and it macerated plant tissue. The Mr of the PNL on sodium dodecyl sulfate-polyacrylamide gels was 32,000 +/- 1,000, and the isoelectric point was 9.4 as determined by isoelectric focusing. The enzyme was constitutively produced, since the highest yields were obtained when glycerol was used as a sole carbon source, and addition of pectin to the medium did not increase its production. Antibodies against purified PNL reacted in Western blots (immunoblots) with a pectate lyase (PLb) produced by Erwinia chrysanthemi EC16. The PNL appeared to be the only factor secreted into the culture medium by P. fluorescens W51 which macerated plant tissue and is probably involved in the soft rot disease caused by the bacterium.
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PMID:Purification and characterization of a pectin lyase produced by Pseudomonas fluorescens W51. 311 58

Pectate lyase was purified approximately 29-fold to electrophoretic homogeneity from Pseudomonas marginalis N6301. A pectate lyase (PL; EC4.2.2.2) gene of the strain was cloned and expressed in Escherichia coli. The nucleotides of the PL gene (pel) were sequenced. An open reading frame that encodes a polypeptide (molecular weight: 40,812) composed of 380 amino acids including a 29 amino acid signal peptide was assigned. The structural gene of pel consisted of 1140 base pairs. The nucleotide sequence of the 5'-flanking region of pel showed a consensus sequence of the promoter region of the pectin lyase gene (pnl) in P. marginalis N6301, a Pribnow box, and a ribosome binding site as found in E. coli.
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PMID:Molecular cloning and nucleotide sequence of the pectate lyase gene from Pseudomonas marginalis N6301. 776 84

We constructed deletion mutant clones of a pectin lyase gene, and measured their pectin lyase activities in Escherichia coli. Pectin lyase activities were detected only in a recA+ strain but not in a recA- strain of E. coli. We also cloned and sequenced recA from Pseudomonas marginalis N6301. The recA from P. marginalis N6301 can complement recA- to form recA+ in the phenotype of E. coli. Highly conserved sequences of recA are observed among E. coli, P. fluorescens, and P. marginalis. From these results, we presume that recA is required for the expression of the pectin lyase gene in P. marginalis N6301.
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PMID:Expression of a pectin lyase gene in Escherichia coli from Pseudomonas marginalis N6301. 776 26

Two pectinolytic enzymes were purified from the culture broth of Pseudomonas marginalis pv. marginalis MAFF 03-01173 with total 33% recovery of the initial activity. From the substrate specificities against pectin and polygalacturonic acid, the requirement of calcium ion for the enzymatic activity, and the N-terminal sequences, the enzymes were identified as pectin lyase and pectate lyase. The M,s of pectin lyase and pectate lyase were estimated to be 34,000 and 43,000, respectively, by SDS polyacrylamide gel electrophoresis. Both enzymes showed almost the same pH dependent activity curves with the highest activity at pH 8.3
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PMID:Pectinolytic enzymes from Pseudomonas marginalis MAFF 03-01173. 923 99

The authors have characterized a chromosomally localized two-gene operon, cueAR, which encodes a putative P1-type ATPase, CueA, and a MerR-type metalloregulatory protein, CueR, in Pseudomonas putida PNL-MK25. Disruption of cueAR by the insertion of mini-Tn5::gfp into the wild-type strain led to a mutant strain with a sixfold reduction in its tolerance to copper; however, the tolerance of this mutant strain to the other seven related transition metals tested was not affected. The sensitivity of the mutant strain was attributed to a higher level of accumulation of intracellular copper, suggesting the involvement of CueA in copper export. Insertion of the cloned cueAR operon into the copper-sensitive mutant strain fully restored its tolerance to copper. cueA::gfp expression studies confirmed that the cueAR operon was transcriptionally regulated by copper and CueR. Studies done on the mutant strain complemented with cueR and cueA revealed partial functional redundancy of cueA and cueR, respectively, in copper tolerance. Thus, the results of this study clearly suggest the involvement of cueAR in copper homeostasis in P. putida.
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PMID:Molecular characterization of an operon, cueAR, encoding a putative P1-type ATPase and a MerR-type regulatory protein involved in copper homeostasis in Pseudomonas putida. 1221 31

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.
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PMID:Inhibition of hydrolytic enzyme activities and plant pathogen growth by invertase inhibitors. 1236 59

A pectin lyase gene (pnl) of Pseudomonas marginalis was cloned and overexpressed in Escherichia coli BL21(DE3). The pnl gene was amplified by PCR, inserted into pET29c with a six-His tag and the overproduced active enzyme was purified almost to homogeneity using a Ni(2+)-nitrilotriacetate-agarose column. The purified pectin lyase (PNL; EC 4.2.2.10, family 1) is inhibited by NAD+ (at concentrations above 0.25 mM), NADH or dithiothreitol. Evidence for the existence of a heat-labile protein inhibitor of PNL is also reported. The DNA-binding ability of PNL was demonstrated by DNA-retardation experiments. The partially purified enzyme was incubated with plasmid DNA and the complex was shifted to a higher molecular mass. Analysis of the electroeluted proteins from the protein-DNA complex revealed that one of the electroeluted protein bands was PNL. Antibodies against the overexpressed PNL were also prepared and partially purified.
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PMID:Overexpression of the pectin lyase gene of Pseudomonas marginalis in Escherichia coli and purification of the active enzyme. 1263 Sep 8

Tissue maceration was generally elucidated by the action of endo-polygalacturonase and endo-pectate or -pectin lyase (endo-PAL or -PNL). In a process of screening of Erwinia and Pseudomonas strains for enzymatic pulping of pectocellulosic bast fibers, it was found that their PAL productivity was not completely related with defibration activity, i.e., the fact that an E.chrysanthemi strain showed high PAL productivity but possessed rather low defibration activity. Moreover, defibration activity was parallel to the amount of neutral sugars released during pulping. Based on these fact, the maceration or enzymatic pulping of basts was estimated to proceed not only by cleavage of interfiber bonding cause by PAL action but also another factors. Among three possibilities proposed on the maceration mechanism of basts, it was elucidated by a concerted action of PAL and PNL with an aid of xylanase. In addition, a quantitative determinative method of maceration activity toward basts was also presented.
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PMID:Approach to maceration mechanism in enzymatic pulping of bast fibers by alkalophilic pectinolytic enzymes produced by Erwinia species. 1454 40

The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation and biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high availability of nutrients and oxygen. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 were examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7 and NRM17, were selected for identification of the tagged genes. In strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased 4.9-26.4-fold under various low-nutrient conditions. In NRM7, expression of the novel NADPH : quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low-nutrient and anoxic conditions. The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low-nutrient conditions but its absolute expression level was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that in Escherichia coli.
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PMID:Characterization of Pseudomonas putida genes responsive to nutrient limitation. 1518 52


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