scholarly journals Pectate lyase C from Bacillus subtilis: a novel endo-cleaving enzyme with activity on highly methylated pectin

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 617-625 ◽  
Author(s):  
Margarita Soriano ◽  
Pilar Diaz ◽  
Francisco I. Javier Pastor
Keyword(s):  
Bacillus Subtilis ◽  
Pectate Lyase ◽  
Maximum Activity ◽  
Methyl Esterification ◽  
Pectate Lyases ◽  
Action Analysis ◽  
Paenibacillus Barcinonensis ◽  
Esterification Degree ◽  
Micro Organisms ◽  
High Degree

The gene yvpA from Bacillus subtilis was cloned and expressed in Escherichia coli. It encoded a pectate lyase of 221 amino acids that was denominated PelC. The heterologously expressed enzyme was purified by His-tag affinity chromatography and characterized. PelC depolymerized polygalacturonate and pectins of methyl esterification degree from 22 % to 89 %, exhibiting maximum activity on 22 % esterified citrus pectin. It showed an absolute Ca2+ requirement and the optimum temperature and pH were 65 °C and pH 10, respectively. The deduced amino acid sequence of PelC showed 53 % identity to pectate lyase PelA from Paenibacillus barcinonensis, which was also characterized. Similarly to PelC, purified PelA showed activity on polygalacturonate and pectins with a high degree of methyl esterification. The two enzymes cleaved pectic polymers to a mixture of oligogalacturonates, indicating an endo mode of action. Analysis of activity on trigalacturonate showed that PelC cleaved it to galacturonic acid and unsaturated digalacturonate, whereas PelA did not show activity on this substrate. PelC and PelA showed high homology to a few recently identified pectate lyases of family 3 and form with them a cluster of small-sized pectate lyases from non-pathogenic micro-organisms.

scholarly journals The Exopolygalacturonate Lyase PelW and the Oligogalacturonate Lyase Ogl, Two Cytoplasmic Enzymes of Pectin Catabolism in Erwinia chrysanthemi 3937

1999 ◽  
Vol 181 (13) ◽  
pp. 3912-3919 ◽  
Author(s):  
Vladimir E. Shevchik ◽  
Guy Condemine ◽  
Janine Robert-Baudouy ◽  
Nicole Hugouvieux-Cotte-Pattat
Keyword(s):  
Yersinia Pseudotuberculosis ◽  
Divalent Cations ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Erwinia Chrysanthemi ◽  
Pectate Lyases ◽  
Acid Protein ◽  
Lyase Activity ◽  
Bacterial Cytoplasm ◽  
High Degree

ABSTRACT Erwinia chrysanthemi 3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of E. chrysanthemi. The E. chrysanthemi 3937 gene kdgC has been shown to exhibit a high degree of similarity to the genes pelY ofYersinia pseudotuberculosis and pelB ofErwinia carotovora, which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the kdgC gene was renamed pelW. PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite −2 to +1, while it is probably two for Ogl, extending from subsite −1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca2+ is unable to restore the activity of PelW or Ogl, while several other cations, including Co2+, Mn2+, and Ni2+, can activate both cytoplasmic lyases.

scholarly journals Modeling the bioconversion of polysaccharides in a continuous reactor: A case study of the production of oligogalacturonates by Dickeya dadantii

2018 ◽  
Vol 294 (5) ◽  
pp. 1753-1762 ◽  
Author(s):  
Jacques-Alexandre Sepulchre ◽  
Sylvie Reverchon ◽  
Jean-Luc Gouzé ◽  
William Nasser
Keyword(s):  
A Priori ◽  
Pectate Lyase ◽  
Degradation Process ◽  
Continuous Reactor ◽  
Practical Implementation ◽  
Dickeya Dadantii ◽  
Pectate Lyases ◽  
Upper Limit ◽  
Promising Avenue

In the quest for a sustainable economy of the Earth's resources and for renewable sources of energy, a promising avenue is to exploit the vast quantity of polysaccharide molecules contained in green wastes. To that end, the decomposition of pectin appears to be an interesting target because this polymeric carbohydrate is abundant in many fruit pulps and soft vegetables. To quantitatively study this degradation process, here we designed a bioreactor that is continuously fed with de-esterified pectin (PGA). Thanks to the pectate lyases produced by bacteria cultivated in the vessel, the PGA is depolymerized into oligogalacturonates (UGA), which are continuously extracted from the tank. A mathematical model of our system predicted that the conversion efficiency of PGA into UGA increases in a range of coefficients of dilution until reaching an upper limit where the fraction of UGA that is extracted from the bioreactor is maximized. Results from experiments with a continuous reactor hosting a strain of the plant pathogenic bacterium Dickeya dadantii and in which the dilution coefficients were varied quantitatively validated the predictions of our model. A further theoretical analysis of the system enabled an a priori comparison of the efficiency of eight other pectate lyase–producing microorganisms with that of D. dadantii. Our findings suggest that D. dadantii is the most efficient microorganism and therefore the best candidate for a practical implementation of our scheme for the bioproduction of UGA from PGA.

Bioconvective dynamics: dependence on organism behaviour

2000 ◽  
Vol 203 (21) ◽  
pp. 3345-3354 ◽  
Author(s):  
A. Czirok ◽  
I.M. Janosi ◽  
J.O. Kessler
Keyword(s):  
Bacillus Subtilis ◽  
Cell Density ◽  
Microbial Populations ◽  
Dominant Wavelength ◽  
Nonlinear Convection ◽  
Convection Regime ◽  
Term Evolution ◽  
Convection Patterns ◽  
Micro Organisms

Bioconvection occurs when a macroscopic nonuniformity of the concentration of microbial populations is generated and maintained by the directional swimming of the organisms. This study investigated the properties of the patterns near the onset of the instability and later during its evolution into a fully nonlinear convection regime. In suspensions of the bacteria Bacillus subtilis, which tend to swim upwards in a gradient of oxygen concentration that they create by consumption, we discovered that the dominant wavelength at the onset of the instability is determined primarily by the cell density and is influenced only weakly by the fluid depth. This observation contrasts strongly with previous observations on the gravitactic alga Chlamydomonas nivalis, in which the opposite dependence was found. Considerable differences were also found in the long-term evolution of the convection patterns. These results demonstrate the existence of readily distinguishable types of bioconvection systems, even at early stages of the instability. The observed differences are clearly and causally correlated with disparate reasons for upward swimming by these micro-organisms, leading to different geometric distributions of the density of the suspension.

scholarly journals Periplasmic depolymerase provides insight into ABC transporter-dependent secretion of bacterial capsular polysaccharides

2018 ◽  
Vol 115 (21) ◽  
pp. E4870-E4879 ◽  
Author(s):  
Sean D. Liston ◽  
Stephen A. McMahon ◽  
Audrey Le Bas ◽  
Michael D. L. Suits ◽  
James H. Naismith ◽  
...  
Keyword(s):  
Cell Surface ◽  
Abc Transporter ◽  
Pectate Lyase ◽  
Capsular Polysaccharides ◽  
Ph Optimum ◽  
Pectate Lyases ◽  
Surface Assembly ◽  
Binding Groove ◽  
Biochemical Analyses ◽  
Vi Antigen

Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogenSalmonella entericaserovar Typhi produces “Vi antigen” CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of theBurkholderiales, which are paradoxically distinguished fromS.Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel β-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced intoS. Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.

Rapid detection of microbial contamination in grape juice by flow cytometry

OENO One ◽  
1999 ◽  
Vol 33 (1) ◽  
pp. 31 ◽  
Author(s):  
Marielle Bouix ◽  
Agnès Grabowski ◽  
Monique Charpentier ◽  
Jean-Yves Leveau ◽  
Bruno Duteurtre
Keyword(s):  
Flow Cytometry ◽  
Reference Method ◽  
Grape Juice ◽  
Plate Count ◽  
Contamination Level ◽  
Cell Counts ◽  
Direct Cell ◽  
Micro Organisms ◽  
High Degree

<p style="text-align: justify;">This study presents an application of flow cytometry to evaluate rapidly the viable micro-organisms in grape juice. In this method, viable cells are firstly specitically labelled with a fluorescent reagent. The sample is then injected into the flow cytometer where the labelled micro-organisms are individually illuminated by a laser beam. The emission of fluorescence is measured. The system counts the number of fluorescent events and prints out a histogram of the fluorescence intensity which is characteristic of the micro-organism being analysed. In laboratory conditions, preliminary trials have been undertaken with an artificially inoculated grape juice with pure yeast and bacteria cultures. This method succeeded in counting simultaneously yeasts and bacteria within 15 minutes, with a high degree of sensitivity, 5.10<sup>3</sup> yeasts perml and 5.10<sup>4</sup> bacteria per ml. This technique can also be applied to the detection of mould contamination and the test has been done with <em>Botrytis</em> spores. The method makes direct cell counts possible and is capable of analysing 30 samples per hour. It can be automatised and easily used in industrial laboratory. During the last harvest, more than a thousand of must samples were controled using this technique. The results let to determine the yeast contamination level of a grape juice tank even before unloading. The results obtained by flow cytometry were compared to the plate count reference method. The correlation between cytometry and count by plate culture was 99 p. cent for the threshold of 5.1 0<sup>4</sup> yeasts/ml which seemed to point out a high contamination. By using this flow cytometry method during the harvest period, the results were supplied in real time. This allowed a rapid selection of the musts, depending upon the scale of their contamination and improved the quality of the wine by corrective actions.</p>

Pénétration d'une endo-pectate lyase tritiée dans les cellules corticales de racines de courge

1984 ◽  
Vol 62 (8) ◽  
pp. 1621-1628
Author(s):  
G. F. Vogt ◽  
J. Coulon
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
External Surface ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Cortical Cells ◽  
Pectate Lyases ◽  
Autoradiographic Analysis ◽  
Host Tissues

Erwinia carotovora produces pectate lyases (endo-PGTE) on sterilized beans hypocotyls. Two endo-PGTE fractions were isolated and purified by electrofocusing. The action of these enzymes on the ultrastructure of cortical cells of pumpkin roots was very similar to the action of the whole bacterium. Erwinia carotovora grown on tritiated amino acid supplemented medium produced [3H]endo-PGTE. By incubating the host tissues with 3H-labelled enzymes and by subsequent autoradiographic analysis it was possible to localize the endo-PGTE inside the cells. Thus, it was shown that the enzyme (the complete molecule or only a peptide part thereof) was transported towards the vacuole. It is suggested that the endo-PGTE acts on the filamentous polysaccharide extensions which bind the external surface of the plasmalemma to the cell wall.

scholarly journals Cloning and Characterization of an Esophageal-Gland-Specific Pectate Lyase from the Root-Knot Nematode Meloidogyne javanica

2002 ◽  
Vol 15 (6) ◽  
pp. 549-556 ◽  
Author(s):  
Elizabeth A. Doyle ◽  
Kris N. Lambert
Keyword(s):  
Plant Root ◽  
Middle Lamella ◽  
Pectate Lyase ◽  
Meloidogyne Javanica ◽  
Basal Cells ◽  
Class Iii ◽  
Root Knot Nematode ◽  
Ph Optimum ◽  
Pectate Lyases ◽  
Esophageal Gland

Root-knot nematodes (Meloidogyne javanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle. Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases. Using differential screening, a class III pectate lyase, Mj-pel-1 (M. javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes. DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family. In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted. When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active. The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin. Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+. The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.

Sign in / Sign up

Export Citation Format

Share Document