Pectate Lyase Activity Research Articles

Thermodynamic characterization of a highly thermoactive extracellular pectate lyase from a new isolate Bacillus pumilus DKS1

An extracellular pectate lyase (EC 4.2.2.2) was purified from the culture filtrate of a newly isolated Bacillus pumilus DKS1 grown in pectin containing medium. Using ion-exchange and gel filtration chromatography, this enzyme was purified and found to have a molecular weight of around 35 kDa. The purified enzyme exhibited maximal activity at a temperature of 75 °C and pH 8.5. The presence of 1 mM calcium and manganese enhanced pectate lyase activity and was strongly inhibited by zinc, nickel and EDTA. The thermal inactivation studies revealed an entropy–enthalpy compensation pattern below a critical temperature. The alkaliphilicity and high thermostability of this pectate lyase may have potential implications in fibre degumming.

Influence of gallic and tannic acids on enzymatic activity and growth of Pectobacterium chrysanthemi (Dickeya chrysanthemi bv. chrysanthemi)

The effect of phenolic acids (gallic and tannic acids) on growth of Pectobacterium chrysanhemi, and its protease and pectate lyase activities was tested. The results obtained showed a significant inhibiting effect of the tannic and gallic acids on the growth of this strain. The growth rate decreases in the presence of 400
g/ml for gallic acid and 100 ig/ml fortannic acid. The enzymatic activity retardation was observed with the two phenolic compounds as well but the rate of inhibition varied from one compound to another. The highest antimicrobial potentials and the highest effect on enzymatic activities were observed with the tannic acid at 200
g/ml, which inhibited 91% of the tested microorganisms, and 88% of pectate lyase activity.

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg 2+ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.

RelA-dependent (p)ppGpp production controls exoenzyme synthesis in Erwinia carotovora subsp. atroseptica.

In this report, we investigate the link between nutrient limitation, RelA-mediated (p)ppGpp production, and virulence in the phytopathogen Erwinia carotovora subsp. atroseptica. A relA null mutant (JWC7) was constructed by allelic exchange, and we confirmed that, unlike the wild-type progenitor, this mutant did not produce elevated levels of (p)ppGpp upon nutrient downshift. However, (p)ppGpp production could be restored in strain JWC7 during nutrient limitation by supplying relA in trans. During growth on exoenzyme-inducing minimal medium, the relA mutant showed a diminution in secreted pectate lyase and protease activities and a severe defect in motility. The relA mutant was also impaired in its ability to cause rot in potato tubers. In the presence of serine hydroxamate (a competitive inhibitor of seryl tRNA synthase and a potent inducer of the stringent response in wild-type E. carotovora subsp. atroseptica), exoenzyme production was essentially abolished in JWC7 but could be restored in the presence of plasmid-borne relA. The inhibition of exoenzyme production in JWC7 caused by serine hydroxamate could not be overcome by addition of the quorum-sensing signal molecule, N-3-oxohexanoyl-l-homoserine lactone. Quantitative reverse transcription-PCR analysis of selected RNA species confirmed that the effects of relA on secreted pectate lyase activity and motility could be attributed to a reduction in transcription of the corresponding genes. We conclude that nutrient limitation is a potent environmental cue that triggers (p)ppGpp-dependent exoenzyme production in E. carotovora subsp. atroseptica. Furthermore, our data suggest that nutrient limitation [or rather, (p)ppGpp accumulation] is a prerequisite for effective quorum-sensing-dependent activation of exoenzyme production.

Modulation of mango ripening by chemicals: physiological and biochemical aspects

During ripening, fleshy fruits undergo textural changes that lead to loss of tissue firmness and consequent softening due to cell wall dismantling carried out by different and specifically expressed enzymes. The effect of various chemical treatments on the ripening of mango fruit (Mangifera indica) was investigated at physiological and biochemical level. Based on changes in respiration, firmness, pH, total soluble sugar and a cell wall degrading enzyme pectate lyase (PEL) activity, treatment with 1-methylcyclopropene (1-MCP), silver nitrate (AgNO3), gibberlic acid (GA3), sodium metabisulphite (SMS) and ascorbic acid led to delaying of ripening process while those of ethrel and calcium chloride (CaCl2) enhanced the process. PEL of mango was found to be inhibited by certain metabolites present in dialysed ammonium sulphate enzyme extract as well as EDTA. Mango PEL activity exhibited an absolute requirement for Ca2+ and an optimum pH of 8.5.

Arabidopsis thaliana Expresses Multiple Lines of Defense to Counterattack Erwinia chrysanthemi

Many taxonomically diverse plant species are attacked by Erwinia chrysanthemi, a member of the causal agents of soft-rotting diseases. Symptom development is due to the collective action of pectin-degrading enzymes secreted by the bacterium through a type II secretion system (T2SS). Using Arabidopsis thaliana as a susceptible host, we show that plants respond to E. chrysanthemi 3937 by expressing cell-wall reactions, production of an oxidative burst, and activation of salicylic acid (SA) and jasmonic acid (JA) or ethylene (ET) signaling pathways. We found that the oxidative burst is mainly generated via the expression of the AtrbohD gene, constitutes a barrier of resistance to bacterial attack, and acts independently of the SA-mediated response. To determine the importance of T2SS-secreted proteins in elicitation of these defenses, we used a T2SS deficient mutant and purified enzymatic preparations of representative members of strain 3937 pectate lyase activity. The T2SS-secreted proteins were responsible only partially for the activation of SA and JA or ET signaling pathways observed after infection with the wild-type bacterium and were not involved in the expression of other identified defense reactions. Our study shows the differential role played by pectate lyases isoenzymes in this process and highlights the complexity of the host immune network, which is finely controlled by the bacterium.

Structural and functional characterization of a novel, host penetration‐related pectate lyase from the potato cyst nematode Globodera rostochiensis

SUMMARY The cell wall, a strong extraprotoplasmic layer surrounding plant cells that mainly consists of a variety of polysaccharides, constitutes a major barrier for potential parasites. Plant-parasitic nematodes are well equipped to overcome this barrier as they produce and secrete cell-wall-degrading enzymes. Expression profiling of various life stages of the potato cyst nematode Globodera rostochiensis revealed a novel pectate lyase gene (Gr-pel2, 759 bp). The Gr-PEL2 protein showed highest similarity to pectate lyases from the facultative plant-parasitic nematodes Bursaphelenchus mucronatus and B. xylophilus and the soil-inhabiting saprophytic Streptomyces and Frankia species (i.e. 40-42% identity and 58-60% similarity), whereas only a remote relatedness to the previously identified Gr-PEL1 was observed (i.e. 28% identity and 43% similarity). Transient expression of Gr-pel2 in leaves of Nicotiana benthamiana resulted in severe malformations of the infiltrated tissues, not relating to maceration and soft rot symptoms. Ca(2+) is known to be essential for pectate lyase activity, and the most likely calcium-binding site was identified in the Gr-PEL2 protein by combining homology modelling of the three-dimensional structure, site-directed mutagenesis and transient expression in leaves. A highly charged cleft in Gr-PEL2, which is likely to be involved in substrate binding and which is also significantly more hydrophobic in Gr-PEL1, was shown to be essential for protein activity. Our results underline the broad spectrum of pectate lyases and cell-wall-degrading enzymes necessary for successful parasitism by cyst nematodes.

Effects of relative humidity on banana fruit drop

Commercial ripening of banana fruit occurs at high relative humidity (RH), which prevents browning of damaged skin areas. In experiments with ripening at high RH (94 ± 1%) the individual fruit (fingers) of ‘Sucrier’ ( Musa acuminata, AA Group) banana exhibited a high rate of drop. The falling off of the fingers is due to rupture of the peel at the pedicel. In contrast, if the fruit was held at low RH (68 ± 3%) finger drop was absent. Water-soluble pectin in the peel at the rupture area was higher at high RH, indicating increased pectin degradation. However, the activities of polygalacturonase (PG) in the peel at the rupture zone were the same in both treatments. The activity of pectinmethylesterase (PME) was only slightly higher prior to rupture in the high RH treatment, whereas, that of pectate lyase (PL) was considerably higher. The lower rate of pectin degradation at low RH may explain, at least partially, why finger drop is inhibited. The decrease in pectin degradation was not accounted for by the measured PG activity, but could be partially accounted for by the measured PME and PL activities.

The Role of Pectate Lyase and the Jasmonic Acid Defense Response in Pseudomonas viridiflava Virulence

Pseudomonas viridiflava is a common pathogen of Arabidopsis thaliana in wild populations, yet very little is known about mechanisms of resistance and virulence in this interaction. We examined the induced defense response of A. thaliana to several strains of P. viridiflava collected from this host by quantifying the expression of PR-1 and LOX2/PDF1.2, which serve as markers for induction of the salicylic and jasmonic acid (JA) pathways, respectively. Growth of these strains then was assessed on Col-0, the fad3/7/8 and coil-1 mutants deficient in JA- and ethylene (ET)-induced defense responses, and the sid2-1 mutant deficient in salicylic acid-induced defense responses. All strains of P. viridiflava induced high expression of LOX2 and PDF1.2 on Col-0. In contrast, PR-1 expression was delayed and reduced relative to PDF1.2 expression. Additionally, three of four P. viridiflava strains were more virulent on fad3/7/8 relative to Col-0, whereas all strains were more virulent on coil-1 relative to Col-0, indicating that P. viridiflava generally may be suppressed by JA/ET-mediated defense responses. In contrast, no increase in the growth of P. viridiflava strains was observed in the sid2-1 mutant relative to Col-0. Parallel experiments were performed with the closely related P. syringae pv. tomato for comparative purposes. In addition, we assessed the role of pectate lyase and the alternative sigma factor HrpL in P. viridiflava virulence on A. thaliana and found that pectate lyase activity is correlated with virulence, whereas the removal of pectate lyase or HrpL significantly reduced virulence.

Patterns of enzymatic activity of cell wall-modifying enzymes during growth and ripening of apples

Fruit softening is thought to result from extensive cell wall modifications that occur during ripening. These modifications are the result, at least in part, of the activity of members of cell wall-modifying enzymes from the same families involved in the cell wall loosening which promote tissue extension and growth. In this work, the activities of a set of pectolytic and non-pectolytic cell wall-modifying enzymes, namely polygalacturonase (PG; endo-and exo-acting), pectin methylesterase (PME), pectate lyase (PL), β-galactosidase (β-Gal), α- l-arabinofuranosidase (AFase), endo-1,4-β-glucanase (EGase), xyloglucan endotransglycosylase (XET) and expansin, were monitored during growth and ripening of ‘Mondial Gala’ apple ( Malus × domestica Borkh.) fruit. After optimisation of protein extraction protocols and standard activity assays, activity could be detected in all the assays, except for endo-PG. The overall results suggest that fruit growth and ripening are possibly coordinated by members of the same families of cell wall-modifying enzymes, although different isoforms may be involved in distinct developmental processes. Based on the trend of total activity measured in vitro using equal amounts of protein per developmental stage, the role of EGase seems to be more prominent during growth than during ripening, and XET activity is most important only after the fruit stopped growing and is maintained throughout ripening. β-Gal and AFase activities increased after harvest as the fruit became over-ripe. On the other hand, exo-PG, PL and expansin activities increase from that in unripe fruit to fruit at harvest but are maintained at similar levels thereafter, throughout the over-ripe stages. The patterns of activity observed are discussed in relation to published information about ripening of apples and to results reported using other species.

Expression, purification and characterization of pectate lyase A from Aspergillus nidulans in Escherichia coli

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni2+-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml−1 medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn2+, Ca2+, Fe2+, Mg2+ and Fe3+ ions stimulated the pectate lyase activity, but Cu2+ and Zn2+ inhibited it. The recombinant PelA had a V max of 77 μmol min−1 mg−1 and an apparent K m of 0.50 mg ml−1 for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.

Application of neutral-alkaline oectate lyases to cotton fabric boil off

Commercial and pilot pectate lyase preparations (EC 4.2.2.2) have been compared. They differ in their effect on pectins with different esterification degrees (ED). The activity of the pilot preparation with respect to a substrate with ED = 70% is tenfold lower than with respect to unesterified polygalacturonic acid. For commercial preparations, this activity ratio ranged within 1.5-2. At equal pectate lyase activities, the commercial preparations better remove pectin from crude cotton fabric during its boil off. The laboratory preparation is more efficient for improving the capillarity (wettability) of the fabric owing to the cooperative effect of the pectate lyase, cellulase, and hemicellulase present in the preparation.

N-acylhomoserine lactonase producingRhodococcusspp. with different AHL-degrading activities

N-acylhomoserine lactones (AHLs) are conserved signal molecules that control diverse biological activities in quorum sensing system of Gram-negative bacteria. Recently, several soil bacteria were found to degrade AHLs, thereby interfering with the quorum sensing system. Previously, Rhodococcus erythropolis W2 was reported to degrade AHLs by both oxido-reductase and AHL-acylase. In the present study, two AHL-utilizing bacteria, strains LS31 and PI33, were isolated and identified as the genus Rhodococcus. They exhibited different AHL-utilization abilities: Rhodococcus sp. strain LS31 rapidly degraded a wide range of AHLs, including N-3-oxo-hexanoyl-l-homoserine lactone (OHHL), whereas Rhodococcus sp. strain PI33 showed relatively less activity towards 3-oxo substituents. Coculture of strain LS31 with Erwinia carotovora effectively reduced the amount of OHHL and pectate lyase activity, compared with coculture of strain PI33 with E. carotovora. A mass spectrometry analysis indicated that both strains hydrolyzed the lactone ring of AHL to generate acylhomoserine, suggesting that AHL-lactonases (AHLases) from the two Rhodococcus strains are involved in the degradation of AHL, in contrast to R. erythropolis W2. To the best of our knowledge, this is the first report on AHLases of Rhodococcus spp.

Optimization of chemical and physical parameters affecting the activity of pectin lyase and pectate lyase from Debaryomyces nepalensis: A statistical approach

Debaryomyces nepalensis was previously isolated, which is capable of producing pectin lyase and pectate lyase using pectin as sole source of carbon. The parameters affecting the activity of these enzymes are categorized into two, viz., chemical (amount of substrate and enzyme) and physical (pH of reaction mixture and temperature of incubation). The effect of these parameters on pectin lyase and pectate lyase was studied using central composite design. The optimal conditions of amount of substrate and enzyme (culture broth) were found to be 545 μl (∼1 g/l pectin) and 123 μl for pectin lyase and 707 μl (∼1.1 g/l polygalacturonic acid) and 96 μl for pectate lyase. The optimum pH and temperature for reaction was found to be 6.4 and 35 °C for pectin lyase and 7.5 and 32 °C for pectate lyase. After optimization, the activity of pectin lyase and pectate lyase was increased by 1.3- and 1.4-fold, respectively.

Role of glucose in production and repression of polygalacturonase and pectate lyase from phytopathogenic fungus Fusarium moniliforme NCIM 1276

A mangrove fungal isolate Fusarium moniliforme NCIM 1276 was found to produce the maximum extracellular endo-polygalacturonase and pectate lyase activities in liquid medium containing 1% citrus pectin and 0.2% glucose at pH 5 and pH 8, respectively. The enzyme secretion started after 10 h, when the spores were completely germinated, and maximum production occurred after 72 h in the extracellular medium. The production of polygalacturonase was enhanced by 0.2% glucose and completely repressed by 1% glucose. Pectate lyase secretion was also induced by a similar concentration of glucose in the extracellular medium and significantly decreased at 1% glucose. Quantification of extracellular and intracellular polygalacturonase and pectate lyase protein concentrations by ELISA and immunocytolocalization data suggested that glucose may control the expression of both enzymes at a transcriptional level.

Physiological and biochemical changes during banana ripening and finger drop

Fruit drop of banana is due to breaking at the junction of the pedicel and pulp, and we found no true abscission zone. The breakage seems therefore due to weakening of the peel. We investigated pectin hydrolysis and some properties at the rupture zone, using ‘Hom Thong’ ( Musa acuminata, AAA Group) and ‘Namwa’ ( Musa x paradisiaca, ABB Group) fruit, which show massive drop and no drop, respectively. During the period of finger drop, the water content of the peel in ‘Namwa’ was similar to that of ‘Hom Thong’ and thus water content does not account for the high breakage in ‘Hom Thong’. The peel thickness at the rupture area in the two cultivars was not significantly different. During the period of finger drop, the level of water-soluble pectin in the peel at the rupture area of ‘Hom Thong’ was higher than that of ‘Namwa’, indicating pectin degradation. CDTA soluble pectin and insoluble pectin was lower in ‘Hom Thong’, also indicating more pectin breakdown in this cultivar. Polygalacturonase activity in the peel at the rupture area of ‘Hom Thong’ bananas rapidly increased, but not clearly more than in ‘Namwa’ bananas. Pectinesterase activity in the peel at the rupture area of ‘Hom Thong’ was much lower than that of ‘Namwa’ bananas, and thus does not account for the breakage. Pectate lyase in the peel of ‘Hom Thong’ was considerably higher than in that of ‘Namwa’. The present data indicate that the much higher pectate lyase activity in ‘Hom Thong’ might be responsible, at least partially, for the finger drop.

Detection of uronic oxidase activity in ripening peaches

Uronic acid oxidase activity was found in an extract from harvested peaches that was incubated with citrus pectin at pH 8.5. The product of this reaction was identified by GC–MS analysis to be galactaric acid. The reaction was linear at 37 °C for up to 20 h, and the pH optimum was 8.5. The activity found in firm peaches one day after harvest did not change as the peaches softened over 5 days to eating softness. The incubation conditions were those suitable for monitoring the activity of pectate lyase, but instead of finding an increase in galacturonosyl residue reducing groups due to generation of pectin-derived oligosaccharides, uronic acid oxidase catalyzed the oxidation of the aldehyde reducing functions to carboxyl groups.

Phenotypic characterisation of Phaeoacremonium and Phaeomoniella strains isolated from grapevines: enzyme production and virulence of extra-cellular filtrate on grapevine calluses

Extra-cellular enzyme production of different Phaeoacremonium spp. and Phaemoniella chlamydospora isolates were used to assay the possibility of inter-specific characterisation. Isolates of Phaeoacremonium aleophilum, Phaeoacremonium angustius, P. viticola and Ph. chlamydospora were grown on solid media and the activities of extra-cellular amylases, lipases, proteases, cellulases, xylanases, laccase, polygalacturonase, pectate lyase, lignin peroxidase, manganese peroxidase, urease and chitinase were assayed. Phaeoacremonium species showed activities of a larger number of enzymes and also enzyme activity was frequently higher suggesting that Phaeoacremonium can be more virulent. To assay if the produced extra-cellular enzymes could reflect the virulence capacity of the two genera, calluses of Vitis vinifera L. (cvs. Baga and Maria Gomes) and of a rootstock (R3309) were inoculated with filtrated culture liquid medium of three isolates of Ph. chlamydospora and one of P. angustius. Filtrates from all strains decreased callus growth and membrane integrity, while soluble protein content of calluses decreased with the strains CAP 054 and 1AS. P. angustius (CAP 054) induced the more severe symptoms in all genotypes. Water content decreased together with an increase of osmolality in both cultivars but not in rootstock suggesting that osmorregulatory capacity is more affected in cultivars. Data show that: (1) Phaeoacremonium and Phaeomoniella genera have different patterns of extra- cellular enzymatic production; (2) these fungi produce extra-cellular compound(s) that induce(s) senescence symptoms in plant cells inhibiting callus proliferation; (3) among the strains tested in plant calluses the most virulent isolate (CAP 054) also produced higher amounts of some extra-cellular enzymes; (5) rootstock calluses were less sensitive to inoculation than grapevine calluses.

Dyeing behaviour of cotton fabric bioscoured with pectate lyase and polygalacturonase

Bioscouring of cotton is normally carried out using pectinases having pectate lyase activity. The present study has examined the influence of pectate lyase and hydrolase on the surface of cotton fibre. Dye uptake by cotton scoured with polygalacturonase is found to be much lower than that scoured with pectate lyase, Pectate lyase gave better dye exhaustion at 90 °C. The difference in dye exhaustion after scouring with sodium hydroxide, polygalacturonase or pectate lyase, may be due to variation in the amount of surfactant retained by the fibre, as this hinders dye uptake. This was particularly the case with polygalacturonase. Reduced numbers of free carboxy groups on the fibre surface could indicate lower pectin content. On the other hand it could, however, be due to the esterification of carboxy groups in the pectin by hydroxy groups in the surfactant.

Involvement of Bacterial Quorum-Sensing Signals in Spoilage of Bean Sprouts

Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 10(8) to 10(9) CFU/g. AHL-producing bacteria (Enterobacteriaceae and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts. Thin-layer chromatography and liquid chromatography-high-resolution mass spectrometry revealed the presence of N-3-oxo-hexanoyl-l-homoserine lactone in spoiled bean sprouts and in extracts from pure cultures of bacteria. During normal spoilage, the pH of the sprouts increased due to proteolytic activity, and the higher pH probably facilitated the activity of pectate lyase. The AHL synthetase gene (I gene) from a spoilage Pectobacterium was cloned, sequenced, and inactivated in the parent strain. The predicted amino acid sequence showed 97% homology to HslI and CarI in Erwinia carotovora. Spoilage of laboratory bean sprouts inoculated with the AHL-negative mutant was delayed compared to sprouts inoculated with the wild type, and the AHL-negative mutant did not cause the pH to rise. Compared to the wild-type strain, the AHL-negative mutant had significantly reduced protease and pectinase activities and was negative in an iron chelation (siderophore) assay. This is the first study demonstrating AHL regulation of iron chelation in Enterobacteriaceae. The present study clearly demonstrates that the bacterial spoilage of some food products is influenced by quorum-sensing-regulated phenotypes, and understanding these processes may be useful in the development of novel food preservation additives that specifically block the quorum-sensing systems.