Pectate Lyase Activity Research Articles

Pectin induces an in vitro rumen microbial population shift attributed to the pectinolytic Treponema group.

Pectin is a non-fiber carbohydrate (NFC) that exists in forages, but it is not clear how pectin exerts its effect on populations of either known microbial species or uncultured ruminal bacteria. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR analysis were used in the present study to investigate the effects of pectin on microbial communities in an in vitro rumen fermentation system. The fermentations were conducted using forage (corn stover or alfalfa), an NFC source (pectin or corn starch), or their combination as the substrates. Addition of pectin increased acetate (P < 0.05), whereas inclusion of starch increased butyrate production (P < 0.05). The pectate lyase activity was higher with alfalfa than with corn straw, or with pectin than with corn starch (P < 0.05), while the amylase activity was higher in corn starch-included treatments than the others (P < 0.05). The cluster analysis of the bacterial 16S rRNA gene showed that the DGGE banding patterns differed significantly between the treatments and led to the identification of three groups that were highly associated with the NFC sources. The specific bands associated with pectin-rich treatments were identified to be dominated by members of the Treponema genus. The growth of the Treponema genus was remarkably supported by the inclusion of pectin, highlighting their specific ability to degrade pectin. The results from the present study expand our knowledge of the microbial populations associated with pectin digestion, which may not only facilitate future research on utilization of pectin in feeds, but also improve our understanding of pectin digestion with respect to the rumen micro-ecosystem.

Process optimization of high-level extracellular production of alkaline pectate lyase in recombinant Escherichia coli BL21 (DE3)

Eco-effective high-level production of alkaline pectate lyase (PL) is a necessary prerequisite for the application of this enzyme in large-scale industries, such as bioscouring. For this purpose, in this work, a multi-step glycerol feeding strategy based on specific growth rate for high cell density cultivation of recombinant Escherichia coli was first established, which could effectively control cell growth and acetate yield. Next, the effects of induction time, temperature, and inducer concentration on cell growth and extracellular secretory production of recombinant PL were investigated. When induced by IPTG (using either low IPTG concentration at 25°C or high IPTG concentration at 30°C), two different extracellular secretion strategies were found to be appropriate for extracellular PL production. On the other hand, when cultivations were induced by mild continuous lactose feeding strategy, the extracellular and total PL activities reached 4478 and 5337U/mL, respectively, representing the highest PL yield reported to date.

Effect of heavy metals on enzymes production by Hebeloma crustuliniforme

Studies were carried out in order to dętermine the effect of some heavy metals (Cu, Cd, Pb, Zn) on the production of enzymes (cellulases, peetinases. proteases) by ectomycorrhizal fungus &lt;i&gt;Hebeloma crusliliniforme&lt;/i&gt; (Buli.: Fr.) Quél. All the heavy metals inhibited the general enzymatic activity regardless of the source of carbon used. The metals reduced the egzocellulolytic activity more in media with cellulose powder than with CMC (carboxymethylocellulosc). Among pectolytic enzymes heavy metals most strongly inhibited polygalacturonase (PG). The heavy metals did not harmful affect the activity of pectate lyase (PGL). Proteolytic activity of &lt;i&gt;Hebeloma crustuliniforme&lt;/i&gt; was leasi affected by zinc (Zn). The degree of inhibition of enzymes by heavy metals can be presented in the following order Pb &lt; Zn &lt; Cd &lt;Cu.

수확 후 고농도 CO2단기처리가 '매향' 딸기의 저장에 미치는 영향

This experiment was conducted to examine the effects of a short-term treatment of high pressure CO₂ on shelf-life of strawberry fruit. A short-term treatment (12 hr) of 100% CO₂ resulted in the increase of fruit firmness up to 71.9% compared to that at harvest. The firmness of CO₂ treated fruit remained a significantly higher than that of control (air) up to 15 days. The alteration of pectic polymers was observed by CO₂ treatment such as an increase of EDTA soluble pectins and decrease of water soluble ones. The CO₂ treatment resulted in the increase of total amount of wall bound calcium. Pectate lyase activity, an important agent of strawberry fruit softening, was also significantly reduced by CO₂ treatment. Contents of soluble solids and acids of CO₂ treated fruit were higher than those of control fruit. Short-term treatment of high pressure CO₂ affected shelf-life through firmness increase whereas the visual quality and decay incidence of strawberry fruit were not affected.

Potential Role of Pectate Lyase and Ca2+ in the Increase in Strawberry Fruit Firmness Induced by Short‐Term Treatment with High‐Pressure CO2

Postharvest treatment with high-pressure CO2 helps to control decay and increase firmness in strawberries. Increases in firmness occurred through modification of calcium binding to cell wall. However, the mechanism(s) involved in Ca(2+) migration to pectic polymers and other physiological events associated with the maintenance of increased firmness are not clearly understood. The focus of this study was to find potential mechanism(s) that are associated with calcium movement, increases in firmness, or maintenance of firmness in strawberry fruit after high-pressure CO2 treatment. An increase in firmness was induced by high-pressure CO2 treatment, but not by high-pressure N2 treatment. This indicates that CO2 stimulates a change in firmness. The increase in firmness induced by high-pressure CO2 seems to involve calcium efflux. Using membrane Ca(2+) -dependent ATPase inhibitors sodium vanadate (250 μM) and erythrosin B (100 μM) delayed both the increase in firmness and calcium binding to wall polymers. Exogenous application of CaCl2 (10 mM) enhanced the firmness increase of fruit slices only when they were exposed to high-pressure CO2 . The activity of pectate lyase was downregulated by CO2 treatment, but β-galactosidase activity was not affected. The increase in strawberry firmness induced by high-pressure CO2 treatment primarily involves the efflux of calcium ions and their binding to wall polymers. These physiological changes are not induced by an anaerobic environment. The downregulation of wall-modifying enzymes, such as pectate lyase, appeared to contribute to the maintenance of firmness that was induced by high-pressure CO2 treatment.

Effect of Dionaea muscipula extract and plumbagin on maceration of potato tissue by Pectobacterium atrosepticum

AbstractPectobacterium atrosepticum (Pba) is a plant pathogen that causes major crop losses. Dionaea muscipula extracts and their antibacterial constituent, plumbagin, inhibit Pba growth in vitro. However, this effect is reduced when the extracts are added to bacterial cultures present on potato tubers or suspended in potato tuber filtrate (PF). To explain this, we examined the response mechanism of Pba cells to Dionaea extract and plumbagin and compared it with the effect of a bactericidal peptide – CAMEL. The addition of the extract and plumbagin to a Pba1043 culture in stationary phase increased the extracellular pectate lyase (Pel) activity in the presence of PF. While the addition of the Dionaea extract and plumbagin caused a dramatic reduction in RNA and protein synthesis in Pba1043, it did not result in cellular damage. PF alone increased the expression of Pba genes encoding protein components of cellular efflux pump systems: ompX, acrA and emrA. Application of both PF and plumbagin resulted in a synergistic stimulation of acrA gene expression. Plumbagin added to potato tubers inoculated with a field isolate Pba5A/1/2005 increased extracellular Pel activity and reduced tissue maceration but did not affect bacterial counts per gram of tissue. These results show that plumbagin in the presence of compounds from potato tuber stimulates Pel production/secretion in Pba cells and increases the expression of the acrA gene. This may be the molecular basis for the less pronounced effects of Dionaea extract on Pba in planta relative to those observed in vitro.

The HrpW protein of Lonsdalea quercina N‐5‐1 has pectate lyase activity and is required for full bacterial virulence

Lonsdalea quercina N-5-1 is a bacterial pathogen that causes poplar bark cankers. It has been isolated from the branch of Populus × euramericana cv. "74/76" in Henan, China. Previous studies have revealed that the Type III secretion system (T3SS) acts as an essential pathogenic factor in L. quercina N-5-1. HrpW is a putative effector of T3SS in strain N-5-1, which has a typical harpin domain at the amino terminal and a pectate lyase (Pel) domain at its carboxyl terminal. Genetic evidence had shown that, compared to the wild-type and the complementary strain, the hrpW mutation causes a small but significant reduction in virulence when inoculated on the poplar branches. The amino terminal domain of HrpW was found to trigger tobacco hypersensitive response, but the carboxyl terminal domain of HrpW was not. Unlike most HrpW homologs in other bacteria, the carboxyl terminal domain of HrpW of strain N-5-1 exhibited detectable pectate lyase activity. Site-direction mutations (W104A, W171M) further demonstrated that two tryptophan residues were essential to its pectate lyase activity. The results of the present work suggest that HrpW in L. quercina N-5-1 possesses pectate lyase activity and acts as a nonessential but important pathogenic factor in poplar bark canker disease.

Cold-Active Acid Pectinolytic System from PsychrotolerantBacillus: Color Extraction from Red Grape Skin

Cold-active enzymes are potentially relevant to food processing and, among them, pectinases belong to the most important enzymes in the fruit juice and wine industry. <i>Bacillus</i> sp. CH15 from grape berries was screened for the production of cold-active acidic pectinolytic enzymes. Taxonomic studies and 16S rDNA analysis revealed that the isolate was closely related to the <i>Bacillus subtilis</i> group. Maximum pectinolytic activity under acidic conditions (pH 5.0) and at low temperature (20°C) was obtained after 24 hr of incubation in medium containing citrus pectin as sole carbon source (0.305 U/mL). Polymethylgalacturonase activity was the predominant pectinase under the given assay conditions, whereas highest levels of pectate lyase activity were found at 60°C. At 5 and 10°C, the enzymatic system maintained 15 and 30% of the maximum activity, respectively. This is the first report on a pectinolytic enzyme system produced by a <i>Bacillus</i> strain and active at 20°C and pH 3.6, conditions similar to those in winemaking. According to classical and CIELab color parameters of short macerations with red grape skins at low temperature, the bacterial pectinolytic system produced a rapid color extraction and the macerates exhibited better chromatic characteristics than those obtained with commercial pectinases or after natural extraction. Total anthocyanin contents of the macerates with <i>Bacillus</i> enzyme system and after natural extraction were significantly different, whereas relative individual pigment quantities did not show any significant difference between the two treatments. In conclusion, <i>Bacillus</i> sp. CH15 could be used as a microbial source of cold-active acidic pectinases in red winemaking.

Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range.

BackgroundCitrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain Aw12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime.ResultsTo characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306.ConclusionsComparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri.

The pectate lyase encoded by the pecCl1 gene is an important determinant for the aggressiveness of Colletotrichum lindemuthianum

Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean, and the genes that encode its cell-wall-degrading enzymes are crucial for the development of the disease. Pectinases are the most important group of cell wall-degrading enzymes produced by phytopathogenic fungi. The pecC1l gene, which encodes a pectate lyase in C. lindemuthianum, was isolated and characterized. Possible cis-regulatory elements and transcription factor binding sites that may be involved in the regulation of genetic expression were detected in the promoter region of the gene. pecCl1 is represented by a single copy in the genome of C. lindemuthianum, though in silico analyses of the genomes of Colletotrichum graminicola and Colletotrichum higginsianum suggest that the genome of C. lindemuthianum includes other genes that encode pectate lyases. Phylogenetic analysis detected two groups that clustered based on different members of the pectate lyase family. Analysis of the differential expression of pecCl1 during different stages of infection showed a significant increase in pecCl1 expression five days after infection, at the onset of the necrotrophic phase. The split-maker technique proved to be an efficient method for inactivation of the pecCl1 gene, which allowed functional study of a mutant with a site-specific integration. Though gene inactivation did not result in complete loss of pectate lyase activity, the symptoms of anthracnose were reduced. Analysis of pectate lyases might not only contribute to the understanding of anthracnose in the common bean but might also lead to the discovery of an additional target for controlling anthracnose.

Cell wall enzymes and changes in sugar content in pollinated orchids treated with ethylene inhibitors and sugars

The pollinated flowers of Dendrobium Pompadour orchids exhibited rapid morphological changes, which resulted in full flower closure. In contrast, unpollinated flowers remained fresh and turgid during the identical time period. The aim of this study was to examine the pollination-induced changes in the cell wall enzyme activity and sugar content. Cell wall hydrolase assays showed that pollination induces significant increases in polygalacturonase, pectate lyase and cellulase activity, whereas pectin methyl esterase showed insignificant changes in activity. Membrane leakage was rapid in pollinated flowers. The total and reducing sugar content increased in the pollinated flowers and was most profound in the column of the flowers. Treatment with ethylene inhibitors and sugars suppressed cell wall hydrolase activity and membrane leakage to a varying extent. The sugar content was more stable in the treated flowers. Altogether, the results show that ethylene inhibitors effectively delay pollination-induced floral destruction.

Effects of nitric oxide treatment on the cell wall softening related enzymes and several hormones of papaya fruit during storage

Papaya fruits (Carica papaya L. cv 'Sui you 2') harvested with < 5% yellow surface at the blossom end were fumigated with 60 microL/L of nitric oxide for 3 h and then stored at 20 degrees C with 85% relative humility for 20 days. The effects of nitric oxide treatment on ethylene production rate, the activities of cell wall softening related enzymes including polygalacturonase, pectin methyl esterase, pectate lyase and cellulase and the levels of hormones including indole acetic acid, abscisic acid, gibberellin and zeatin riboside were examined. The results showed that papaya fruits treated with nitric oxide had a significantly lower rate of ethylene production and a lesser loss of firmness during storage. A decrease in polygalacturonase, pectin methyl esterase, pectate lyase and cellulase activities was observed in nitric oxide treated fruit. In addition, the contents of indole acetic acid, abscisic acid and zeatin riboside were reduced in nitric oxide treated fruit, but no significant reduction in the level of gibberellin was found. These results indicate that nitric oxide treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of cell wall softening related enzymes and certain hormones.

Potato signal molecules that activate pectate lyase synthesis in Pectobacterium atrosepticum SCRI1043

A new type of plant-derived signal molecules that activate extracellular pectate lyase activity in phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 was revealed. These compounds were characterized and partially purified by means of several approaches including RT-PCR analysis, luminescence bioassay and HPLC fractionation. They were smaller than 1 kDa, thermoresistant, nonproteinaceous, hydrophilic, and slightly negatively charged molecules. Using gene expression analysis and bacterial biosensor assay the mode of activity of revealed compounds was studied. The possibility of their action through quorum sensing- and KdgR-mediated pathways was analyzed.

Cell wall disassembly and post-harvest deterioration of ‘sweetheart’ sweet cherry fruit: involvement of enzymic and non-enzymic factors

Sweet cherries are very perishable, owing to rapid softening rates associated to significant deterioration of fruit. Ripening-related firmness loss is generally attributed to enzyme-catalysed modifications in cell wall composition and structure, but some experimental evidence suggests that non-enzymic mechanisms may be also involved in the process. In this study, ‘Sweet Heart’ cherries were picked at commercial maturity and kept at 0 oC during 15 or 30 days, followed by up to 3 days at 20 oC to simulate commercial shelf life. Cell wall materials were extracted, fractionated and analysed, and some cell wall-modifying enzyme activities were assessed for a possible role on firmness loss and overall deterioration of fruit. Radical scavenging activity (RSA) and both reduced and oxidised ascorbate contents were also analysed in each case. Regression analysis revealed that firmness was associated to reduced ascorbate and RSA, in turn inversely correlated to decay and weight loss. Higher firmness levels were found for fruit displaying higher RSA and yields of the cell wall fractions containing covalently-bound pectins and hemicelluloses, while decay correlated inversely with RSA and reduced ascorbate content. Polygalacturonase (PG) and pectate lyase (PL) activities were related to solubilisation of cell wall pectins and inversely to reduced ascorbate, but not to firmness-contributing cell wall fractions, which correlated rather to enzyme activities acting on pectin side-chains that

Cloning, Expression, Purification and Initial Analysis of a Novel Pectate Lyase Pcpel1 from Phytophthora capsici

AbstractPhytophthora capsici inflicts damage on numerous crop plants by secreting a series of pectinase including pectate lyase (PEL). Here, we report a pectate lyase gene (Pcpel1) from a genomic library of a highly virulent P. capsici strain SD33. Pcpel1 was identified as an open reading frame of 1233 bp encoding a protein of 410 amino acids with a predicted amino‐terminal signal sequence of 21 amino acids. The predicted protein of Pcpel1 has a calculated molecular mass of 43.8 kDa and a pI value of 6.8. Analysis of the amino acid sequence suggested that it was a member of the polysaccharide lyase family 1 that shows pectate lyase activity. Moreover, heterologous expression of Pcpel1 in Pichia pastoris produced proteins with molecular mass 66 kDa, very likely due to differential glycosylation by the yeast. By western blotting and northern blotting analysis, Pcpel1 was strongly expressed during interaction of P. capsici with the host plant, suggesting its involvement in the process of host infection. The role of Pcpel1 in cell wall disassembly and host/parasite interaction is discussed.

Characterization of a family 3 polysaccharide lyase with broad temperature adaptability, thermo-alkali stability, and ethanol tolerance

The 774-bp pectate lyase gene plyAI4 from Bacillus sp. I4 was cloned and expressed in E. coli. The gene encodes a 257-residue polypeptide (PlyAI4, 28.3 kDa) with the highest identities of 97.3% with a putative pectate lyase from Bacillus subtilis BSn5 (ADV94306) and 60.3% with an identified pectate lyase of the polysaccharide lyase family (PL) 3 from Paenibacillus amylolyticus 27C64 (ADB78774). The purified recombinant PlyAI4 (rPlyAI4) exhibited apparently optimal activity at pH 10.5 ∼ 11.0 and 50°C. Compared with the majority of reported alkaline pectate lyases, rPlyAI4 exhibited more residual enzyme activity at 20°C (∼45%) or at 70°C (∼50%) and better thermostability at 70°C (∼60 min half-life at 70°C). In the presence of 20% (v/v) ethanol, pectate lyase activity was enhanced by 0.2 fold. After incubation in 40% (v/v) ethanol at 37°C and pH 8.5 for 1 h, the purified rPelAI4 retained more than 75% of the initial activity. Sequence analysis proposed a new signature block, A-D-G-[V/I]-H, for PL 3 pectate lyases. These properties may prove to be important with regards to PlyAI4 for basic research and industrial application.

Changes in activities and gene expression of enzymes associated with cell wall modification in peels of hot water treated bananas

Fruit softening is an important event during ripening that affects quality and postharvest shelf life. Cell wall degrading enzymes are considered to be the main factor responsible for cell wall disassembly and texture changes in fruit, both pulp and peel. To elucidate the effect of hot water treatment (HWT, 50°C, 10min) on the softening of ‘Hom Thong’ banana, firmness, the activities of cell wall degrading enzymes, and the expression of related genes were investigated in the peel. The firmness of banana fruit decreased throughout the storage period; however, the decrease in firmness was delayed in hot water treated fruit as compare to control fruit during storage at 25°C. Treatment of intact fruit with hot water reduced activities of pectate lyase (PL) and β-galactosidase (β-Gal) in the peel, and also decreased the accumulation of their mRNAs (MaPL1 and MaGAL, respectively). Polygalacturonase (PG) enzyme activity was inhibited by heat treatment, and the increase in MaPG1 expression was delayed. HWT had little effect on pectin methylesterase (PME) or β-1,3 glucanase (Glu). After treatment, reduced PME activity was measured in the peels, but activity returned to levels similar to that of control fruit. PME activity varied during storage and did not correlate well with MaPME1 expression. There was no significant difference in Glu enzyme activity or MaGLU expression between treated and control fruit. Postharvest HWT was effective in delaying softening of ‘Hom Thong’ banana and reduced activities and gene expression of some enzymes associated with cell wall modification in the peel.

Improvement of thermostability and activity of pectate lyase in the presence of hydroxyapatite nanoparticles

The activity and half-life of pectate lyase (PL) from Bacillus megaterium were nine- and 60-fold, respectively, higher at 90°C in the presence of hydroxyapatite nanoparticles (NP-PLs) than in the presence of 1mM CaCl2. Thermodynamic analysis of the nanoparticle-induced stability revealed an enhanced entropy–enthalpy compensation by the NP-PLs since a reciprocal linearity of the enthalpy–entropy change to 90°C was observed. Without nanoparticles, the linearity range was 70°C. Such compensation reflected the maintenance of the native structure of proteins. The remarkable enhancement of activity and stability of the NP-PL system at high temperatures may be utilized commercially e.g. in the food industry or the processing of natural fibers that may require a thermotolerant enzyme.

Involvement of bacterial TonB-dependent signaling in the generation of an oligogalacturonide damage-associated molecular pattern from plant cell walls exposed to Xanthomonas campestris pv. campestris pectate lyases

BackgroundEfficient perception of attacking pathogens is essential for plants. Plant defense is evoked by molecules termed elicitors. Endogenous elicitors or damage-associated molecular patterns (DAMPs) originate from plant materials upon injury or pathogen activity. While there are comparably well-characterized examples for DAMPs, often oligogalacturonides (OGAs), generated by the activity of fungal pathogens, endogenous elicitors evoked by bacterial pathogens have been rarely described. In particular, the signal perception and transduction processes involved in DAMP generation are poorly characterized.ResultsA mutant strain of the phytopathogenic bacterium Xanthomonas campestris pv. campestris deficient in exbD2, which encodes a component of its unusual elaborate TonB system, had impaired pectate lyase activity and caused no visible symptoms for defense on the non-host plant pepper (Capsicum annuum). A co-incubation of X. campestris pv. campestris with isolated cell wall material from C. annuum led to the release of compounds which induced an oxidative burst in cell suspension cultures of the non-host plant. Lipopolysaccharides and proteins were ruled out as elicitors by polymyxin B and heat treatment, respectively. After hydrolysis with trifluoroacetic acid and subsequent HPAE chromatography, the elicitor preparation contained galacturonic acid, the monosaccharide constituent of pectate. OGAs were isolated from this crude elicitor preparation by HPAEC and tested for their biological activity. While small OGAs were unable to induce an oxidative burst, the elicitor activity in cell suspension cultures of the non-host plants tobacco and pepper increased with the degree of polymerization (DP). Maximal elicitor activity was observed for DPs exceeding 8. In contrast to the X. campestris pv. campestris wild type B100, the exbD2 mutant was unable to generate elicitor activity from plant cell wall material or from pectin.ConclusionsTo our knowledge, this is the second report on a DAMP generated by bacterial features. The generation of the OGA elicitor is embedded in a complex exchange of signals within the framework of the plant-microbe interaction of C. annuum and X. campestris pv. campestris. The bacterial TonB-system is essential for the substrate-induced generation of extracellular pectate lyase activity. This is the first demonstration that a TonB-system is involved in bacterial trans-envelope signaling in the context of a pathogenic interaction with a plant.

Expression and Bioinformatics Analysis of Pectate Lyase Gene from Bacillus subtilis521

Abstract In order to exploit new genetic resources, Pectate lyase(PEL) gene was amplified by PCR using the genome DNA from an alkaline Bacillus subtilis521. The PCR product was inserted into pET22b(+) vector. The recombinant plasmids were cloned in E.coli DH5α and then expressed in E.coli BL21. When cultured in the optimized medium, the positive clones E.coli BL21(pET22b(+)pel)showed intracellular pectate lyase activity of 90.0 U/mL. It was indicated that we had obtained the correct PEL gene. The pel has an open reading frame of 1263 nucleotides and codes for a product of 420 amino acids with a calculated molecular mass of 45.5 kD. Based on computer assisted analysis, a signal peptides and two conserved domains were revealed. The sequence analysis for PEL showed that it shares 26-82% homology with other strains in GenBank. In addition, the advanced structure of PEL were also predicted and analysed. This study will help to the experimental design of PEL fermentation and production purification and enzyme evolution.