Production of a cell wall-associated endopolygalacturonase by Colletotrichum lindemuthianum and pectin degradation during bean infection

The bean pathogen Colletotrichum lindemuthianum expresses two endopolygalacturonase genes, CLPG1 and CLPG2, during interaction with its host plant. However, only CLPG1 was found to be secreted to the extracellular medium during saprophytic growth of the fungus on pectin. To localize CLPG2, a FLAG epitope sequence was inserted in the C-terminal sequence of CLPG2 and the modified gene was introduced into C. lindemuthianum. Western blot analysis using a FLAG monoclonal antibody allowed the detection of CLPG2 in intracellular protein extracts and in the cell wall fraction, but not in the culture medium. Indirect immunofluorescence microscopy was performed to detect CLPG2 during saprophytic or parasitic growth. According to the expression pattern of CLPG2, it was found that CLPG2 accumulates in the fungal cell wall during growth on pectin medium and during appressorium formation, both in vitro and during interaction with the plant. Pectin degradation was not detected around the infection peg using the monoclonal antibody JIM7, specific for methyl-esterified galacturonan. However, extensive pectin dissolution was observed during the development of secondary hyphae.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of pectin lyase and pectinase sequences

A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.     

Wax removal for accelerated cotton scouring with alkaline pectinase

A rational approach has been applied to design a new environmentally acceptable and industrially viable enzymatic scouring process. Owing to the substrate specificity, the selection of enzymes depends on the structure and composition of the substrate, i.e. cotton fibre. The structure and composition of the outer layers of cotton fibre has been established on the basis of thorough literature study, which identifies wax and pectin removal to be the key steps for successful scouring process. Three main issues are discussed here, i.e. benchmarking of the existing alkaline scouring process, an evaluation of several selected acidic and alkaline pectinases for scouring, and the effect of wax removal treatment on pectinase performance. It has been found that the pectinolytic capability of alkaline pectinases on cotton pectin is nearly 75% higher than that of acidic pectinases. It is concluded that an efficient wax removal prior to pectinase treatment indeed results in improved performance in terms of hydrophilicity and pectin removal. To evaluate the hydrophilicity, the structural contact angle (theta) was measured using an auto-porosimeter.     

Molecular characterization of CLPT1, a SEC4-like Rab/GTPase of the phytopathogenic fungus Colletotrichum lindemuthianum which is regulated by the carbon source

The gene CLPT1 (Colletotrichum lindemuthianum Protein Transport 1) encoding a Rab/GTPase was isolated from the filamentous fungus Colletotrichum lindemuthianum, the causal agent of bean anthracnose. At the amino acid level, CLPT1 shows between 54 and 80% identity to SEC4-like proteins, a class of molecules required for intracellular vesicular transport in yeasts. In particular, typical SEC4 domains involved in nucleotide binding and membrane attachment are present in the CLPT1 sequence. Functional identity of CLPT1 with SEC4 was confirmed by complementation of the Saccharomyces cerevisiae sec4-8 mutation. This is the first report of a gene involved in the control of intracellular vesicular trafficking in a phytopathogenic fungus. RNA blot analyses of CLPT1 expression were performed during in vitro growth of the fungus on synthetic media containing glucose or pectin, as single carbon source. The accumulation of CLPT1 mRNA was strongly increased on pectin, a plant cell wall polysaccharide that induces the production of extracellular pectinases, whereas the level of CLPT1 mRNA was below the detection threshold on glucose. These results suggest that CLPT1 is mainly involved in protein secretion and that the production of extracellular enzymes potentially involved in pathogenesis in filamentous fungi is sustained by induction of the genes involved in the secretory machinery.     

Validation of leaf and microbial pectinases: commercial launching of a new platform technology

Almost all current genetically modified plant commercial products are derived from seeds. The first protein product made in leaves for commercial use is reported here. Leaf pectinases are validated here with eight liquid commercial microbial enzyme products for textile or juice industry applications. Leaf pectinases are functional in broad pH/temperature ranges as crude leaf extracts, while most commercial enzyme products showed significant loss at alkaline pH or higher temperature, essential for various textile applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, leaf pectinase powder was stored up to 16 months at ambient temperature without loss of enzyme activity. Commercial pectinase products showed much higher enzyme protein PAGE than crude leaf extracts with comparable enzyme activity without protease inhibitors. Natural cotton fibre does not absorb water due to hydrophobic nature of waxes and pectins. After bioscouring with pectinase, measurement of contact‐angle water droplet absorption by the FAMAS videos showed 33 or 63 (leaf pectinase), 61 or 64 (commercial pectinase) milliseconds , well below the 10‐second industry requirements. First marker‐free lettuce plants expressing pectinases were also created by removal of the antibiotic resistance aadA gene. Leaf pectinase powder efficiently clarified orange juice pulp similar to several microbial enzyme products. Commercial pilot scale biomass production of tobacco leaves expressing different pectinases showed that hydroponic growth at Fraunhofer yielded 10 times lower leaf biomass per plant than soil‐grown plants in the greenhouse. Pectinase enzyme yield from the greenhouse plants was double that of Fraunhofer. Thus, this leaf‐production platform offers a novel, low‐cost approach for enzyme production by elimination of fermentation, purification, concentration, formulation and cold chain.     

Microbial pectinase: sources, characterization and applications

Today pectinases are upcoming industrially important bacterial enzymes. It can be produced by a variety of microorganisms. These enzymes act on pectin, which is the major component of middle lamella in plant cell wall. Pectinolytic enzymes are classified according to their mode of attack on the galacturonan part of the pectin molecules such as protopectinases, esterase’s and depolymerases. As we know that microbial enzymes work depends up on the type of enzymes application, temperature, concentration, and pH and so on, therefore, pectinase enzyme also differentiated according to their physical and chemical factors too. The biochemical structures of pectinases include members of all the major classes and the structure–function relationship, studies of a few available complexes of pectinases with substrate/analogs could be considered as prototypes for related family member and the molecular characterization of pectinolytic enzymes is also well documented. Furthermore, it provides a bird’s eye view of the possible application of these enzymes in commercial sector.     

Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937

The secretion of extracellular pectinases, among which there are least six isoenzymes of pectate lyase and one pectin methylesterase, allows the phytopathogenic bacterium Erwinia chrysanthemi to degrade pectin. A gene coding for a novel pectin methylesterase has been cloned from an E. chrysanthemi strain 3937 gene library. This gene, pemB , codes for a 433-amino-acid protein. The PemB N-terminal region has the characteristics of lipoprotein signal sequences. We have shown that the PemB precursor is processed and that palmitate is incorporated into the mature protein. The PemB lipoprotein is not released into the extracellular medium and is localized in the outer membrane. The PemB sequence presents homology with other pectin methylesterases from bacterial and plant origin. pemB -like proteins were detected in four other E. chrysanthemi strains but not in Erwinia carotovora strains. PemB was overproduced in Escherichia coli and purified to homogeneity. PemB activity is strongly increased by non-ionic detergents. The enzyme is more active on methylated oligogalacturonides than on pectin, and it is necessary for the growth of the bacteria on oligomeric substrates. PemB is more probably involved in the degradation of methylated oligogalacturonides present in the periplasm of the bacteria, rather than in a direct action on extracellular pectin. pemB expression is inducible in the presence of pectin and is controlled by the negative regulator KdgR.     

Autophagy vitalizes the pathogenicity of pathogenic fungi

Plant pathogenic fungi utilize a series of complex infection structures, in particular the appressorium, to gain entry to and colonize plant tissue. As a consequence of the accumulation of huge quantities of glycerol in the cell the appressorium generates immense intracellular turgor pressure allowing the penetration peg of the appressorium to penetrate the leaf cuticle. Autophagic processes are ubiquitous in eukaryotic cells and facilitate the bulk degradation of macromolecules and organelles. The study of autophagic processes has been extended from the model yeast Saccharomyces cerevisiae to pathogenic fungi such as the rice blast fungus Magnaporthe oryzae. Significantly, null mutants for the expression of M. oryzae autophagy gene homologs lose their pathogenicity for infection of host plants. Clarification of the functions and network of interactions between the proteins expressed by M. oryzae autophagy genes will lead to a better understanding of the role of autophagy in fungal pathogenesis and help in the development of new strategies for disease control.     

Pectinolytic activity of bacteria isolated from soil and two fungal strains during submerged fermentation

Seven different strains were selected for their ability to degrade citrus pectin. Alkaline pectinases were produced by five bacterial soil isolates, whereas two fungal strains produced pectinase in an acidic environment. The bacteria were isolated from soil of a plum orchard in Northern Ireland. These isolates produced significant amounts of pectin lyase (PL) and polygalacturonase (PG) with maximum activities of 30.1 and 29.1 U/ml respectively. Fungal strains Aspergillus sp. and PN-1 produced four different pectinolytic activities; endo-PG, exo-PG, pectin esterase (PE) and PL. The Aspergillus sp. produced higher amounts of pectinase than PN-1. The Aspergillus sp. excreted highly stable pectinases, which may be of importance for industrial applications.     

Anaerobic degradation of pectin by mixed consortia and optimization of fermentation parameters for higher pectinase activity

Samples from biogas digesters, sewage ponds, animal house effluents and food processing wastes were used in enrichment systems seeking anaerobic bacteria producing pectinases. Among the 46 anaerobic consortia developed from various samples, four showed high pectinase activity under static anaerobic conditions. Investigation of fermentation variables showed the optimum conditions for pectinase activity were pH 7.0, 45°C and 72 h of growth with 0.5% pectin in the cultivation medium. A 1.4- to 1.6-fold increase in the pectinase activity was achieved under these conditions. The maximum yield of enzymes (62.72 U ml-1 of pectinase, 4.74 U ml^-1 of polygalacturonase, 113.30 U ml^-1 of pectin lyase, 2.10 U ml^-1 of pectinesterase, 0.75 U ml^-1 of total cellulase and 9.27 U ml^-1 of xylanase) was recorded with the consortia C-S2 developed from decomposed plant samples collected from a pond.