Cell Wall Hydrolase Enzyme Research Articles

Visualization of a Cell Wall Hydrolase Inhibitor in Fusobacterium nucleatum by Immunofluorescence Microscopy.

Innately present in tears, saliva and mucosal secretions, lysozyme provides a critical defensive strategy to the host by cleaving the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetyl-D-glucosamine residues of peptidoglycan of invading bacteria, leading to bacterial lysis. To counter this class of cell wall hydrolase enzymes, bacteria produce several lysozyme inhibitors, a representative of which, MliC, was identified in Escherichia coli, Pseudomonas aeruginosa, and various bacterial species. The Gram-negative oral anaerobe Fusobacterium nucleatum encodes an uncharacterized lipoprotein homologous to MliC, whose localization is unknown. Here, we provide an experimental procedure to localize this MliC-like lipoprotein by employing immunofluorescence microscopy. In principle, this protocol can be used for any bacterial system to monitor protein localization.

Polygalacturonase (PG) gene expression in Musa acuminata cultivars from Kerala

Banana cannot be preserved for a long time after harvesting due to a short shelf life. Fruit softening is associated with textural changes due to disassembly of the primary cell wall and modification of the structure and composition of various polysaccharides. Cell wall degradation is caused by the action of various cell wall hydrolase enzymes. Polygalacturonase (PG) is the key enzyme involved in this process. The ripening period is different in cultivars maintained under domesticated cultivation in Kerala. PG activity was profiled in eight Musa acuminata cultivars from Kerala and expression analysis of the PG gene was accomplished using semi-quantitative RT-PCR. Maximum PG activity was observed in Palayankodan and minimum activity was observed in Kadali. Gene expression analysis showed variation between ethylene treated fruits and controls in Palayankodan, whereas the expression patterns in Kadali were similar. The fruit softening process is cultivar specific.

Comparative Secretome Investigation of Magnaporthe oryzae Proteins Responsive to Nitrogen Starvation

Magnaporthe oryzae is a fungal pathogen that causes blast disease in rice. During its early infection process, during which starvation of nutrients, including nitrogen, prevails before establishment of successful infection, the fungally secreted proteins play an important role in the pathogenicity and stress response. In this study, M. oryzae-secreted proteins were investigated in an N-deficient minimal medium using two-dimensional gel electrophoresis (2-DGE) coupled with mass spectrometry analysis (MALDI-TOF-MS and μLC-ESI-MS/MS). The 2-DGE analysis of secreted proteins detected 89 differentially expressed protein spots (14 downregulated and 75 upregulated) responsive to N starvation. Eighty five of the protein spots were identified by mass spectrometry analyses. Identified proteins were mainly cell wall hydrolase enzymes (22.4%), protein and lipid hydrolases (24.7%), reactive oxygen species detoxifying proteins (22.4%), and proteins with unknown function (14.1%), suggesting early production of prerequisite proteins for successful infection of the host. SignalP analysis predicted the presence of signal peptides in 67% of the identified proteins, suggesting that in addition to the classical Golgi/endoplasmic reticulum secretory pathway, M. oryzae might possess other, as yet undefined, secretory pathways. Those nonclassical or leaderless secretion proteins accounted for 25.9% of the total identified proteins by TatP and SecretomeP predictions. Semiquantitative reverse transcriptase polymerase chain reaction of seven randomly selected N-responsive secreted proteins also revealed a good correlation between RNA and protein levels. Taken together, the establishment of the M. oryzae secretome that is responsive to N starvation provides the first evidence of the secretion of 60 unreported and 25 previously known proteins. This developed protein inventory could be exploited to improve our understanding of the secretory mechanisms of M. oryzae and its invasive growth process in rice tissue.

BIOCHEMICAL AND MOLECULAR CHARACTERISATION OF PECTATE LYASE IN RIPENING NANGKA (AAB) BANANAS

Biochemical and molecular analysis were carried on Nangka (AAB) banana, a cooking variety banana which is widely distributed in Malaysia. Analysis was focused on pectate lyase which is a cell wall hydrolase enzyme. The enzyme was assayed from crude extract to measure its activity in pulp softening during ripening of Nangka banana. Pectate lyase showed trends of increasing activity which may contribute to cell wall degradation during ripening. This may give insight into the function or the redundancy of pectate lyase enzymes in pulp softening during ripening. Total DNA was extracted from the pulp and pectate lyase gene was amplified and sequenced. Further characterisation of the isolated gene using bioinformatics tools showed more than 90% homology with PL1 gene from 'Williams' (AAA) cultivar and 'Dwarf Cavendish' (AAA).

Changes in pectic substances and cell wall hydrolase enzymes of mangosteen (Garcinia mangostana) fruit during storage

Mangosteen (Garcinia mangostana) fruits at mature green stage with 5% red spotting were held at room temperature (31–35°C, 84 ± 2% relative humidity (RH)) and at the storage temperature of 13°C (90 ± 2% RH). Firmness, pectin content, and activities of pectin methylesterase and polygalacturonase in the pulp (aril) were monitored every 2 days. Pulp firmness rapidly decreased at room temperature from day 0 to day 4 after storage and slightly decreased thereafter, whereas pulp firmness slowly decreased at 13°C. Water soluble pectin content in pulp increased more rapidly at room temperature than at 13°C. Insoluble pectin content in pulp decreased throughout the storage time at both storage conditions. Both pectin methylesterase and polygalacturonase activities in pulp increased more rapidly at room temperature than at 13°C. Changes in water soluble pectin and cell wall hydrolase enzymes in relation to pulp softening of mangosteen fruits are discussed.