Purification, characterization and physiological significance of a chitinase from the pilei of<i>Coprinopsis cinerea</i>fruiting bodies

Zhou, Yajun; Kang, Liqin; Niu, Xinhuan; Wang, Jun; Liu, Zhonghua; Yuan, Sheng

doi:10.1093/femsle/fnw120

Cited by 29 publications

(32 citation statements)

References 31 publications

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“…Chitinases ChiIII, ChiEn1, ChiB1, ChiE1, and ChiEn3 were prepared according to previous reports (17,(25)(26)(27)(28). Chitinase activity and protein amount were analyzed as described in Niu et al (17).…”

Section: Chemicals and Enzymesmentioning

confidence: 99%

“…There are eight putative chitinases in the C. cinerea genome (18,25). We isolated, purified, and characterized a class V exochitinase, ChiB1, from C. cinerea pilei (26) and heterologously expressed, The ϳ10-mm length of heat-inactivated apical stipe fragments clamped to an extensometer with an applied force of 3 ϫ g was suspended in bathing solution (50 mM sodium acetate, pH 4.5) for 30 min. The bathing solution was then replaced by the same solution (arrows), with or without protein extractions from apical stipes or chitinases from C. cinerea for the next 120 min.…”

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Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea

Zhou

Kang

Liu

et al. 2019

Appl Environ Microbiol

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The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heatinactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1-or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1-or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heatsensitive cell wall extension of native stipes. These results indicate a chitinasehydrolyzing mechanism for stipe cell wall extension. IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by ␤-1,6-branched ␤-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and ␤-1,3-glucans under turgor pressure in vivo.O ne of the remarkable characteristics of the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation (1-5). The ...

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Section: Chemicals and Enzymesmentioning

confidence: 99%

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confidence: 99%

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea

Zhou

Kang

Liu

et al. 2019

Appl Environ Microbiol

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“…ATG11 is essential for selective autophagy and mitophagy in yeast 32 . Mutation of SCH9 has been reported to extend the lifespan of yeast 33 , nematodes 16,19 , flies 20 , and mice 21 .…”

Section: Validation Of Transcriptome Data By Qrt-pcr To Validate the Expression Profiles Obtainedmentioning

confidence: 99%

“…Glucan is a primary structural component of the fungal cell wall 20 . β-1,3-glucanases contribute greatly to the autolysis of fruiting bodies 37 .…”

Section: Validation Of Transcriptome Data By Qrt-pcr To Validate the Expression Profiles Obtainedmentioning

confidence: 99%

“…To date, the studies of mushroom autolysis have mainly focused on storage [16][17][18] , and the underlying molecular processes have been infrequently assessed. Genes that encode the exp1 transcriptional regulator 19 and glucan hydrolases 20 are believed to be involved in the autolysis of the fruiting bodies of the edible mushroom Coprinopsis cinerea (Schaeff.) Redhead.…”

Section: Introductionmentioning

confidence: 99%

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Transcriptome Analysis of Genes Associated with Autolysis of Coprinus Comatus

Guo¹,

Zhang

Wang

et al. 2021

Preprint

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Coprinus comatus, widely known as “Jituigu”, is an important commodity and food in China. The yield of C. comatus, however, is substantially reduced by the autolysis of the fruiting bodies after harvest. To gain insight into the molecular mechanism underlying this autolysis, we divided the growth of C. comatus fruiting bodies into four stages: infant stage (I), mature stage (M), discolored stage (D), and autolysis stage (A). We then subjected these stages to de novo transcriptomic analysis using high-throughput Illumina sequencing. A total of 12,946 unigenes were annotated and analyzed with the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG). We analyzed the differentially expressed genes (DEGs) between stages I and M, M and D, and D and A. Because the changes from M to D are thought to be related to autolysis, we focused on the DEGs between these two stages. We found that the pathways related to metabolic activity began to decline in the transition from M to D. In contrast, expression levels of chitinases, β-1,3-glucanases, and the ubiquitin-conjugating enzyme E2 related to degradation of cell walls were higher in D than in M. In addition, 20 genes of interest were analyzed by quantitative real-time PCR to verify their expression profiles at the four developmental stages. This study, which is the first to describe the transcriptome of C. comatus, provides a foundation for future studies concerning the molecular basis of the autolysis of its fruiting bodies.

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The use of matrix‐assisted laser desorption/ionization mass spectrometry in enzyme activity assays and its position in the context of other available methods

Šebela

2021

Mass Spectrometry Reviews

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Activity assays are indispensable for studying biochemical properties of enzymes. The purposes of measuring activity are wide ranging from a simple detection of the presence of an enzyme to kinetic experiments evaluating the substrate specificity, reaction mechanisms, and susceptibility to inhibitors. Common activity assay methods include spectroscopy, electrochemical sensors, or liquid chromatography coupled with various detection techniques. This review focuses on the use of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a growing and modern alternative, which offers high speed of analysis, sensitivity, versatility, possibility of automation, and cost‐effectiveness. It may reveal reaction intermediates, side products or measure more enzymes at once. The addition of an internal standard or calculating the ratios of the substrate and product peak intensities and areas overcome the inherent inhomogeneous distribution of analyte and matrix in the sample spot, which otherwise results in a poor reproducibility. Examples of the application of MALDI‐TOF MS for assaying hydrolases (including peptidases and β‐lactamases for antibiotic resistance tests) and other enzymes are provided. Concluding remarks summarize advantages and challenges coming from the present experience, and draw future perspectives such as a screening of large libraries of chemical compounds for their substrate or inhibitory properties towards enzymes.

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Purification, characterization and physiological significance of a chitinase from the pilei ofCoprinopsis cinereafruiting bodies

Cited by 29 publications

References 31 publications

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea

Transcriptome Analysis of Genes Associated with Autolysis of Coprinus Comatus

The use of matrix‐assisted laser desorption/ionization mass spectrometry in enzyme activity assays and its position in the context of other available methods

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