Identification and enzymatic characterization of an endo-1,3-β-glucanase from Euglena gracilis

Takeda, Takumi; Nakano, Yüki; Takahashi, Machiko; Konno, Naotake; Sakamoto, Yuichi; Arashida, Ryo; Marukawa, Yuka; Yoshida, Eriko; Ishikawa, Takahiro; Suzuki, Kengo

doi:10.1016/j.phytochem.2015.05.010

Cited by 35 publications

(19 citation statements)

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“…XTH expression in ‘Qiu JinXing’ fruits was higher than that in ‘Ruanrou Shanlihong 3 Hao’ fruits in early developmental stageI; however, the level of XTH expression in ‘Ruanrou Shanlihong 3 Hao’ fruits was higher than that in ‘Qiu JinXing’ fruits in the other developmental stages. Takeda et al (2015) found that Gns plays a significant role in the degradation and modification of β-1, 3-glucan in fungal cell walls, and this is consistent with our results. The expression level of Gns in our study was high in soft-fleshed hawthorn fruits but weak in hard-flesh hawthorns at the late developmental stage.…”

Section: Discussionsupporting

confidence: 93%

Transcriptome Analysis and Ultrastructure Observation Reveal that Hawthorn Fruit Softening Is due to Cellulose/Hemicellulose Degradation

Zhao

Zhang

et al. 2016

Front. Plant Sci.

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Softening, a common phenomenon in many fruits, is a well coordinated and genetically determined process. However, the process of flesh softening during ripening has rarely been described in hawthorn. In this study, we found that ‘Ruanrou Shanlihong 3 Hao’ fruits became softer during ripening, whereas ‘Qiu JinXing’ fruits remained hard. At late developmental stages, the firmness of ‘Ruanrou Shanlihong 3 Hao’ fruits rapidly declined, and that of ‘Qiu JinXing’ fruits remained essentially unchanged. According to transmission electron microscopy, the middle lamella of ‘Qiu JinXing’ and ‘Ruanrou Shanlihong 3 Hao’ fruit flesh was largely degraded as the fruits matured. Microfilaments in ‘Qiu JinXing’ flesh were arranged close together and were deep in color, whereas those in ‘Ruanrou Shanlihong 3 Hao’ fruit flesh were arranged loosely, partially degraded and light in color. RNA-Seq analysis yielded approximately 46.72 Gb of clean data and 72,837 unigenes. Galactose metabolism and pentose and glucuronate interconversions are involved in cell wall metabolism, play an important role in hawthorn texture. We identified 85 unigenes related to the cell wall between hard- and soft-fleshed hawthorn fruits. Based on data analysis and real-time PCR, we suggest that β-GAL and PE4 have important functions in early fruit softening. The genes Ffase, Gns,α-GAL, PE63, XTH, and CWP, which are involved in cell wall degradation, are responsible for the different textures of hawthorn fruits. Thus, we hypothesize that the different textures of ‘Qiu JinXing’ and ‘Ruanrou Shanlihong 3 Hao’ fruits at maturity mainly result from cellulose/hemicelluloses degradation rather than from lamella degradation. Overall, we propose that different types of hydrolytic enzymes in cells interact to degrade the cell wall, resulting in ultramicroscopic Structure changes in the cell wall and, consequently, fruit softening. These results provide fundamental insight regarding the mechanisms by which hawthorn fruits acquire different textures and also lay a solid foundation for further research.

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Section: Discussionsupporting

confidence: 93%

Transcriptome Analysis and Ultrastructure Observation Reveal that Hawthorn Fruit Softening Is due to Cellulose/Hemicellulose Degradation

Zhao

Zhang

et al. 2016

Front. Plant Sci.

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“…Previous studies reported that glucanase and phosphorylase were both involved in the degradation of paramylon [ 23 , 24 ]. Seven components were annotated as β-1,3-glucanase; three of these components were recently purified from Euglena extract and identified as endo-form glucanase [ 25 ]. On the other hand, no components with significant similarity to 1,3-beta-D-glucan phosphorylase were found in our assembled database.…”

Section: Resultsmentioning

confidence: 99%

De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions

et al. 2016

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BackgroundThe phytoflagellated protozoan, Euglena gracilis, has been proposed as an attractive feedstock for the accumulation of valuable compounds such as β-1,3-glucan, also known as paramylon, and wax esters. The production of wax esters proceeds under anaerobic conditions, designated as wax ester fermentation. In spite of the importance and usefulness of Euglena, the genome and transcriptome data are currently unavailable, though another research group has recently published E.gracilis transcriptome study during our submission. We herein performed an RNA-Seq analysis to provide a comprehensive sequence resource and some insights into the regulation of genes including wax ester metabolism by comparative transcriptome analysis of E.gracilis under aerobic and anaerobic conditions.ResultsThe E.gracilis transcriptome analysis was performed using the Illumina platform and yielded 90.3 million reads after the filtering steps. A total of 49,826 components were assembled and identified as a reference sequence of E.gracilis, of which 26,479 sequences were considered to be potentially expressed (having FPKM value of greater than 1). Approximately half of all components were estimated to be regulated in a trans-splicing manner, with the addition of protruding spliced leader sequences. Nearly 40 % of 26,479 sequences were annotated by similarity to Swiss-Prot database using the BLASTX program. A total of 2080 transcripts were identified as differentially expressed genes (DEGs) in response to anaerobic treatment for 24 h. A comprehensive pathway enrichment analysis using the KEGG pathway revealed that the majority of DEGs were involved in photosynthesis, nucleotide metabolism, oxidative phosphorylation, fatty acid metabolism. We successfully identified a candidate gene set of paramylon and wax esters, including novel β-1,3-glucan and wax ester synthases. A comparative expression analysis of aerobic- and anaerobic-treated E.gracilis cells indicated that gene expression changes in these components were not extensive or dynamic during the anaerobic treatment.ConclusionThe RNA-Seq analysis provided comprehensive transcriptome information on E.gracilis for the first time, and this information will advance our understanding of this unique organism. The comprehensive analysis indicated that paramylon and wax ester metabolic pathways are regulated at post-transcriptional rather than the transcriptional level in response to anaerobic conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2540-6) contains supplementary material, which is available to authorized users.

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“…Some ␤-1,3-1,4-endoglucanases can act as a glucan synthase by transferring oligosaccharides with different DPs to sugar acceptors (44). An ␤-1,3-endoglucanase, EgCel17A from Euglena gracilis, catalyzed such transglycosylations but, ultimately, all of the intermediates were degraded to glucose, laminaribiose, and laminaritriose (45). In this study, Gly5M was suggested to recognize laminarioligosaccharides as both donors and acceptors for the synthesis of high-DP ␤-1,3-glucan oligosaccharides.…”

Section: Discussionmentioning

confidence: 99%

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 ^T and Its Transglycosylase Activity

Wang

Kim

Seo

et al. 2016

Appl Environ Microbiol

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In this study, we characterized Gly5M, originating from a marine bacterium, as a novel ␤-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. The gly5M gene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) in Saccharophagus degradans 2-40 T . The gly5M gene was cloned and overexpressed in Escherichia coli. Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry, Gly5M was identified as a novel ␤-1,3-endoglucanase (EC 3.2.1.39) and bacterial ␤-1,6-glucanase (EC 3.2.1.75) in GH5. The ␤-1,3-endoglucanase and ␤-1,6-endoglucanase activities were detected by using laminarin (a ␤-1,3-glucan with ␤-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a ␤-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward ␤-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes. IMPORTANCEIn this study, we have discovered a novel ␤-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40 T . Gly5M was identified as the newly found ␤-1,3-endoglucanase and bacterial ␤-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both ␤-1,3-glucans and ␤-1,6-glucans. Gly5M also possesses a transglycosylase activity toward ␤-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value ␤-1,3-and/or ␤-1,6-oligosaccharides. Marine macroalgae have been considered as potential sources of carbohydrates for the production of biofuels and biologically based products (1). Among the marine macroalgae, over 70 million tons of brown macroalgae are produced annually (2). The major carbon storage compounds of brown algae are alginate, laminarin, fucoidan, and mannitol (3). Laminarin is a linear polysaccharide composed of D-glucose with ␤-1,3-glycosidic linkages in the main backbone and degrees of polymerization (DPs) of 20 to 25 (4), with some 6-O-branching from D-glucose in the main chain and some ␤-1,6-glycosidic linkages as branches; the ␤-1,3-glucan/␤-1,6-glucan ratio is approximately 3:1, depending on the species of brown macroalgae (5). Among brown macroalgae, the main sources of laminarin are Laminaria and Saccharina spp. For instance, Laminaria hyperborean and Saccharina latissima contain laminarin at up to 32% of dry weight (6), and 84% of total carbohydrates are laminarin in Fucus vesiculosus (7). Unlike other marine polysaccharides, such as alginate and agar, from which monomeric sugars are not fermentable by terrestrial microorganisms (8, 9), the final hydrolys...

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Identification and enzymatic characterization of an endo-1,3-β-glucanase from Euglena gracilis

Cited by 35 publications

References 33 publications

Transcriptome Analysis and Ultrastructure Observation Reveal that Hawthorn Fruit Softening Is due to Cellulose/Hemicellulose Degradation

Transcriptome Analysis and Ultrastructure Observation Reveal that Hawthorn Fruit Softening Is due to Cellulose/Hemicellulose Degradation

De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 ^T and Its Transglycosylase Activity

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Identification and enzymatic characterization of an endo-1,3-β-glucanase from Euglena gracilis

Cited by 35 publications

References 33 publications

Transcriptome Analysis and Ultrastructure Observation Reveal that Hawthorn Fruit Softening Is due to Cellulose/Hemicellulose Degradation

Transcriptome Analysis and Ultrastructure Observation Reveal that Hawthorn Fruit Softening Is due to Cellulose/Hemicellulose Degradation

De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 T and Its Transglycosylase Activity

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A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 ^T and Its Transglycosylase Activity