Identification and characterization of GH11 xylanase and GH43 xylosidase from the chytridiomycetous fungus, Rhizophlyctis rosea

Huang, Yuhong; Zheng, Xianliang; Pilgaard, Bo; Holck, Jesper; Muschiol, Jan; Li, Shengying; Lange, Lene

doi:10.1007/s00253-018-9431-5

Cited by 29 publications

(15 citation statements)

References 57 publications

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“…The specific activity of the intracellularly located GH43_1 (PUL 15) on p-NP-Ara and p-NP-Xyl was alike, coinciding with a dual specificity previously reported for some enzymes in this subfamily (Huang et al 2019;Matsuzawa et al 2017;Mewis et al 2016). This enzyme presented a specific activity over xylan substrates higher (~ one order of magnitude) than those of GH43_12 ( 3).…”

Section: Biochemical Characterization Of the Recombinant Proteinssupporting

confidence: 83%

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Novel xylan degrading enzymes from polysaccharide utilizing loci ofPrevotella copriDSM18205

Linares-Pastén

Hero

Pisa

et al. 2020

Preprint

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Prevotella copri DSM18205 is a bacterium, classified under Bacteroidetes that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers in P. copri, which currently are unexplored. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight GH-encoding genes, respectively. Three of the eight gene products were successfully produced in Escherichia coli: One monomeric two-domain extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two dimeric single module enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 subfamily 1 enzyme (37 kDa). The GH43_12 enzyme was hydrolysing arabinofuranose residues from different substrates, and a model of the 3D-structure revealed a single arabinose binding pocket. The GH10 (1) and GH43_1 are cleaving the xylan backbone. Hydrolysis products of GH10 (1) were DP2-4, and seven subsites (−3 to +4) were predicted in the 3D-model of the GH10 active site. GH43_1 mainly produced xylose (in line with its intracellular location). Based on our results we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-β-xylanase, that hydrolyses mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular β-xylosidase, catalysing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalysing removal of arabinose-residues on xylan polymers.

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Section: Biochemical Characterization Of the Recombinant Proteinssupporting

confidence: 83%

“…On the other hand, the β-xylosidase GH43_12 from PUL 10 was confirmed to be a monomer in solution, with a molecular mass of 60.1 kDa ( ions in its proposed model that further supports such structure. However, the enzyme remained partially active as a monomer (Huang et al 2019).…”

Section: Cloning and Production Of The Xylan Degrading Enzymes From Pmentioning

confidence: 98%

Novel xylan degrading enzymes from polysaccharide utilizing loci ofPrevotella copriDSM18205

Linares-Pastén

Hero

Pisa

et al. 2020

Preprint

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“…A reduced amount of X 3 , combined with an increase of X 2 was concomitant to the production of xylose by the Bh Xyl43. This accumulation of xylose revealed the specificity of the xylosidase, which is able to hydrolase small oligosaccharides produced by the xylanase [ 58 ]. Intermediate xylooligosaccharides ranging from X 4 to X 6 were not accumulated as they are substrate for both enzymes.…”

Section: Resultsmentioning

confidence: 99%

“…The arc shape or the “M” shape of the complexes resulting in a different positioning of the Jo module fused to Bh Xyl43 impacted the activity of each chimeric enzyme. Of course, a xylanase supplemented with a xylosidase increases the total activity, as measured by total reducing end sugar compared to xylanase alone [ 58 ]. However, by covalently associating the two GHs, we were able to modulate the SA or to increase the amount of hydrolytic events, as measured by DNSA.…”

Section: Discussionmentioning

confidence: 99%

Characterisation of the Effect of the Spatial Organisation of Hemicellulases on the Hydrolysis of Plant Biomass Polymer

Enjalbert

Mare

Roblin

et al. 2020

IJMS

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Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature in the recycling of the carbon derived from plant cell wall polymers. By using an innovative covalent association process using two protein fragments, Jo and In, we produced two bi-modular chimeric complexes connecting a xylanase and a xylosidase, involved in the deconstruction of xylose-based plant cell wall polymer. We first show that the intrinsic activity of the individual enzymes was preserved. Small Angle X-rays Scattering (SAXS) analysis of the complexes highlighted two different spatial organisations in solution, affecting both the distance between the enzymes (53 Å and 28 Å) and the distance between the catalytic pockets (94 Å and 75 Å). Reducing sugar and HPAEC-PAD analysis revealed different behaviour regarding the hydrolysis of Beechwood xylan. After 24 h of hydrolysis, one complex was able to release a higher amount of reducing sugar compare to the free enzymes (i.e., 15,640 and 14,549 µM of equivalent xylose, respectively). However, more interestingly, the two complexes were able to release variable percentages of xylooligosaccharides compared to the free enzymes. The structure of the complexes revealed some putative steric hindrance, which impacted both enzymatic efficiency and the product profile. This report shows that controlling the spatial geometry between two enzymes would help to better investigate synergism effect within complex multi-enzymatic machinery and control the final product.

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“…Beta-1,4-endoglucanase has been successfully produced in Pichia pastoris (Pilgaard 2014; Lange et al 2018). Furthermore, Huang et al (2018) recombinantly produced both GH11 and GH43 proteins from R. rosea in P. pastoris . Production of enzymes from rumen fungi has also been shown to be possible for genes from Piromyces and Orpinomyces (Gruninger et al 2014).…”

Section: Recombinant Production Of Enzymes Of Zoosporic Fungal Originmentioning

confidence: 99%

Enzymes of early-diverging, zoosporic fungi

Lange¹,

Barrett

Pilgaard

et al. 2019

Appl Microbiol Biotechnol

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The secretome, the complement of extracellular proteins, is a reflection of the interaction of an organism with its host or substrate, thus a determining factor for the organism’s fitness and competitiveness. Hence, the secretome impacts speciation and organismal evolution. The zoosporic Chytridiomycota , Blastocladiomycota , Neocallimastigomycota , and Cryptomycota represent the earliest diverging lineages of the Fungal Kingdom. The review describes the enzyme compositions of these zoosporic fungi, underscoring the enzymes involved in biomass degradation. The review connects the lifestyle and substrate affinities of the zoosporic fungi to the secretome composition by examining both classical phenotypic investigations and molecular/genomic-based studies. The carbohydrate-active enzyme profiles of 19 genome-sequenced species are summarized. Emphasis is given to recent advances in understanding the functional role of rumen fungi, the basis for the devastating chytridiomycosis, and the structure of fungal cellulosome. The approach taken by the review enables comparison of the secretome enzyme composition of anaerobic versus aerobic early-diverging fungi and comparison of enzyme portfolio of specialized parasites, pathogens, and saprotrophs. Early-diverging fungi digest most major types of biopolymers: cellulose, hemicellulose, pectin, chitin, and keratin. It is thus to be expected that early-diverging fungi in its entirety represents a rich and diverse pool of secreted, metabolic enzymes. The review presents the methods used for enzyme discovery, the diversity of enzymes found, the status and outlook for recombinant production, and the potential for applications. Comparative studies on the composition of secretome enzymes of early-diverging fungi would contribute to unraveling the basal lineages of fungi.

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Identification and characterization of GH11 xylanase and GH43 xylosidase from the chytridiomycetous fungus, Rhizophlyctis rosea

Cited by 29 publications

References 57 publications

Novel xylan degrading enzymes from polysaccharide utilizing loci ofPrevotella copriDSM18205

Novel xylan degrading enzymes from polysaccharide utilizing loci ofPrevotella copriDSM18205

Characterisation of the Effect of the Spatial Organisation of Hemicellulases on the Hydrolysis of Plant Biomass Polymer

Enzymes of early-diverging, zoosporic fungi

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