Synergistic degradation of arabinoxylan by free and immobilized xylanases and arabinofuranosidase

Jia, Lili; Budinova, Geisa A.L.G.; Takasugi, Yusaku; Noda, Shinshi; Tanaka, Tsutomu; Ichinose, Hirofumi; Goto, Masahiro; Kamiya, Noriho

doi:10.1016/j.bej.2016.07.013

Cited by 24 publications

(4 citation statements)

References 38 publications

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“…The main substituted sugar released had the structure A 2 X, which is arabinose substituted at the non‐reducing end of xylobiose . Consistent with previous research, the arabinofuranosidase used in the current study (AraF‐ST) only displays activity toward short oligomers rather than long polymer substrates . Thus, the ability of endo‐type Xyn‐ST to depolymerize the arabinoxylan chain played a major role in the release of short oligomers in the hydrolysis of arabinoxylan.…”

Section: Resultssupporting

confidence: 86%

Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio‐Controllable Manner

Jia

Minamihata

Ichinose

et al. 2017

Biotechnology Journal

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Conjugating enzymes into a large protein assembly often results in an enhancement of overall catalytic activity, especially when different types of enzymes that work cooperatively are assembled together. However, exploring the proper method to achieve protein assemblies with high stability and also to avoid loss of the function of each component for efficient enzyme clustering is remained challenging. Assembling proteins onto synthetic scaffolds through varied post-translational modification methods is particularly favored since the proteins can be site-specifically conjugated together with less activity loss. Here, a SpyCatcher polymer is prepared through catalytic reaction of horseradish peroxidase (HRP) and serves as a polymeric proteinaceous scaffold for construction of protein assemblies. Taking advantage of the favorable SpyCatcher-SpyTag interaction, SpyTagged proteins can be easily assembled onto the polymeric SpyCatcher scaffold with controllable binding ratio and site specificity. Firstly, the feasibility of construction of ratio-controllable binary artificial hemicellulosomes by assembling endoxylanase and arabinofuranosidase is explored. This construct achieves higher sugar conversion than that of the free enzymes when the proportion of arabinofuranosidase is high, because the close spatial proximity of the enzymes allows them to work in a synergistic manner. Another application for biosensing is developed by conjugating SpyTagged Nanoluc and protein G onto SpyCatcher polymer. Due to the protein clustering effect, an amplified luminescent intensity is achieved by the resulting conjugates than chimera protein of Nanoluc and protein G in ovalbumin detection in ELISA.

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Section: Resultssupporting

confidence: 86%

Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio‐Controllable Manner

Jia

Minamihata

Ichinose

et al. 2017

Biotechnology Journal

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“…Yeasts in this clade were also found to contain genes for different xylanolytic de-branching enzymes, e.g., likely GH43, GH51 and GH62 α- l -arabinofuranosidases and GH67 α-glucuronidases. These could enable de-branching of complex xylans and reduce steric hindrances thus promoting the access to the xylan backbone for main-chain cleaving xylanases [ 60 ].…”

Section: Discussionmentioning

confidence: 99%

CAZyme prediction in ascomycetous yeast genomes guides discovery of novel xylanolytic species with diverse capacities for hemicellulose hydrolysis

Ravn

Engqvist

Larsbrink

et al. 2021

Biotechnol Biofuels

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Background Ascomycetous yeasts from the kingdom fungi inhabit every biome in nature. While filamentous fungi have been studied extensively regarding their enzymatic degradation of the complex polymers comprising lignocellulose, yeasts have been largely overlooked. As yeasts are key organisms used in industry, understanding their enzymatic strategies for biomass conversion is an important factor in developing new and more efficient cell factories. The aim of this study was to identify polysaccharide-degrading yeasts by mining CAZymes in 332 yeast genomes from the phylum Ascomycota. Selected CAZyme-rich yeasts were then characterized in more detail through growth and enzymatic activity assays. Results The CAZyme analysis revealed a large spread in the number of CAZyme-encoding genes in the ascomycetous yeast genomes. We identified a total of 217 predicted CAZyme families, including several CAZymes likely involved in degradation of plant polysaccharides. Growth characterization of 40 CAZyme-rich yeasts revealed no cellulolytic yeasts, but several species from the Trichomonascaceae and CUG-Ser1 clades were able to grow on xylan, mixed-linkage β-glucan and xyloglucan. Blastobotrys mokoenaii, Sugiyamaella lignohabitans, Spencermartinsiella europaea and several Scheffersomyces species displayed superior growth on xylan and well as high enzymatic activities. These species possess genes for several putative xylanolytic enzymes, including ones from the well-studied xylanase-containing glycoside hydrolase families GH10 and GH30, which appear to be attached to the cell surface. B. mokoenaii was the only species containing a GH11 xylanase, which was shown to be secreted. Surprisingly, no known xylanases were predicted in the xylanolytic species Wickerhamomyces canadensis, suggesting that this yeast possesses novel xylanases. In addition, by examining non-sequenced yeasts closely related to the xylanolytic yeasts, we were able to identify novel species with high xylanolytic capacities. Conclusions Our approach of combining high-throughput bioinformatic CAZyme-prediction with growth and enzyme characterization proved to be a powerful pipeline for discovery of novel xylan-degrading yeasts and enzymes. The identified yeasts display diverse profiles in terms of growth, enzymatic activities and xylan substrate preferences, pointing towards different strategies for degradation and utilization of xylan. Together, the results provide novel insights into how yeast degrade xylan, which can be used to improve cell factory design and industrial bioconversion processes.

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“…Literature indicates that ABFs are one of the rate-limiting enzymes in xylan degradation (Saha 2000 ), and their application showed a strong synergistic role with endo-xylanase in degradation of arabinoxylans into arabinose, xylose, and xylooligosaccharides (XOS) (Goncalves et al 2012 ; Jia et al 2016 ; Ravn et al 2018 ). Compared with the individual enzymes, the total amount of released sugar from wheat arabinoxylan was increased to 2.92-fold with simultaneous addition of a GH51 ABF and the GH10 endoxylanase XynBE18 (Yang et al 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Characterization and functional analysis of two novel thermotolerant α-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

Long

Sun

Lin

et al. 2020

Appl Microbiol Biotechnol

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Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-l-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-l-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. 1H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone. Key points • Two novel α-l-arabinofuranosidases (ABFs) displayed high thermal stability. • The thermal stability of GH62 family EpABF62C was dependent on calcium. • Buffer pH affects the thermal stability of the two ABFs. • Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

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Synergistic degradation of arabinoxylan by free and immobilized xylanases and arabinofuranosidase

Cited by 24 publications

References 38 publications

Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio‐Controllable Manner

Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio‐Controllable Manner

CAZyme prediction in ascomycetous yeast genomes guides discovery of novel xylanolytic species with diverse capacities for hemicellulose hydrolysis

Characterization and functional analysis of two novel thermotolerant α-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

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