Electrospray multistage mass spectrometry in the negative ion mode for the unambiguous molecular and structural characterization of acidic hydrolysates from 4‐<i>O</i>‐methylglucuronoxylan generated by endoxylanases

Fouquet, Thierry; Sato, Hiroaki; Nakamichi, Yusuke; Matsushika, Akinori; Inoue, Hiroyuki

doi:10.1002/jms.4321

Cited by 8 publications

(7 citation statements)

References 21 publications

(42 reference statements)

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“…The structural characteristics of the acidic XOSs obtained from the TcXyn30C hydrolysis of beech wood glucuronoxylan were analyzed by ESI(Ϫ)-MS n (n ϭ 1 to 3) and an amaZon SL-STT2 ion trap instrument (Bruker, Bremen, Germany), as described previously (30). Instrument control, data acquisition, and data processing were performed using the Compass 1.3 SR2 software provided by Bruker.…”

Section: Methodsmentioning

confidence: 99%

“…Multistage mass spectrometry (MS n ) in the negative-ion mode has been used for the structural analysis of acidic XOSs produced by GH10, GH11, and GH30-7 xylanases (21,24,30). This technique relies on the one-way and stepwise depolymerization of the xylose chain via glycosidic bond cleavage at the reducing end of the ions, triggered by the consecutive selection/activation steps in MS n (30).…”

Section: Hydrolysis Of Xylan Time-course Analysis Of Xylan Degradatimentioning

confidence: 99%

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GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus

Nakamichi

Fujii

Fouquet³

et al. 2019

Appl Environ Microbiol

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Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C). TcXyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that TcXyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4-O-methyl-α-d-glucuronic acid (MeGlcA) substituent. TcXyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 22-(MeGlcA)-xylobiose (U4m2X). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 22-(MeGlcA)-XOSs (XnU4m2X, n ≥ 0) in the initial stages of the hydrolysis and by specific release of U4m2X from a mixture containing XnU4m2X. Our results suggest that the unique endoxylanase activity of TcXyn30C may be applicable to the production of linear and acidic XOSs. The gene xyn30C was located adjacent to the putative GH62 arabinofuranosidase gene (abf62C) in the T. cellulolyticus genome. The expression of both genes was induced by cellulose. The results suggest that TcXyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the T. cellulolyticus cellulolytic system. IMPORTANCE Xylooligosaccharides (XOSs), which are composed of xylose units with a β-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase TcXyn30C from Talaromyces cellulolyticus was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 22-(4-O-methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.

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Section: Methodsmentioning

confidence: 99%

Section: Hydrolysis Of Xylan Time-course Analysis Of Xylan Degradatimentioning

confidence: 99%

GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus

Nakamichi

Fujii

Fouquet³

et al. 2019

Appl Environ Microbiol

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“…Multistage mass spectrometry in negative-ion mode is used to analyze the structures of acidic XOSs. This technique relies on one-way and stepwise depolymerization of the xylose chain by glycosidic bond cleavage at the reducing ends of ions triggered by MS n selection/activation (6). Pairs of diagnostic product ions instantly reveal the shape of each activated species and the original architecture of the acidic XOS; this provides insights into the mode of action of the enzyme.…”

Section: Fig 3 Hydrolysis Of Xylan and Xyl 6 Bymentioning

confidence: 99%

“…Endoxylanases randomly cleave the ␤-1,4 linkage in the xylan main chain in an endo manner. This activity, which generates xylose, xylobiose (Xyl 2 ), and xylooligosaccharides (XOSs), including branched XOSs, depends on the type of side chain residues present in xylan (3)(4)(5)(6). Xyl 2 and XOSs are further cleaved into xylose by ␤-xylosidase.…”

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confidence: 99%

Mode of Action of GH30-7 Reducing-End Xylose-Releasing Exoxylanase A (Xyn30A) from the Filamentous Fungus Talaromyces cellulolyticus

Nakamichi

Fouquet

Ito

et al. 2019

Appl Environ Microbiol

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In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p-nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 22-(4-O-methyl-α-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyln (n = 2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyln (n = 2 and 3) in an exo manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of the reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite −2b, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems. IMPORTANCE Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. β-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.

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“…Since all product ions are linked to specific precursor ions in MS n experiments, the reconstruction of fragmentation pathway can give additional information on the genealogy of the ions and thus the potential identity of precursor (sub)structures. Moreover, since MS n can yield additional diagnostic ions, it is often used to differentiate constitutional isomers that are not distinguishable by MS/MS [66][67][68]. A drawback of MS n -based strategies is that the accumulation of product ion spectra is time-consuming and therefore difficult to combine with rapid UHPLC methods that result in narrow elution windows.…”

Section: Technological Advancements Facilitating Data Acquisitionmentioning

confidence: 99%

Mass spectrometry-based structure elucidation of small molecule impurities and degradation products in pharmaceutical development

Liu

Romijn

Verniest

et al. 2019

TrAC Trends in Analytical Chemistry

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Mass spectrometry (MS) has played a critical role in the identification of unknown small molecules for many years. As such, it is also a pivotal technique in the structure elucidation of drug impurities and degradation products during pharmaceutical development. In this review we discuss the regulatory expectations and some general practices regarding the identification of process-related impurities and degradation products of small molecule therapeutics; with an emphasis on the role of MS in this process. Recent advances in instrumentation, facilitating MS-based structure elucidation are covered. Over the last few years, many software tools have been developed that aid in the annotation and identification of small molecules by their precursor and product ion spectra. This evolution is promising since it is key to alleviate the current data interpretation bottleneck. The main approaches used in these bioinformatics tools are critically surveyed. This review aims to give meaningful insight in the current state of MS-based structure elucidation of small molecule impurities and degradation products.

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Electrospray multistage mass spectrometry in the negative ion mode for the unambiguous molecular and structural characterization of acidic hydrolysates from 4‐O‐methylglucuronoxylan generated by endoxylanases

Cited by 8 publications

References 21 publications

GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus

GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus

Mode of Action of GH30-7 Reducing-End Xylose-Releasing Exoxylanase A (Xyn30A) from the Filamentous Fungus Talaromyces cellulolyticus

Mass spectrometry-based structure elucidation of small molecule impurities and degradation products in pharmaceutical development

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