Feruloyl Esterases for Biorefineries: Subfamily Classified Specificity for Natural Substrates

Underlin, Emilie Nørmølle; Frommhagen, Matthias; Dilokpimol, Adiphol; Erven, Gijs van; Kabel, Mirjam A.

doi:10.3389/fbioe.2020.00332

Cited by 42 publications

(32 citation statements)

References 62 publications

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“…Few CE family members have been biochemically characterized and the number of available crystal structures is limited [ 19 , 22 , 61 , 62 ]. The sequences of CEs with similar catalytic activities often show low identity, which has hampered sequence-based classification [ 18 , 19 , 22 , 63 ]. Moreover, many sequence-based esterase families show low substrate specificity, frequently including members that act on substrates beyond carbohydrates [ 61 ].…”

Section: Discussionmentioning

confidence: 99%

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans

Hameleers

Penttinen

Ikonen

et al. 2021

Biotechnol Biofuels

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Background Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes. Results We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. Conclusions The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.

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Section: Discussionmentioning

confidence: 99%

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans

Hameleers

Penttinen

Ikonen

et al. 2021

Biotechnol Biofuels

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“…Apparently, the hemicellulosic substrate used in the current cultivations without lignin already induced these enzymes and the presence of lignin did not further increase their production. Although not changed in relative abundance, the FAEs were still most likely responsible for the reduction in ferulate and p-coumarate moieties in the residual lignin after growth of P. anserina ( Table 2) [23]. The highly significant increase in H 2 O 2 -producing enzymes (2.9-fold) was particularly due to a single putative AA5_1 glyoxal oxidase (GLOX, #15405), which increased 7.3-fold (Fig.…”

Section: Structural Changes In Light Of the Fungal Secretomementioning

confidence: 93%

“…HSQC NMR analysis of the GAX failed to show any ferulate-specific correlation peaks, presumably due to poor solubilization/gelling of the sample. A total esterified content of (di) ferulates in the GAX sample was estimated at 2.9% (w/w) by our research group in earlier research [23].…”

Section: Cultivation Setupmentioning

confidence: 99%

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Evidence for ligninolytic activity of the ascomycete fungus Podospora anserina

Erven

Kleijn

Patyshakuliyeva

et al. 2020

Biotechnol Biofuels

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Background: The ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome.Results: Quantitative 13 C lignin internal standard py-GC-MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC-MS and HSQC NMR analyses demonstrated that C α -oxidized substructures significantly increased through fungal action, while intact β-O-4′ aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H 2 O 2 -producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis. Conclusions:For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms.

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“…These structurally more complex compounds are obtained using multi-step synthesis, considerably limiting availability. Moreover, they might be specific for certain subcategories of feruloyl esterases [18][19][20] and their use involves a tricky tandem reaction [21]. Finally, the synthesis of other more generic esters that can be used to assay esterases, including Faes, and lipases have been reported [22,23].…”

Section: Introductionmentioning

confidence: 99%

Regioselective chemo-enzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

Gherbovet¹,

Ferreira²,

Clément³

et al. 2020

Preprint

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Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. In the case of feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl O-5-feruloyl-α-l-arabinofuranosides 1a and 1b were significantly shortened (7-8 steps reduced to 4-6) and transesterification yields enhanced (from 46 to 73% for 1a and 47 to 86 % for 1b). This was achieved using enzymatic (immobilized Lipolase 100T from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxyl group of α‐l‐arabinofuranosides. Moreover, a novel feruloylated-butanetriol 4-nitrocatechol-1-yl analog 12, containing a cleavable hydroxylated linker was also synthesized in 29% overall yield in 3 steps (convergent synthesis). The latter route combined regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of 12 as a substrate to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases.

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Feruloyl Esterases for Biorefineries: Subfamily Classified Specificity for Natural Substrates

Cited by 42 publications

References 62 publications

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans

Evidence for ligninolytic activity of the ascomycete fungus Podospora anserina

Regioselective chemo-enzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

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