Novel xylanolytic triple domain enzyme targeted at feruloylated arabinoxylan degradation

Holck, Jesper; Djajadi, Demi T.; Brask, Jesper; Pilgaard, Bo; Krogh, Kristian B. R. M.; Meyer, Anne S.; Lange, Lene; Wilkens, Casper

doi:10.1016/j.enzmictec.2019.05.010

Cited by 15 publications

(13 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2A-B), but the properties and the topology of the active site pocket and the binding cleft are very different ( Fig. 8A-E BK010417.1) for which the above mentioned characteristics were previously described (22).…”

Section: The Role Of Cbm48 In Relation To Substrate Distortion To Assmentioning

confidence: 78%

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan

Holck

Fredslund

Møller

et al. 2019

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μm and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1–CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

show abstract

Section: The Role Of Cbm48 In Relation To Substrate Distortion To Assmentioning

confidence: 78%

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan

Holck

Fredslund

Møller

et al. 2019

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…A unique feature has been reported for the first time from an exceptional metagenomic xylanase (UX66) that comprises two CBDs and two catalytic domains (Zhao et al, 2010). Another unique three catalytic domain multi-enzyme (a CE1 ferulic acid esterase, a GH62 α-L-arabinofuranosidase, and a GH10 β-D-1,4-xylanase) has been recovered from the metagenome of wastewater treatment sludge (Holck et al, 2019). Such attributes enhance the applicability of xylanase manifold over the xylanases having one single catalytic domain.…”

Section: Unusual Metagenomic Xylanolytic Enzymesmentioning

confidence: 99%

Xylanolytic Extremozymes Retrieved From Environmental Metagenomes: Characteristics, Genetic Engineering, and Applications

Verma

Satyanarayana

2020

Front. Microbiol.

View full text Add to dashboard Cite

Xylanolytic enzymes have extensive applications in paper, food, and feed, pharmaceutical, and biofuel industries. These industries demand xylanases that are functional under extreme conditions, such as high temperature, acidic/alkaline pH, and others, which are prevailing in bioprocessing industries. Despite the availability of several xylan-hydrolyzing enzymes from cultured microbes, there is a huge gap between what is available and what industries require. DNA manipulations as well as protein-engineering techniques are also not quite satisfactory in generating xylanhydrolyzing extremozymes. With a compound annual growth rate of 6.6% of xylanhydrolyzing enzymes in the global market, there is a need for xylanolytic extremozymes. Therefore, metagenomic approaches have been employed to uncover hidden xylanolytic genes that were earlier inaccessible in culture-dependent approaches. Appreciable success has been achieved in retrieving several unusual xylanolytic enzymes with novel and desirable characteristics from different extreme environments using functional and sequence-based metagenomic approaches. Moreover, the Carbohydrate Active Enzymes database includes approximately 400 GH-10 and GH-11 unclassified xylanases. This review discusses sources, characteristics, and applications of xylanolytic enzymes obtained through metagenomic approaches and their amelioration by genetic engineering techniques.

show abstract

“…The electrospray was operated in negative mode with UltraScan mode and a scan range from 100 to 1000 m/z, smart parameter setting of 190 m/z, capillary voltage at 4.5 kV, endplate off-set 0.5 kV, nebulizer pressure at 3.0 bar, dry gas flow at 12.0 L/min, and dry gas temperature at 280 °C. CID fragmentation of deprotonated ions was performed by Multiple Reaction Monitoring using SmartFrag enhanced amplitude ramping from 80 to 120%, fragmentation time 20 ms. Quantification was performed in Compass QuantAnalysis 2.2 (Bruker Daltonics, Bremen Germany) using trans-ferulic acid as external standard and adjusted according to the internal standard as described previously [ 38 ].…”

Section: Methodsmentioning

confidence: 99%

High throughput in vitro characterization of pectins for pig(let) nutrition

et al. 2021

Self Cite

View full text Add to dashboard Cite

Background Fiber-rich feed components possess prebiotic potential to enhance pig health and are considered a potential solution to the high prevalence of post-weaning diarrhea in pig production under the phased suspension of antibiotics and zinc oxide use. Methods We screened the gut microbiota modulatory properties of pectin substrates prepared from sugar beet within the freshly weaned piglet gut microbiome using an in vitro colon model, the CoMiniGut. We focused on testing a variety (13) of sugar beet-derived pectin substrates with defined structures, as well as known prebiotics such as inulin, fructooligosaccharide (FOS) and galactooligosaccharide (GOS), to gain insights on the structure–function related properties of specific substrates on the weaner gut microbial composition as well as shortchain fatty acid production (SCFA). Results Sugar beet-derived pectin and rhamnogalacturonan-I selectively increased the relative abundance of Bacteroidetes, specifically Prevotella copri, Bacteroides ovatus, Bacteroides acidificiens, and an unclassified Bacteroides member. The degree of esterification impacted the relative abundance of these species and the SCFA production during the in vitro fermentations. Modified arabinans derived from sugar beet promoted the growth of Blautia, P. copri, Lachnospiraceae members and Limosilactobacillus mucosae and amongst all oligosaccharides tested yielded the highest amount of total SCFA produced after 24 h of fermentation. Sugar beet-derived substrates yielded higher total SCFA concentrations (especially acetic and propionic acid) relative to the known prebiotics inulin, FOS and GOS. Conclusion Our results indicate that the molecular structures of pectin, that can be prepared form just one plant source (sugar beet) can selectively stimulate different GM members, highlighting the potential of utilizing pectin substrates as targeted GM modulatory ingredients.

show abstract

Novel xylanolytic triple domain enzyme targeted at feruloylated arabinoxylan degradation

Abstract: releasing soluble oligosaccharides that CE1 can utilize as substrate. CE1 also displayed activity towards solubilized 5-O-trans-feruloyl-α-L-Araf (kcat = 36.35 s-1). This suggests that CE1 preferably acts on soluble oligosaccharides.

Cited by 15 publications

References 30 publications

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan

Xylanolytic Extremozymes Retrieved From Environmental Metagenomes: Characteristics, Genetic Engineering, and Applications

High throughput in vitro characterization of pectins for pig(let) nutrition

Contact Info

Product

Resources

About