Affinity Electrophoresis for Analysis of Catalytic Module–Carbohydrate Interactions

Cockburn, Darrell; Wilkens, Casper; Svensson, Birte

doi:10.1007/978-1-0716-3151-5_6

Cited by 3 publications

(2 citation statements)

References 22 publications

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“…There is also a high molecular weight band found in both the recombinant protein mixture and the maltose-grown C. butyricum supernatant. This is likely to be proteins that were not fully unfolded during the milder than typical incubation in loading buffer (performed at 60 °C) whose progress through the gel was then slowed through interaction with the incorporated amylopectin as is typically seen in affinity electrophoresis experiments (53). These results suggest that maltose serves as an inducer of C. butyricum's starch digestion system and that Amy13A, Amy13B and Amy13C are the key α-amylases of that system.…”

Section: Zymogram Analysismentioning

confidence: 82%

Clostridium butyricum Prazmowski can degrade and utilize resistant starch via a set of synergistically acting enzymes

Cockburn,

Pickens

2023

Preprint

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Resistant starch is a prebiotic fiber that is best known for its ability to increase butyrate production by the gut microbiota. This butyrate then plays an important role modulating the immune system and inflammation. However, the ability to use this resistant starch appears to be a rare trait within the gut microbiota, with only a few species such as Ruminococcus bromii and Bifidobacterium adolescentis having been demonstrated to possess this ability. Furthermore, these bacteria do not directly produce butyrate themselves, rather they rely on cross-feeding interactions with other gut bacteria for its production. Here we demonstrate that the often-used probiotic organism Clostridium butyricum also possesses the ability to utilize resistant starch from a number of sources, with direct production of butyrate. We further explore the enzymes responsible for this trait, demonstrating that they exhibit significant synergy, though with different enzymes exhibiting more or less importance depending on the source of the resistant starch. Thus, the co-administration of Clostridium butyricum may have the ability to improve the beneficial effects of resistant starch.

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Section: Zymogram Analysismentioning

confidence: 82%

Clostridium butyricum Prazmowski can degrade and utilize resistant starch via a set of synergistically acting enzymes

Cockburn,

Pickens

2023

Preprint

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show abstract

“…There is also a high molecular weight band found in both the recombinant protein mixture and the maltose-grown C. butyricum supernatant. This is likely to be proteins that were not fully unfolded during the milder than typical incubation in loading buffer (performed at 60°C) whose progress through the gel was then slowed through interaction with the incorporated amylopectin as is typically seen in affinity electrophoresis experiments ( 31 ). These results suggest that maltose serves as an inducer of C. butyricum ’s starch digestion system and that Amy13A, Amy13B, and Amy13C are the key α-amylases of that system.…”

Section: Resultsmentioning

confidence: 99%

Clostridium butyricum Prazmowski can degrade and utilize resistant starch via a set of synergistically acting enzymes

Pickens,

Cockburn

2024

mSphere

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Resistant starch is a prebiotic fiber that is best known for its ability to increase butyrate production by the gut microbiota. This butyrate then plays an important role in modulating the immune system and inflammation. However, the ability to use this resistant starch appears to be a rare trait within the gut microbiota, with only a few species such as Ruminococcus bromii and Bifidobacterium adolescentis having been demonstrated to possess this ability. Furthermore, these bacteria do not directly produce butyrate themselves, rather they rely on cross-feeding interactions with other gut bacteria for its production. Here, we demonstrate that the often-used probiotic organism Clostridium butyricum also possesses the ability to utilize resistant starch from a number of sources, with direct production of butyrate. We further explore the enzymes responsible for this trait, demonstrating that they exhibit significant synergy, though with different enzymes exhibiting more or less importance depending on the source of the resistant starch. Thus, the co-administration of Clostridium butyricum may have the ability to improve the beneficial effects of resistant starch. IMPORTANCE Clostridium butyricum is seeing increased use as a probiotic, due to potential health benefits tied to its ability to produce butyrate. Here, we demonstrate that this organism can use a variety of resistant starch sources and characterize the enzymes it uses to accomplish this. Given the relative rarity of resistant starch utilizing ability within the gut and the health benefits tied to resistant starch, the combined use of this organism with resistant starch in synbiotic formulations may prove beneficial.

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Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile

Norlander,

Jasilionis,

Allahgholi

et al. 2024

Glycobiology

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Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC–PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.

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Affinity Electrophoresis for Analysis of Catalytic Module–Carbohydrate Interactions

Cited by 3 publications

References 22 publications

Clostridium butyricum Prazmowski can degrade and utilize resistant starch via a set of synergistically acting enzymes

Clostridium butyricum Prazmowski can degrade and utilize resistant starch via a set of synergistically acting enzymes

Clostridium butyricum Prazmowski can degrade and utilize resistant starch via a set of synergistically acting enzymes

Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile

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