Cloning of theRuminococcus albus cel5Dandcel9AGenes Encoding Dockerin Module-containing Endoglucanases and Expression ofcel5DinEscherichia coli

Taguchi, Hidenori; Hagiwara, Daisuke; Genma, Tsutomu; Karita, Shuichi; Kimura, Takeshi; Sakka, Kazuo; Ohmiya, Kunio

doi:10.1271/bbb.68.1557

“…albus appears to use a variety of modes of attachment to cellulosic substrates. A cellulosome-like complex that contained at least 15 proteins was identified in strain F-40 (Ohara et al, 2000a), and characterised F-40 fibrolytic enzymes contain the requisite dockerin and CBMs suggestive of bacterial cell attachment to cellulose (Ohara et al, 2000b, Taguchi et al, 2004a. Cellulose adhesion defective R. albus 8 mutants have been generated that lack two cell-bound cellulolytic enzymes, neither of which contain dockerin domains, which suggests that non-cellulosome associated secreted fibrolytic enzymes may play a direct role in cell attachment to cellulose (Devillard et al, 2004).…”

Section: Prevotella Bryantii and Prevotella Ruminicolamentioning

confidence: 99%

A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

Dunne¹

0

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Butyrivibrio proteoclasticus B316T is a Gram-positive, lignocellulose degrading bacterium that is prevalent in the rumen of animals grazing pasture, and is one of only a few rumen microbes known to degrade and utilise xylan in vitro. Xylan is a hemicellulose that comprises up to 45% of the polysaccharide component of ruminant forages. Often as little as 30% of the total energy content of forages is utilised by the ruminant due to poor hemicellulose degradation by the fibrolytic rumen microbes. An opportunity exists to improve forage degradation in the rumen, which is predicted to improve the productivity of forage fed ruminants. A clearer understanding of the strategies employed by fibrolytic rumen microbes to degrade and utilise lignocellulose is important in realising this goal. Almost 10% of the B. proteoclasticus genome encodes proteins involved in polysaccharide metabolism and transport, which includes 134 fibrolytic enzymes that are active upon plant fibre. Many of these are clustered into one of 36 polysaccharide utilisation loci that also contain transmembrane transporters, transcriptional regulators, environmental sensors and genes involved in further polysaccharide metabolism. Gel-based and gel-free proteomic analyses of the cytosolic, cell-associated, and secreted fractions of cells grown on xylan were used to identify proteins involved in the degradation, assimilation, and metabolism of hemicellulose. A set of 416 non-redundant proteins were identified, which included 12 extracellular and 24 cytosolic polysaccharidases, and 59 proteins involved in the uptake and further metabolism of polysaccharide degradation products, many of which were substrate-binding protein components of ATP-driven transporter systems. In cells grown on xylan, several of these proteins displayed significant protein abundance changes relative to cells grown on the monomeric sugar xylose, in a pattern that reflected the growth substrates used. A model of xylan degradation by B. proteoclasticus based on these results hypothesises that B. proteoclasticus attacks the xylan backbone and main substituent groups of hemicellulose in the extracellular space, assimilates the xylooligosaccharides and performs the final stages of degradation within the cell. These results provide insight into a xylan degrading enzyme system that has evolved to efficiently degrade and utilise hemicellulose, extend our understanding of the enzymes that are likely to play important roles in hemicellulose degradation, and support the notion that Butyrivibrio species are important contributors to rumen fibre degradation.

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“…albus appears to use a variety of modes of attachment to cellulosic substrates. A cellulosome-like complex that contained at least 15 proteins was identified in strain F-40 (Ohara et al, 2000a), and characterised F-40 fibrolytic enzymes contain the requisite dockerin and CBMs suggestive of bacterial cell attachment to cellulose (Ohara et al, 2000b, Taguchi et al, 2004a. Cellulose adhesion defective R. albus 8 mutants have been generated that lack two cell-bound cellulolytic enzymes, neither of which contain dockerin domains, which suggests that non-cellulosome associated secreted fibrolytic enzymes may play a direct role in cell attachment to cellulose (Devillard et al, 2004).…”

Section: Prevotella Bryantii and Prevotella Ruminicolamentioning

confidence: 99%

A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

Dunne¹

0

View full text Add to dashboard Cite

Butyrivibrio proteoclasticus B316T is a Gram-positive, lignocellulose degrading bacterium that is prevalent in the rumen of animals grazing pasture, and is one of only a few rumen microbes known to degrade and utilise xylan in vitro. Xylan is a hemicellulose that comprises up to 45% of the polysaccharide component of ruminant forages. Often as little as 30% of the total energy content of forages is utilised by the ruminant due to poor hemicellulose degradation by the fibrolytic rumen microbes. An opportunity exists to improve forage degradation in the rumen, which is predicted to improve the productivity of forage fed ruminants. A clearer understanding of the strategies employed by fibrolytic rumen microbes to degrade and utilise lignocellulose is important in realising this goal. Almost 10% of the B. proteoclasticus genome encodes proteins involved in polysaccharide metabolism and transport, which includes 134 fibrolytic enzymes that are active upon plant fibre. Many of these are clustered into one of 36 polysaccharide utilisation loci that also contain transmembrane transporters, transcriptional regulators, environmental sensors and genes involved in further polysaccharide metabolism. Gel-based and gel-free proteomic analyses of the cytosolic, cell-associated, and secreted fractions of cells grown on xylan were used to identify proteins involved in the degradation, assimilation, and metabolism of hemicellulose. A set of 416 non-redundant proteins were identified, which included 12 extracellular and 24 cytosolic polysaccharidases, and 59 proteins involved in the uptake and further metabolism of polysaccharide degradation products, many of which were substrate-binding protein components of ATP-driven transporter systems. In cells grown on xylan, several of these proteins displayed significant protein abundance changes relative to cells grown on the monomeric sugar xylose, in a pattern that reflected the growth substrates used. A model of xylan degradation by B. proteoclasticus based on these results hypothesises that B. proteoclasticus attacks the xylan backbone and main substituent groups of hemicellulose in the extracellular space, assimilates the xylooligosaccharides and performs the final stages of degradation within the cell. These results provide insight into a xylan degrading enzyme system that has evolved to efficiently degrade and utilise hemicellulose, extend our understanding of the enzymes that are likely to play important roles in hemicellulose degradation, and support the notion that Butyrivibrio species are important contributors to rumen fibre degradation.

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Heterologous expression and characterization of two novel glucanases derived from sheep rumen microbiota

Gao

¹

,

Sun

²

,

Fang

³

et al. 2022

World J Microbiol Biotechnol

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β-Glucanases are a suite of glycoside hydrolases that depolymerize β-glucan into cellooligosaccharides and/or monosaccharides and have been widely used as feed additives in livestock. In this study, two novel glucanase genes, IDSGluc5-26 and IDSGluc5-37, derived from sheep rumen microbiota, were expressed and functionally characterized. The optimal temperatures/pH of recombinant IDSGLUC5-26 and IDSGLUC5-37 were 50°C/5.0 and 40°C/6.0, respectively. Notably, IDSGLUC5-26 showed considerable stability under acidic conditions. Both IDSGLUC5-26 and IDSGLUC5-37 showed the highest activities toward barley β-glucan, with V max values of 89.96 ± 9.19 µmol/min/mg and 459.50 ± 25.02 µmol/min/mg, respectively. Additionally, these two glucanases demonstrated hydrolysis of barley βglucan and Icelandic moss lichenan, IDSGLUC5-26 releasing cellobiose (G2; occupying 17.37% of total reducing sugars), cellotriose (G3; 23.97%), and cellotetraose (G4; 30.93%) from β-glucan and lichenan after 10 min and suggestive of a typical endo-β-1,4-glucanase (EC.3.2.1.4). In contrast, IDSGLUC5-37 was capable of liberating dominant G3 (64.11% or 67.55%) from β-glucan or lichenan, suggesting that the enzyme was likely an endo-β-1,3-1,4-glucanases/lichenase (EC3.2.1.73). These ndings describe the expression and characterization of two novel glucanase genes from sheep rumen microbiota. The two recombinant enzymes, particularly the acid-adapted IDSGLUC5-26, will be of interest for potential application in food-/feed-additive development.

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Cloning of theRuminococcus albus cel5Dandcel9AGenes Encoding Dockerin Module-containing Endoglucanases and Expression ofcel5DinEscherichia coli

Cited by 5 publications

References 22 publications

A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

Heterologous expression and characterization of two novel glucanases derived from sheep rumen microbiota

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