Kinetic Studies of a Recombinant Cellobiose Phosphorylase (CBP) of the Clostridium thermocellum YM4 Strain Expressed in Escherichia coli

Kim, Yeon-Kye; Kitaoka, Motomitsu; Krishnareddy, M.; Mori, Yutaka; Hayashi, Kiyoshi

doi:10.1093/oxfordjournals.jbchem.a003210

Cited by 51 publications

(48 citation statements)

References 35 publications

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“…Cellobiose phosphorylase was first found in Clostridium thermocellum, then later in other cellulolytic bacteria, including Cellvibrio gilvus [12,31], Ruminococcus flavefaciens [3], Ruminococcus albus [18], Prevotella ruminicola [19], Clostridium thermocellum [1,16,22,42], Clostridium stercorarium [29], Clostridium cellulolyticum [10], Thermotoga neapolitana [41], and Thermotoga maritime [28]. Most of these bacteria are anaerobes.…”

Section: Discussionmentioning

confidence: 99%

Hydrolytic and phosphorolytic metabolism of cellobiose by the marine aerobic bacterium Saccharophagus degradans 2-40T

Zhang

Moon

Watson

et al. 2011

J Ind Microbiol Biotechnol

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Saccharophagus degradans 2-40 is a marine gamma proteobacterium that can produce polyhydroxyalkanoates from lignocellulosic biomass using a complex cellulolytic system. This bacterium has been annotated to express three surface-associated β-glucosidases (Bgl3C, Ced3A, and Ced3B), two cytoplasmic β-glucosidases (Bgl1A and Bgl1B), and unusual for an aerobic bacterium, two cytoplasmic cellobiose/cellodextrin phosphorylases (Cep94A and Cep94B). Expression of the genes for each of the above enzymes was induced when cells were transferred into a medium containing Avicel as the major carbon source except for Bgl1B. Both hydrolytic and phosphorolytic degradation of cellobiose by crude cell lysates obtained from cellulose-grown cells were demonstrated and all of these activities were cell-associated. With the exception of Cep94B, each purified enzyme exhibited their annotated activity upon cloning and expression in E. coli. The five β-glucosidases hydrolyzed a variety of glucose derivatives containing β-1, (2, 4, or 6) linkages but did not act on any α-linked glucose derivatives. All but one β-glucosidases exhibited transglycosylation activity consistent with the formation of an enzyme-substrate intermediate. The biochemistry and expression of these cellobiases indicate that external hydrolysis by surface-associated β-glucosidases coupled with internal hydrolysis and phosphorolysis are all involved in the metabolism of cellobiose by this bacterium.

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Section: Discussionmentioning

confidence: 99%

Hydrolytic and phosphorolytic metabolism of cellobiose by the marine aerobic bacterium Saccharophagus degradans 2-40T

Zhang

Moon

Watson

et al. 2011

J Ind Microbiol Biotechnol

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“…Observed K m values were substantially lower for cellopentaose (mean value for Avicel-and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM). These should be regarded as apparent K m values, since they were measured at a given concentration of inorganic phosphate (8 mM) and it is expected in light of mechanistic considerations that the K m for ␤-glucans will be a function of the phosphate concentration (15,22). As reviewed elsewhere (29), the potential bioenergetic benefit from phosphorolytic bond cleavage is greater, and the bioenergetic cost of substrate transport is smaller, for cellodextins than for cellobiose.…”

Section: Discussionmentioning

confidence: 99%

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Zhang¹,

Lynd

2004

Appl Environ Microbiol

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Rates of phosphorolytic cleavage of ␤-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)-and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by >20-fold for both cellobiose and cellopentaose over a 10-fold range of ␤-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of ␤-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V max values indicated that cellobiose-and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel-and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).

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“…2). These results indicate that the phosphorolytic reaction of GalGlyNAcP Vv followed the sequential bi-bi mechanism, like the reactions of inverting phosphorylases, such as GalGlyNAcP from B. bifidum (5), GalGlyNAcP Bl (27), GalGlyNAcP Cp (27), maltose phosphorylase (39), cellobiose phosphorylase (15,17,28,34), chitobiose phosphorylase (12), and laminaribiose phosphorylase (18).…”

Section: Resultsmentioning

confidence: 88%

Identification of Lacto-N-Biose I Phosphorylase fromVibrio vulnificusCMCP6

Nakajima

Kitaoka

2008

Appl Environ Microbiol

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A ␤-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalGlyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GlcNAc and GalNAc as acceptors in the presence of ␣-D-galactose-1-phosphate as a donor to form lacto-N-biose I (LNB) (Gal␤1 3 3GlcNAc) and galacto-N-biose (GNB) (Gal␤1 3 3GalNAc), respectively. GlcNAc was a much better acceptor than GalNAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the k cat /K m for LNB was approximately 60 times higher than the k cat /K m for GNB. This result indicated that the enzyme was remarkably different from GalGlyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GalGlyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated lacto-N-biose I phosphorylase. The discovery of an LNB-specific GalGlyNAcP resulted in recategorization of bifidobacterial GalGlyNAcPs as galacto-N-biose/ lacto-N-biose I phosphorylases.

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Kinetic Studies of a Recombinant Cellobiose Phosphorylase (CBP) of the Clostridium thermocellum YM4 Strain Expressed in Escherichia coli

Cited by 51 publications

References 35 publications

Hydrolytic and phosphorolytic metabolism of cellobiose by the marine aerobic bacterium Saccharophagus degradans 2-40T

Hydrolytic and phosphorolytic metabolism of cellobiose by the marine aerobic bacterium Saccharophagus degradans 2-40T

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Identification of Lacto-N-Biose I Phosphorylase fromVibrio vulnificusCMCP6

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