Enhancement of Cellulase Activity by Clones Selected from the Combinatorial Library of the Cellulose-Binding Domain by Cell Surface Engineering

2006 ◽  
Vol 22 (4) ◽  
pp. 933-938 ◽  
Author(s):  
T. Fukuda ◽  
T. Ishikawa ◽  
M. Ogawa ◽  
S. Shiraga ◽  
M. Kato ◽  
...  
Keyword(s):  
Cell Surface ◽  
Combinatorial Library ◽  
Surface Engineering ◽  
Cellulase Activity ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Cell Surface Engineering ◽  
Cellulose Binding

scholarly journals Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: stoichiometry and cellulolytic activity of the complexes

1997 ◽  
Vol 326 (2) ◽  
pp. 617-624 ◽  
Author(s):  
Irina KATAEVA ◽  
Gérard GUGLIELMI ◽  
Pierre BÉGUIN
Keyword(s):  
Clostridium Thermocellum ◽  
Analytical Ultracentrifugation ◽  
Cellulase Activity ◽  
Hydrolytic Activity ◽  
Binding Domain ◽  
Cellulolytic Activity ◽  
Cellulose Binding Domain ◽  
Optimal Ratio ◽  
Cellulose Binding

Four mini-scaffoldins were constructed from modules derived from the Clostridium thermocellum cellulosome-integrating protein CipA. Cip7 and Cip6 contained one and two cohesin modules respectively. Cip14 and Cip16, also containing one and two cohesin modules respectively, were flanked by a cellulose-binding domain. Endoglucanase CelD formed stable complexes with all mini-scaffoldins. Analytical ultracentrifugation of the complexes showed that 1 mol of CelD bound per mol of Cip14, and 2 mol of CelD bound per mol of Cip16. Under the conditions used for assaying cellulase activity, 96% of CelD alone bound to Avicel. Association with Cip14 or Cip16 increased the cellulose binding of CelD to 99%, while association with Cip7 or Cip6 decreased binding to 79 and 75% respectively. The hydrolytic activity of CelD against Avicel was increased 3-fold in complexes with Cip14 and Cip16, but remained substantially the same in complexes with Cip6 and Cip7. Addition of whole CipA also enhanced the efficiency of Avicel hydrolysis by CelD. However, even at an optimal ratio of the components, CelD–CipA complexes were somewhat less active than complexes of CelD with Cip14 or Cip16. These results suggest that the synergism observed between CelD and Cip14 or Cip16 is mostly due to the presence of the cellulose-binding domain, which promotes productive binding of the enzyme.

scholarly journals Isolation of a cDNA Encoding a β-1,4-endoglucanase in the Root-Knot Nematode Meloidogyne incognita and Expression Analysis During Plant Parasitism

1999 ◽  
Vol 12 (7) ◽  
pp. 585-591 ◽  
Author(s):  
Marie-Noëlle Rosso ◽  
Bruno Favery ◽  
Christine Piotte ◽  
Laury Arthaud ◽  
Jan M. De Boer ◽  
...  
Keyword(s):  
Meloidogyne Incognita ◽  
Catalytic Domain ◽  
Cellulase Activity ◽  
Root Surface ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Root Knot Nematode ◽  
Cyst Nematodes ◽  
Adult Females ◽  
Cellulose Binding

A β-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of β-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode β-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a β-1,4-endoglucanase devoid of a cellulose-binding domain.

scholarly journals Specific Adhesion to Cellulose and Hydrolysis of Organophosphate Nerve Agents by a Genetically Engineered Escherichia coli Strain with a Surface-Expressed Cellulose-Binding Domain and Organophosphorus Hydrolase

2002 ◽  
Vol 68 (4) ◽  
pp. 1684-1689 ◽  
Author(s):  
Aijun A. Wang ◽  
Ashok Mulchandani ◽  
Wilfred Chen
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Nerve Agents ◽  
Genetically Engineered ◽  
Binding Domain ◽  
Organophosphorus Hydrolase ◽  
Fusion System ◽  
Cellulose Binding Domain ◽  
Cellulose Binding ◽  
Hydrolysis Of

ABSTRACT A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.

scholarly journals Cellulose as an inert matrix for presenting cytokines to target cells: production and properties of a stem cell factor–cellulose-binding domain fusion protein

1999 ◽  
Vol 339 (2) ◽  
pp. 429-434
Author(s):  
J. Greg DOHENY ◽  
Eric J. JERVIS ◽  
M. Marta GUARNA ◽  
R. Keith HUMPHRIES ◽  
R. Antony J. WARREN ◽  
...  
Keyword(s):  
Stem Cell ◽  
Fusion Protein ◽  
Stem Cell Factor ◽  
Direct Analysis ◽  
Binding Domain ◽  
Target Cells ◽  
Cellulose Binding Domain ◽  
Binding Characteristics ◽  
Cellulose Binding ◽  
Cellulose Surface

A chimaera of stem cell factor (SCF) and a cellulose-binding domain from the xylanase Cex (CBDCex) effectively immobilizes SCF on a cellulose surface. The fusion protein retains both the cytokine properties of SCF and the cellulose-binding characteristics of CBDCex. When adsorbed on cellulose, SCF–CBDCex is up to 7-fold more potent than soluble SCF–CBDCex and than native SCF at stimulating the proliferation of factor-dependent cell lines. When cells are incubated with cellulose-bound SCF–CBDCex, activated receptors and SCF–CBDCex co-localize on the cellulose matrix. The strong binding of SCF–CBDCex to the cellulose surface permits the effective and localized stimulation of target cells; this is potentially significant for long-term perfusion culturing of factor-dependent cells. It also permits the direct analysis of the effects of surface-bound cytokines on target cells.

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