scholarly journals The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain

1991 ◽  
Vol 273 (2) ◽  
pp. 289-293 ◽  
Author(s):  
A J Durrant ◽  
J Hall ◽  
G P Hazlewood ◽  
H J Gilbert
Keyword(s):  
Amino Acids ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Full Length ◽  
Crystalline Cellulose ◽  
Cellulose Binding Domain ◽  
Beta Glucan ◽  
Cellulose Binding

Mature endoglucanase E (EGE) from Clostridium thermocellum consists of 780 amino acid residues and has an Mr of 84,016. The N-terminal 334 amino acids comprise a functional catalytic domain. Full-length EGE bound to crystalline cellulose (Avicel) but not to xylan. Bound enzyme could be eluted with distilled water. The capacity of truncated derivatives of the enzyme to bind cellulose was investigated. EGE lacking 109 C-terminal residues (EGEd) or a derivative in which residues 367-432 of the mature form of the enzyme had been deleted (EGEb), bound to Avicel, whereas EGEa and EGEc, which lack 416 and 246 C-terminal residues respectively, did not. The specific activity of EGEa, consisting of the N-terminal 364 amino acids, was 4-fold higher than that of the full-length enzyme. The truncated derivative also exhibited lower affinity for the substrate beta-glucan than the full-length enzyme. It is concluded that EGE contains a cellulose-binding domain, located between residues 432 and 671, that is distinct from the active site. The role of this substrate-binding domain is discussed.

scholarly journals Properties and Mutation Analysis of the CelK Cellulose-Binding Domain from the Clostridium thermocellum Cellulosome

2001 ◽  
Vol 183 (5) ◽  
pp. 1552-1559 ◽  
Author(s):  
Irina A. Kataeva ◽  
Ronald D. Seidel ◽  
Xin-Liang Li ◽  
Lars G. Ljungdahl
Keyword(s):  
Amino Acids ◽  
Calcium Binding ◽  
Clostridium Thermocellum ◽  
Binding Capacity ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Calcium Binding Site ◽  
Aromatic Residues ◽  
Cellulose Binding ◽  
Family Iv

ABSTRACT The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBDCelK) was expressed inEscherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 μmol/g of cellulose and relative affinities (K r) of 2.33 and 9.87 liters/g, respectively. The CBDCelK is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBDCelKalso binds to the soluble polysaccharides lichenin, glucomannan, and barley β-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBDCelK contains 1 mol of calcium per mol. The CBDCelK has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBDCelK four highly conserved aromatic residues (Trp56, Trp94, Tyr111, and Tyr136) and Asp192 were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-NCelK. The CBD-NCelK and D192A retained binding parameters close to that of the intact CBDCelK, W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K rand Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBDCelK led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBDCelK and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBDCelK are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBDN1Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBDCelK correctly folded.

scholarly journals THE SEPARATION OF CELLULOSE-BINDING DOMAIN FROM ENDOGLUCANASE EGL-II BY PROTEOLYSIS METHOD

2016 ◽  
Vol 3 (01) ◽  
Author(s):  
Rina Masriani ◽  
Taufan Hidayat ◽  
Dewi Christanti Trisulo
Keyword(s):  
Molecular Weight ◽  
Protein Degradation ◽  
Catalytic Domain ◽  
Protein Molecule ◽  
Binding Domain ◽  
Intact Protein ◽  
Crystalline Cellulose ◽  
Cellulose Binding Domain ◽  
Paper Fibers ◽  
Cellulose Binding

Protein molecule of endoglucanase Egl-II is consisted of two domains, namely cellulose-binding domain (CBD) which serves in promoting the adsorption of the enzyme to the insoluble crystalline cellulose and cellulase catalytic domain which is responsible for the hydrolysis reaction. In this study, CBD of endoglucanase Egl-II was separated from the intact protein by degradation using the papain and then separation by ultrafiltration methods. The CBD resulted can be used to modify the waste paper fibers. The results of electrophoresis before degradation showed that endoglucanase Egl-II has a molecular weight about 57.5 kDa. The electrophoregram after protein degradation and separation of CBD from the endoglucanase Egl-II showed that CBD was separated from the intact protein with a molecular weight about 21 kDa. The yield of CBDs were 59.51%. It can be concluded that CBD of endoglucanase Egl-II can be separated from the intact protein.Keywords: endoglucanase Egl-II, cellulose-binding domain, papain, ultrafiltration. ABSTRAKMolekul protein dari endoglukanase Egl-II terdiri dari dua domain, yaitu cellulose-binding domain (CBD) yang berfungsi untuk mempromosikan adsorpsi enzim ke selulosa kristalin dan domain katalitik yang bertanggung jawab dalam reaksi hidrolisis. Dalam studi ini, CBD dari endoglukanase Egl-II telah dipisahkan dari protein utuhnya setelah didegradasi dengan papain. Pemisahan CBD dari campuran hasil degradasi dilakukan dengan metode ultrafiltrasi. CBD yang dihasilkan dapat digunakan untuk memodifikasi serat kertas bekas. Hasil elektroforesis sebelum degradasi menunjukkan endoglukanase Egl-II memiliki massa molekul sekitar 57,5 kDa. Dari elektroforegram setelah degradasi protein dan pemisahan CBD dari endoglukanase Egl-II memperlihatkan bahwa CBD telah terpisah dari protein utuhnya dan memiliki massa molekul sekitar 21 kDa. Rendemen CBD adalah 59,51%. CBD dari endoglukanase Egl-II dapat dipisahkan dengan metode ini.Kata kunci: endoglukanase Egl-II, cellulose-binding domain, papain, ultrafiltrasi.

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 A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulose-binding domain

1993 ◽  
Vol 294 (2) ◽  
pp. 349-355 ◽  
Author(s):  
L M Ferreira ◽  
T M Wood ◽  
G Williamson ◽  
C Faulds ◽  
G P Hazlewood ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Methoxycinnamic Acid ◽  
Cellulose Binding ◽  
Derivatives Of

The 5′ regions of genes xynB and xynC, coding for a xylanase and arabinofuranosidase respectively, are identical and are reiterated four times within the Pseudomonas fluorescens subsp. cellulosa genome. To isolate further copies of the reiterated xynB/C 5′ region, a genomic library of Ps. fluorescens subsp. cellulosa DNA was screened with a probe constructed from the conserved region of xynB. DNA from one phage which hybridized to the probe, but not to sequences upstream or downstream of the reiterated xynB/C locus, was subcloned into pMTL22p to construct pFG1. The recombinant plasmid expressed a protein in Escherichia coli, designated esterase XYLD, of M(r) 58,500 which bound to cellulose but not to xylan. XYLD hydrolysed aryl esters, released acetate groups from acetylxylan and liberated 4-hydroxy-3-methoxycinnamic acid from destarched wheat bran. The nucleotide sequence of the XYLD-encoding gene, xynD, revealed an open reading frame of 1752 bp which directed the synthesis of a protein of M(r) 60,589. The 5′ 817 bp of xynD and the amino acid sequence between residues 37 and 311 of XYLD were almost identical with the corresponding regions of xynB and xynC and their encoded proteins XYLB and XYLC. Truncated derivatives of XYLD lacking the N-terminal conserved sequence retained the capacity to hydrolyse ester linkages, but did not bind cellulose. Expression of truncated derivatives of xynD, comprising the 5′ 817 bp sequence, encoded a non-catalytic polypeptide that bound cellulose. These data indicate that XYLD has a modular structure comprising of a N-terminal cellulose-binding domain and a C-terminal catalytic domain.

Crystallization and Preliminary X-ray Analysis of the Major Cellulose-binding Domain of the Cellulosome from Clostridium thermocellum

1994 ◽  
Vol 244 (2) ◽  
pp. 236-237 ◽  
Author(s):  
Raphael Lamed ◽  
José Tormo ◽  
Arthur J. Chirino ◽  
Ely Morag ◽  
Edward A. Bayer
Keyword(s):  
Clostridium Thermocellum ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
X Ray ◽  
Cellulose Binding

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 An α-l-Arabinofuranosidase fromTrichoderma reesei Containing a Noncatalytic Xylan-Binding Domain

1999 ◽  
Vol 65 (9) ◽  
pp. 3964-3968 ◽  
Author(s):  
Masahiro Nogawa ◽  
Kenji Yatsui ◽  
Akiko Tomioka ◽  
Hirofumi Okada ◽  
Yasushi Morikawa
Keyword(s):  
Molecular Mass ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Crystalline Cellulose ◽  
Terminal Amino Acid ◽  
Catalytic Constant ◽  
Terminal Amino ◽  
Oat Spelt

ABSTRACT l-Sorbose, an excellent cellulase and xylanase inducer from Trichoderma reesei PC-3-7, also induced α-l-arabinofuranosidase (α-AF) activity. An α-AF induced by l-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported α-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported α-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters forp-nitrophenyl-α-l-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported α-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.

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