scholarly journals Characterization of EngF from Clostridium cellulovorans and Identification of a Novel Cellulose Binding Domain

1998 ◽  
Vol 64 (3) ◽  
pp. 1086-1090 ◽  
Author(s):  
Akihiko Ichi-ishi ◽  
Salah Sheweita ◽  
Roy H. Doi
Keyword(s):  
Amino Acids ◽  
Cellulose Degradation ◽  
Binding Activity ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Binding Studies ◽  
Endoglucanase Activity ◽  
Clostridium Cellulovorans ◽  
C Terminus ◽  
Cellulose Binding

ABSTRACT The physical and enzymatic properties of noncellulosomal endoglucanase F (EngF) from Clostridium cellulovorans were studied. Binding studies revealed that the Kd and the maximum amount of protein bound for acid-swollen cellulose were 1.8 μM and 7.1 μmol/g of cellulose, respectively. The presence of cellobiose but not glucose or maltose could dissociate EngF from cellulose. N- and C-terminally truncated enzymes showed that binding activity was located at some site between amino acid residues 356 and 557 and that enzyme activity was still present when 20 amino acids but not 45 amino acids were removed from the N terminus and when 32 amino acids were removed from the C terminus; when 57 amino acids were removed from the C terminus, all activity was lost. EngF showed low endoglucanase activity and could hydrolyze cellotetraose and cellopentaose but not cellotriose. Activity studies suggested that EngF plays a role as an endoglucanase during cellulose degradation. Comparative sequence analyses indicated strongly that the cellulose binding domain (CBD) is different from previously reported CBDs.

scholarly journals Site-Directed Mutagenesis and Expression of the Soluble Form of the Family IIIa Cellulose Binding Domain from the Cellulosomal Scaffolding Protein of Clostridium cellulovorans

2005 ◽  
Vol 187 (20) ◽  
pp. 7146-7149 ◽  
Author(s):  
Koichiro Murashima ◽  
Akihiko Kosugi ◽  
Roy H. Doi
Keyword(s):  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Scaffolding Protein ◽  
Soluble Form ◽  
Directed Mutagenesis ◽  
Cellulose Binding Domain ◽  
Binding Studies ◽  
Clostridium Cellulovorans ◽  
The Family ◽  
Cellulose Binding

ABSTRACT The planar and anchoring residues of the family IIIa cellulose binding domain (CBD) from the cellulosomal scaffolding protein of Clostridium cellulovorans were investigated by site-directed mutagenesis and cellulose binding studies. By fusion with maltose binding protein, the family IIIa recombinant wild-type and mutant CBDs from C. cellulovorans were expressed as soluble forms. Cellulose binding tests of the mutant CBDs indicated that the planar strip residues played a major role in cellulose binding and that the anchoring residues played only a minor role.

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 Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

1993 ◽  
Vol 175 (18) ◽  
pp. 5762-5768 ◽  
Author(s):  
M A Goldstein ◽  
M Takagi ◽  
S Hashida ◽  
O Shoseyov ◽  
R H Doi ◽  
...  
Keyword(s):  
Binding Protein ◽  
Protein A ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Clostridium Cellulovorans ◽  
Cellulose Binding

scholarly journals PERBAIKAN FREENESS DAN MUTU KERTAS BEKAS MENGGUNAKAN CELLULOSE BINDING DOMAIN DARI ENDOGLUKANASE Egl-II

2014 ◽  
Vol 4 (02) ◽  
Author(s):  
Rina Masriani ◽  
Taufan Hidayat ◽  
Henggar Hardiani
Keyword(s):  
Cellulose Degradation ◽  
Binding Domain ◽  
Waste Paper ◽  
Cellulose Binding Domain ◽  
Total Enzyme Activity ◽  
Tensile Index ◽  
Tear Index ◽  
Paper Fibers ◽  
Cellulose Binding ◽  
Total Enzyme

The objective of this research is improving the freeness of waste paper stock and paper quality by using the Cellulose Binding Domain (CBD) of endoglucanase Egl-II. CBD has been separated from endoglucanase Egl-II by proteolysis method. CBD has a molecular weight of approximately 21 kD. The produced CBD contained 0.04 mg / mL protein and did not show the total enzyme activity. Waste paper disintegrated using Niagara beater with no load at the consistency of 1.5%. CBD was applied to the refined waste paper fibers with a freeness of 200 mL CSF (Canadian Standard Freeness). The dosages of CBD used for waste paper treatment were 0.2 and 0.3 mg CBD/g of oven-dried pulp. The result shows that this treatment increases the freeness of fibers by 140 mL CSF (70%). CBD also increases the amount of removed water from the fibers from 290 mL to 390 mL and 370 mL, respectively, using the dynamic drainage jar (DDJ) measurement. The cellobiose assay of the waste paper filtrate treated with CBD shows no sugar dissolution, which indicates no cellulose degradation. The tear index of paper produced by treatment with CBD shows insignificant change. The Concora Medium Test (CMT) of paper produced by treatment with CBD has higher tensile index, burst index, and ring crush.Keywords: cellulose-binding domain, endoglucanase Egl-II, freeness improvement, waste paperABSTRAKTujuan penelitian ini adalah untuk memperbaiki freeness dari stok kertas bekas dan mutu lembaran kertas yang  dihasilkan dengan menggunakan Cellulose Binding Domain (CBD) dari endoglukanase Egl-II. CBD yang  digunakan merupakan hasil pemisahan dari endoglukanase Egl-II dengan metode proteolisis. CBD ini memiliki  berat molekul sekitar 21 kD. CBD yang dihasilkan mengandung kadar protein sebesar 0,04 mg/mL dan tidak  terdeteksi adanya aktivitas total enzim. Kertas bekas diuraikan dengan menggunakan Niagara beater tanpa  beban pada konsistensi 1,5%. CBD diaplikasikan pada serat kertas bekas yang telah digiling dan memiliki freeness 200 mL CSF (Canadian Standard Freeness). Dosis CBD yang digunakan untuk perlakuan terhadap serat kertas  bekas adalah 0,2 dan 0,3 mg CBD/g pulp kering-oven. Hasil penelitian menunjukkan perlakuan dengan CBD  meningkatkan freeness bubur serat kertas bekas sebesar 140 mL CSF (70%). CBD juga meningkatkan volume air yang dihilangkan dari serat kertas bekas dari 290 mL menjadi 390 mL dan 370 mL menggunakan pengukuran dynamic drainage jar (DDJ). %FPR meningkat dari 98,80% menjadi 99,77%. Pengujian selobiosa terlarut pada filtrat serat kertas bekas yang telah mengalami perlakuan dengan CBD memperlihatkan tidak ada gula terlarut, artinya tidak ada degradasi selulosa menjadi gula terlarut. Indeks sobek dari kertas yang dihasilkan melalui perlakuan dengan CBD memperlihatkan tidak ada perubahan yang signifikan. Nilai tensile index, burst index, ring crush dan Concora Medium Test (CMT) dari kertas yang dihasilkan melalui perlakuan dengan CBD meningkat.Kata kunci: cellulose-binding domain, endoglukanase Egl-II, perbaikan freeness, kertas bekas

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.

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