scholarly journals Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cel9A

2007 ◽  
Vol 73 (10) ◽  
pp. 3165-3172 ◽  
Author(s):  
Yongchao Li ◽  
Diana C. Irwin ◽  
David B. Wilson
Keyword(s):  
Catalytic Domain ◽  
Cellulose Hydrolysis ◽  
Site Directed Mutagenesis ◽  
Crystalline Cellulose ◽  
Thermobifida Fusca ◽  
Processive Endoglucanase ◽  
Cellulose Binding ◽  
Increased Activity ◽  
Catalytic Cleft ◽  
Conserved Residue

ABSTRACT Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(−1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(−2) to Glc(−4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(−2) to Glc(−4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.

scholarly journals Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of Thermobifida fusca Cel9A

2010 ◽  
Vol 76 (8) ◽  
pp. 2582-2588 ◽  
Author(s):  
Yongchao Li ◽  
Diana C. Irwin ◽  
David B. Wilson
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Crystalline Cellulose ◽  
Thermobifida Fusca ◽  
Carbohydrate Binding ◽  
Synergistic Activity ◽  
The Family ◽  
Cellulose Binding ◽  
Increased Activity ◽  
Catalytic Cleft

ABSTRACT Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.

scholarly journals The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains

1991 ◽  
Vol 279 (3) ◽  
pp. 793-799 ◽  
Author(s):  
L M A Ferreira ◽  
G P Hazlewood ◽  
P J Barker ◽  
H J Gilbert
Keyword(s):  
Pseudomonas Fluorescens ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Terminal Region ◽  
Crystalline Cellulose ◽  
Nucleotide Sequencing ◽  
Reading Frame ◽  
Strong Homology ◽  
Cellulose Binding

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in pUC18 and Escherichia coli recombinants expressing 4-methylumbelliferyl beta-D-cellobioside-hydrolysing activity (MUCase) were isolated. Enzyme produced by MUCase-positive clones did not hydrolyse either cellobiose or cellotriose but converted cellotetraose into cellobiose and cleaved cellopentaose and cellohexaose, producing a mixture of cellobiose and cellotriose. There was no activity against CM-cellulose, insoluble cellulose or xylan. On this basis, the enzyme is identified as an endo-acting cellodextrinase and is designated cellodextrinase C (CELC). Nucleotide sequencing of the gene (celC) which directs the synthesis of CELC revealed an open reading frame of 2153 bp, encoding a protein of Mr 80,189. The deduced primary sequence of CELC was confirmed by the Mr of purified CELC (77,000) and by the experimentally determined N-terminus of the enzyme which was identical with residues 38-47 of the translated sequence. The N-terminal region of CELC showed strong homology with endoglucanase, xylanases and an arabinofuranosidase of Ps. fluorescens subsp. cellulosa; homologous sequences included highly conserved serine-rich regions. Full-length CELC bound tightly to crystalline cellulose. Truncated forms of celC from which the DNA sequence encoding the conserved domain had been deleted, directed the synthesis of a functional cellodextrinase that did not bind to crystalline cellulose. This is consistent with the N-terminal region of CELC comprising a non-catalytic cellulose-binding domain which is distinct from the catalytic domain. The role of the cellulose-binding region is discussed.

scholarly journals X-Ray Crystal Structure of the Multidomain Endoglucanase Cel9G from Clostridium cellulolyticum Complexed with Natural and Synthetic Cello-Oligosaccharides

2003 ◽  
Vol 185 (14) ◽  
pp. 4127-4135 ◽  
Author(s):  
David Mandelman ◽  
Anne Belaich ◽  
J. P. Belaich ◽  
Nushin Aghajari ◽  
Hugues Driguez ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Structural Basis ◽  
Crystalline Cellulose ◽  
X Ray ◽  
Clostridium Cellulolyticum ◽  
Active Site Cleft ◽  
Cellulose Binding

ABSTRACT Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the β-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-Å resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family IIIc cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 Å, respectively.

scholarly journals Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase

1999 ◽  
Vol 181 (15) ◽  
pp. 4611-4616 ◽  
Author(s):  
Helen D. Simpson ◽  
Frederic Barras
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Erwinia Chrysanthemi ◽  
Dimensional Structure ◽  
Carbohydrate Binding ◽  
Binding Domains ◽  
Cellulose Binding

ABSTRACT The Cel5 cellulase (formerly known as endoglucanase Z) fromErwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBDCel5 has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBDCel5 exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBDCel5 bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.

scholarly journals Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

2010 ◽  
Vol 76 (19) ◽  
pp. 6583-6590 ◽  
Author(s):  
Machiko Takahashi ◽  
Hideyuki Takahashi ◽  
Yuki Nakano ◽  
Teruko Konishi ◽  
Ryohei Terauchi ◽  
...  
Keyword(s):  
Magnaporthe Oryzae ◽  
Magnaporthe Grisea ◽  
Catalytic Domain ◽  
Cellulose Binding Domain ◽  
Enhanced Activity ◽  
Hydrolytic Activities ◽  
Cellulose Binding ◽  
Hydrolysis Of ◽  
Increased Activity

ABSTRACT Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.

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 EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

2001 ◽  
Vol 67 (10) ◽  
pp. 4426-4431 ◽  
Author(s):  
Marco T. Rincón ◽  
Sheila I. McCrae ◽  
James Kirby ◽  
Karen P. Scott ◽  
Harry J. Flint
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Ruminococcus Flavefaciens ◽  
Cellulose Binding Module ◽  
Strong Candidate ◽  
Cellulose Binding ◽  
Enzyme Complexes

ABSTRACT The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase geneendB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression inEscherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.

scholarly journals Processive Endoglucanase Active in Crystalline Cellulose Hydrolysis by the Brown Rot Basidiomycete Gloeophyllum trabeum

2005 ◽  
Vol 71 (5) ◽  
pp. 2412-2417 ◽  
Author(s):  
Roni Cohen ◽  
Melissa R. Suzuki ◽  
Kenneth E. Hammel
Keyword(s):  
Extracellular Enzymes ◽  
Reducing Sugars ◽  
Soluble Sugars ◽  
Brown Rot ◽  
Cellulose Hydrolysis ◽  
Major Product ◽  
Crystalline Cellulose ◽  
Gloeophyllum Trabeum ◽  
Processive Endoglucanase ◽  
Brown Rot Fungus

ABSTRACT Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity—4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a β-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.

scholarly journals Roles of the Catalytic Domain and Two Cellulose Binding Domains of Thermomonospora fusca E4 in Cellulose Hydrolysis

1998 ◽  
Vol 180 (7) ◽  
pp. 1709-1714 ◽  
Author(s):  
Diana Irwin ◽  
Dong-Hoon Shin ◽  
Sheng Zhang ◽  
Brian K. Barr ◽  
Joshua Sakon ◽  
...  
Keyword(s):  
Filter Paper ◽  
Recombinant Dna ◽  
Catalytic Domain ◽  
Cellulose Hydrolysis ◽  
Hydrolytic Activity ◽  
Thermomonospora Fusca ◽  
Binding Domains ◽  
The Family ◽  
Recombinant Dna Techniques ◽  
Cellulose Binding

ABSTRACT Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

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.

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