Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

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.

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Cel9M, a New Family 9 Cellulase of the Clostridium cellulolyticum Cellulosome

Journal of Bacteriology ◽

10.1128/jb.184.5.1378-1384.2002 ◽

2002 ◽

Vol 184 (5) ◽

pp. 1378-1384 ◽

Cited By ~ 40

Author(s):

Anne Belaich ◽

Goetz Parsiegla ◽

Laurent Gal ◽

Claude Villard ◽

Richard Haser ◽

...

Keyword(s):

Microcrystalline Cellulose ◽

Carboxymethyl Cellulose ◽

Catalytic Domain ◽

Soluble Sugar ◽

Significant Activity ◽

Cellulose Binding Domain ◽

Clostridium Cellulolyticum ◽

New Family ◽

Cellulose Binding ◽

Hydrolysis Of

ABSTRACT A new cellulosomal protein from Clostridium cellulolyticum Cel9M was characterized. The protein contains a catalytic domain belonging to family 9 and a dockerin domain. Cel9M is active on carboxymethyl cellulose, and the hydrolysis of this substrate is accompanied by a decrease in viscosity. Cel9M has a slight, albeit significant, activity on both Avicel and bacterial microcrystalline cellulose, and the main soluble sugar released is cellotetraose. Saccharification of bacterial microcrystalline cellulose by Cel9M in association with two other family 9 enzymes from C. cellulolyticum, namely, Cel9E and Cel9G, was measured, and it was found that Cel9M acts synergistically with Cel9E. Complexation of Cel9M with the mini-CipC1 containing the cellulose binding domain, the X2 domain, and the first cohesin domain of the scaffoldin CipC of the bacterium did not significantly increase the hydrolysis of Avicel and bacterial microcrystalline cellulose.

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Characterization of aNeocallimastixpatriciarumxylanase gene and its product

Canadian Journal of Microbiology ◽

10.1139/w99-092 ◽

1999 ◽

Vol 45 (11) ◽

pp. 970-974 ◽

Cited By ~ 14

Author(s):

Jin-Hao Liu ◽

Brent L Selinger ◽

Cheng-Fang Tsai ◽

Kuo-Jaon Cheng

Keyword(s):

Catalytic Domain ◽

Xylanase Activity ◽

Binding Domain ◽

Glycosyl Hydrolases ◽

Cellulose Binding Domain ◽

Xylanase Gene ◽

Neocallimastix Patriciarum ◽

Cellulose Binding ◽

Linker Domain

A xylanase gene (xynC) isolated from the anaerobic ruminal fungus Neocallimastix patriciarum was characterized. The gene consists of an N-terminal catalytic domain that exhibited homology to family 11 of glycosyl hydrolases, a C-terminal cellulose binding domain (CBD) and a putative dockerin domain in between. Each domain was linked by a short linker domain rich in proline and alanine. Deletion analysis demonstrated that the CBD was essential for optimal xylanase activity of the enzyme, while the putative dockerin domain may not be required for enzyme function.Key words: xylanase, cellulose binding domain, Neocallimastix patriciarum.

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Identification and Characterization of the Three Chitin-Binding Domains within the Multidomain Chitinase Chi92 fromAeromonas hydrophila JP101

Applied and Environmental Microbiology ◽

10.1128/aem.67.11.5100-5106.2001 ◽

2001 ◽

Vol 67 (11) ◽

pp. 5100-5106 ◽

Cited By ~ 27

Author(s):

Mei Li Wu ◽

Yin Ching Chuang ◽

Jen Pin Chen ◽

Chin Shuh Chen ◽

Ming Chung Chang

Keyword(s):

Aeromonas Hydrophila ◽

Fusion Proteins ◽

Catalytic Domain ◽

The Third ◽

Binding Domains ◽

Hydrolytic Activities ◽

Cellulose Binding ◽

Identification And Characterization ◽

Gst Fusion Proteins

ABSTRACT The gene (chi92) encoding the extracellular chitinase of Aeromonas hydrophila JP101 has been cloned and expressed in Escherichia coli. The mature form of Chi92 is an 842-amino-acid (89.830-kDa) modular enzyme comprised of a family 18 catalytic domain, an unknown-function region (the A region), and three chitin-binding domains (ChBDs; Chi92-N, ChBDCI, and ChBDCII). The C-terminally repeated ChBDs, ChBDCI and ChBDCII, were grouped into family V of cellulose-binding domains on the basis of sequence homology. Chitin binding and enzyme activity studies with C-terminally truncated Chi92 derivatives lacking ChBDs demonstrated that the ChBDs are responsible for its adhesion to unprocessed and colloidal chitins. Further adsorption experiments with glutathione S-transferase (GST) fusion proteins (GST-CI and GST-CICII) demonstrated that a single ChBD (ChBDCI) could promote efficient chitin and cellulose binding. In contrast to the two C-terminal ChBDs, the Chi92-N domain is similar to ChiN of Serratia marcescens ChiA, which has been proposed to participate in chitin binding. A truncated derivative of Chi92 that contained only a catalytic domain and Chi92-N still exhibited insoluble-chitin-binding and hydrolytic activities. Thus, it appears that Chi92 contains Chi92-N as the third ChBD in addition to two ChBDs (ChBDCI and ChBDCII).

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Gene Cloning and Characterization of a Novel Cellulose-Binding β-Glucosidase from Phanerochaete chrysosporium

Applied and Environmental Microbiology ◽

10.1128/aem.64.7.2748-2754.1998 ◽

1998 ◽

Vol 64 (7) ◽

pp. 2748-2754 ◽

Cited By ~ 25

Author(s):

Bin Li ◽

V. Renganathan

Keyword(s):

Sequence Analysis ◽

Phanerochaete Chrysosporium ◽

Catalytic Domain ◽

Genomic Sequence ◽

Single Gene ◽

Cellulose Binding Domain ◽

Genomic Sequence Analysis ◽

Cellulose Binding ◽

Gene Cloning And Characterization

ABSTRACT Analysis of a 2.4-kb cDNA of the cellulose-binding extracellular β-glucosidase (CBGL) from Phanerochaete chrysosporiumsuggested that CBGL is organized into two domains, an N-terminal cellulose-binding domain and a C-terminal catalytic domain. Genomic sequence analysis suggested that cbgl is encoded by 30 exons. Southern analysis of DNA from homokaryotic cultures indicated that CBGL is encoded by two alleles, cbgl-1 andcbgl-2, of a single gene.

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Structural and Catalytic Characterization of TsBGL, a β-Glucosidase From Thermofilum sp. ex4484_79

Frontiers in Microbiology ◽

10.3389/fmicb.2021.723678 ◽

2021 ◽

Vol 12 ◽

Author(s):

Anke Chen ◽

Dan Wang ◽

Rui Ji ◽

Jixi Li ◽

Shaohua Gu ◽

...

Keyword(s):

Catalytic Domain ◽

Specific Activity ◽

Maximum Activity ◽

Homologous Proteins ◽

Glycosidic Bonds ◽

Catalytic Sites ◽

Potential Applications ◽

Beta Glucosidase ◽

Hydrolysis Of

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable β-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified β-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2μmol/min/mgzne against p-nitrophenyl β-D-glucopyranoside (pNPGlc) and 24.3μmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/β)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.

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Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

Biochemical Journal ◽

10.1042/bj20040942 ◽

2005 ◽

Vol 388 (2) ◽

pp. 493-500 ◽

Cited By ~ 44

Author(s):

Chandra N. PATEL ◽

David W. KOH ◽

Myron K. JACOBSON ◽

Marcos A. OLIVEIRA

Keyword(s):

Catalytic Activity ◽

Catalytic Domain ◽

Functional Characterization ◽

Sequence Alignments ◽

Inhibitor Binding ◽

Conserved Sequence ◽

Binding Residue ◽

Acidic Residues ◽

Hydrolysis Of

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG.

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Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose

BMC Biotechnology ◽

10.1186/1472-6750-13-101 ◽

2013 ◽

Vol 13 (1) ◽

pp. 101 ◽

Cited By ~ 21

Author(s):

Linguo Zhao ◽

Qian Pang ◽

Jingcong Xie ◽

Jianjun Pei ◽

Fei Wang ◽

...

Keyword(s):

Microcrystalline Cellulose ◽

Binding Domain ◽

Enzymatic Properties ◽

Cellulose Binding Domain ◽

Clostridium Cellulovorans ◽

Thermoanaerobacterium Thermosaccharolyticum ◽

Cellulose Binding ◽

Hydrolysis Of

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Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

Biochemical Journal ◽

10.1042/bj2790787 ◽

1991 ◽

Vol 279 (3) ◽

pp. 787-792 ◽

Cited By ~ 22

Author(s):

D M Poole ◽

A J Durrant ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Domain ◽

Binding Capacity ◽

Binding Domains ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus ◽

Fusion Enzymes ◽

Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

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Substrate-binding domains of glycanases from Streptomyces lividans: characterization of a new family of xylan-binding domains

Biochemical Journal ◽

10.1042/bj3300041 ◽

1998 ◽

Vol 330 (1) ◽

pp. 41-45 ◽

Cited By ~ 36

Author(s):

Claude DUPONT ◽

Martin ROBERGE ◽

François SHARECK ◽

Rolf MOROSOLI ◽

Dieter KLUEPFEL

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Streptomyces Lividans ◽

Cellulose Binding Domain ◽

Binding Domains ◽

New Family ◽

Cellulose Binding ◽

Sequence Similarities

The substrate-binding domains of six glycanases from Streptomyces lividans were investigated to determine their specificity towards cellulose and xylan. Based upon amino acid sequence similarities, four of the six domains could be assigned to existing cellulose-binding domain families. However, the binding domains of xylanase A and arabinofuranosidase B could not be classified in any of the known families and should therefore be classified as members of a new family. Evidence is also presented that this new family is one of true xylan-binding domains.

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