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

2001 ◽  
Vol 67 (11) ◽  
pp. 5100-5106 ◽  
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).

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

1991 ◽  
Vol 279 (3) ◽  
pp. 787-792 ◽  
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.

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 type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism

1999 ◽  
Vol 342 (2) ◽  
pp. 473-480 ◽  
Author(s):  
Jaitinder GILL ◽  
Jane E. RIXON ◽  
David N. BOLAM ◽  
Simon MCQUEEN-MASON ◽  
Peter J. SIMPSON ◽  
...  
Keyword(s):  
Microcrystalline Cellulose ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Wall Material ◽  
Common Mechanism ◽  
Type Ii ◽  
Binding Domains ◽  
Covalently Linked ◽  
Cellulose Binding ◽  
Internal Domain

Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10ACD) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBDX) or fused to the catalytic domain (Pf Xyn10A′), bound to amorphous and bacterial microcrystalline cellulose with a Ka of 2.5×105 M-1. CBDX exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A′ was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10ACD; however, Pf Xyn10A′ and Pf Xyn10ACD exhibited the same activity against soluble substrates. CBDX did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10ACD when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A′, and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10ACD via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBDX is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase.

scholarly journals Identification and characterization of virulent Aeromonas hydrophila Ah17 from infected Channa striata in river Cauvery and in vitro evaluation of shrimp chitosan

2020 ◽  
Vol 8 (2) ◽  
pp. 1272-1283 ◽  
Author(s):  
Vignesh Samayanpaulraj ◽  
Muthukumar Sivaramapillai ◽  
Sankara Naynar Palani ◽  
Krishnaveni Govindaraj ◽  
Vijay Velu ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
In Vitro Evaluation ◽  
Channa Striata ◽  
Virulent Aeromonas Hydrophila ◽  
Identification And Characterization

Identification and Characterization of Novel Fusion Proteins in Pediatric Acute Megakaryoblastic Leukemia

2014 ◽  
Vol 14 ◽  
pp. S123-S124
Author(s):  
Jinjun Dang ◽  
Amanda Larson Gedman ◽  
Cary S. Koss ◽  
Suresh Marada ◽  
Stacey K. Ogden ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Acute Megakaryoblastic Leukemia ◽  
Megakaryoblastic Leukemia ◽  
Identification And Characterization

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

1998 ◽  
Vol 330 (1) ◽  
pp. 41-45 ◽  
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.

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.

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