scholarly journals Characterization of an Exo-β-1,3-Galactanase from Clostridium thermocellum

2006 ◽  
Vol 72 (5) ◽  
pp. 3515-3523 ◽  
Author(s):  
Hitomi Ichinose ◽  
Atsushi Kuno ◽  
Toshihisa Kotake ◽  
Makoto Yoshida ◽  
Kazuo Sakka ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
High Performance ◽  
Side Chain ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Gene Encoding ◽  
Chromatography Analysis ◽  
C Terminus ◽  
Affinity Gel Electrophoresis

ABSTRACT A gene encoding an exo-β-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-β-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50�C and catalyzes hydrolysis only of β-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from β-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a β-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-β-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including β-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a β-1,3-galactobiose, β-1,4-galactosyl glucose, or β-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward β-1,3-galactan and α-l-arabinofuranosidase-treated AGP.

scholarly journals Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

2005 ◽  
Vol 71 (12) ◽  
pp. 7670-7678 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomonori Nakai ◽  
Yoshiro Kameda ◽  
Ayako Hiyoshi ◽  
Yasushi Mitsuishi
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
C Terminus ◽  
Paenibacillus Sp ◽  
Genes Encoding ◽  
Molecular Masses

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

scholarly journals Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene fromEscherichia coli

1998 ◽  
Vol 180 (10) ◽  
pp. 2782-2787 ◽  
Author(s):  
K. Suvarna ◽  
D. Stevenson ◽  
R. Meganathan ◽  
M. E. S. Hudspeth
Keyword(s):  
High Performance ◽  
Promoter Sequence ◽  
Anaerobic Growth ◽  
Gene Encoding ◽  
Reading Frame ◽  
Chromatography Analysis ◽  
Naphthoic Acid ◽  
Membrane Bound ◽  
Carbon Side Chain

ABSTRACT A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of amenA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from theorf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into theorf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.

scholarly journals Isolation from Agricultural Soil and Characterization of a Sphingomonas sp. Able To Mineralize the Phenylurea Herbicide Isoproturon

2001 ◽  
Vol 67 (12) ◽  
pp. 5403-5409 ◽  
Author(s):  
Sebastian R. Sørensen ◽  
Zeev Ronen ◽  
Jens Aamand
Keyword(s):  
High Performance ◽  
Agricultural Soil ◽  
Side Chain ◽  
Rrna Gene ◽  
Chromatography Analysis ◽  
Phenylurea Herbicide ◽  
Partial Analysis ◽  
Pure Bacterial Culture ◽  
The 16S Rrna Gene

ABSTRACT A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the α-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.

scholarly journals The family 42 carbohydrate-binding module of family 54 α-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose

2006 ◽  
Vol 399 (3) ◽  
pp. 503-511 ◽  
Author(s):  
Akimasa Miyanaga ◽  
Takuya Koseki ◽  
Yozo Miwa ◽  
Yuichiro Mese ◽  
Sachiko Nakamura ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Side Chain ◽  
Titration Calorimetry ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Binding Assays ◽  
The Family ◽  
Affinity Gel Electrophoresis ◽  
Hydrolysis Of

α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.

scholarly journals Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
Author(s):  
Takane Katayama ◽  
Akiko Sakuma ◽  
Takatoshi Kimura ◽  
Yutaka Makimura ◽  
Jun Hiratake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Sugar Chain ◽  
Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

scholarly journals Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of thepigA Gene of Pseudomonas aeruginosa

2001 ◽  
Vol 183 (21) ◽  
pp. 6394-6403 ◽  
Author(s):  
Melanie Ratliff ◽  
Wenming Zhu ◽  
Rahul Deshmukh ◽  
Angela Wilks ◽  
Igor Stojiljkovic
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heme Oxygenase ◽  
High Performance ◽  
Visible Spectrum ◽  
Inducible Promoter ◽  
Major Product ◽  
Gene Encoding ◽  
Heme Oxygenases ◽  
Chromatography Analysis ◽  
Biliverdin Ix

ABSTRACT The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. ThepigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO ofNeisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced withpigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-β. This differs from the previously characterized heme oxygenases in which biliverdin IX-α is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.

Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase fromYersinia enterocolitica

1999 ◽  
Vol 45 (5) ◽  
pp. 396-403 ◽  
Author(s):  
Ching-Hsing Liao ◽  
Larry Revear ◽  
Arland Hotchkiss ◽  
Brett Savary
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Amperometric Detection ◽  
Pectate Lyase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Pathogen ◽  
Gene Encoding ◽  
Reading Frame ◽  
Chromatography Analysis

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated Mrof 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the Mrand pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.Key words: pectinase activities, human pathogen, HPLC analysis, pehY gene.

scholarly journals Characterization of an Endo-Processive-Type Xyloglucanase Having a β-1,4-Glucan-Binding Module and an Endo-Type Xyloglucanase from Streptomyces avermitilis

2012 ◽  
Vol 78 (22) ◽  
pp. 7939-7945 ◽  
Author(s):  
Hitomi Ichinose ◽  
Yuko Araki ◽  
Mari Michikawa ◽  
Koichi Harazono ◽  
Katsuro Yaoi ◽  
...  
Keyword(s):  
Gene Product ◽  
Recombinant Proteins ◽  
Catalytic Domain ◽  
Streptomyces Avermitilis ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Optimal Activity ◽  
C Terminus ◽  
Ph Range

ABSTRACTWe cloned two glycoside hydrolase family 74 genes, thesav_1856gene and thesav_2574gene, fromStreptomyces avermitilisNBRC14893 and characterized the resultant recombinant proteins. Thesav_1856gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while thesav_2574gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to β-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.

scholarly journals Cloning of the Novel Gene Encoding β-Agarase C from a Marine Bacterium, Vibrio sp. Strain PO-303, and Characterization of the Gene Product

2006 ◽  
Vol 72 (9) ◽  
pp. 6399-6401 ◽  
Author(s):  
Jinhua Dong ◽  
Shinnosuke Hashikawa ◽  
Takafumi Konishi ◽  
Yutaka Tamaru ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Gene Product ◽  
Glycoside Hydrolase ◽  
Marine Bacterium ◽  
Glycoside Hydrolase Family ◽  
The Novel ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Hydrolase Family

ABSTRACT The β-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. β-Agarase C was identified as the first β-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.

Sign in / Sign up

Export Citation Format

Share Document