Characteristics of adjacent family 6 acetylxylan esterases fromFibrobacter succinogenesand the interaction with the Xyn10E xylanase in hydrolysis of acetylated xylan

2005 ◽  
Vol 51 (10) ◽  
pp. 821-832 ◽  
Author(s):  
Dong Keun Kam ◽  
Hyun-Sik Jun ◽  
Jong K Ha ◽  
G Douglas Inglis ◽  
Cecil W Forsberg
Keyword(s):  
Amino Acid ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Amino Acid Sequences ◽  
Carbohydrate Binding ◽  
Fibrobacter Succinogenes ◽  
Acetylxylan Esterase ◽  
Low K ◽  
Hydrolysis Of ◽  
Improve Access

Acetylxylan esterase genes axe6A and axe6B located adjacent to one another on a Fibrobacter succinogenes chromosome have been separately cloned and their properties characterized. The corresponding esterases contained an N-terminal carbohydrate esterase family 6 catalytic domain (CD) and a C-terminal family 6 carbohydrate-binding module (CBM). The amino acid sequences of the CDs and CBMs were found to exhibit 52% and 40% amino acid similarity, respectively. The CDs of the two esterases exhibited the highest similarity to CDs of acetylxylan esterases: AxeA from the ruminal fungi Orpinomyces sp. and BnaA from Neocallimastix patriciarum. Axe6A and Axe6B were optimally active at neutral pH and had low Kmvalues of 0.084 and 0.056 mmol·L–1, respectively. Axe6A and Axe6B were shown to bind to insoluble cellulose and xylan and to soluble arabinoxylan. Axe6A deacetylated acetylated xylan at the same initial rate in the presence and absence of added Xyn10E xylanase from F. succinogenes, but the action of the xylanase on acetylated xylan was dependent upon the initial activity of Axe6A. The capacity of acetylxylan esterases to bind to plant cell wall polymers and to independently deacetylate xylan enabling xylanase to release xylooligo saccharides, documents the central role these enzymes have to improve access of F. succinogenes to cellulose.Key words: Fibrobacter succinogenes S85, acetylxylan esterase, xylanase, synergy.

Characteristics of a cluster of xylanase genes inFibrobacter succinogenesS85

2003 ◽  
Vol 49 (3) ◽  
pp. 171-180 ◽  
Author(s):  
Hyun S Jun ◽  
Jong K Ha ◽  
Laercio M Malburg, Jr. ◽  
Ann M Verrinder Gibbins ◽  
Cecil W Forsberg
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Glycosyl Hydrolase ◽  
Amino Acid Sequences ◽  
Pcr Analysis ◽  
Carbohydrate Binding ◽  
Fibrobacter Succinogenes ◽  
Rt Pcr ◽  
Ph Optimum ◽  
Oat Spelt

Xylanase genes xyn10D, xyn10E, and xyn10B, located sequentially on the Fibrobacter succinogenes S85 chromosome, were separately cloned and their properties characterized. Analysis of the sequences documented that xylanases Xyn10D, Xyn10E, and Xyn10B each consist of an N-terminal catalytic domain (glycosyl hydrolase family 10) and a C-terminal carbohydrate-binding module (CBM, family 6) connected by proline-rich linker sequences. The amino acid sequences exhibited similarities of between 53 and 60%. The xyn10D, xyn10E, and truncated xyn10BΔCBM were expressed in Escherichia coli and purified to homogeneity. The purified Xyn10D, Xyn10E, and Xyn10BΔCBM exhibited the same temperature optimum (40°C) and pH optimum (6.5) and the highest specific activity against arabinoxylan, oat spelt xylan, and birchwood xylan, respectively. Xyn10D exhibited an affinity for cellulose and xylan with 47 and 33% binding, respectively, while the truncated Xyn10DΔCBM did not bind to the substrates. The main hydrolysis products of the three xylanases acting on oat spelt xylan and arabinoxylan were xylose and xylobiose. RT-PCR analysis showed that the three genes were co-transcribed as a single transcript. Western immunoblot analysis revealed that the three xylanases were expressed at a very low level by F. succinogenes grown on either glucose or cellulose as the source of carbohydrate.Key words: Fibrobacter succinogenes S85, xylan, xylanase, clustered genes, RT-PCR.

The amino acid sequences of the carbohydrate-binding peptides of various di-N-acetylchitobiose-binding lectins

1992 ◽  
Vol 11 (4) ◽  
pp. 396-397
Author(s):  
Yukiko Konami ◽  
Kazuo Yamamoto ◽  
Toshiaki Osawa ◽  
Taturo Irimura
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Carbohydrate Binding ◽  
Binding Peptides

scholarly journals Prediction of Carbohydrate-Binding Proteins from Sequences Using Support Vector Machines

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Seizi Someya ◽  
Masanori Kakuta ◽  
Mizuki Morita ◽  
Kazuya Sumikoshi ◽  
Wei Cao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Support Vector Machines ◽  
Binding Proteins ◽  
Prediction Method ◽  
Amino Acid Sequences ◽  
Support Vector ◽  
Carbohydrate Binding ◽  
Genome Database ◽  
Vector Machines ◽  
Carbohydrate Binding Proteins

Carbohydrate-binding proteins are proteins that can interact with sugar chains but do not modify them. They are involved in many physiological functions, and we have developed a method for predicting them from their amino acid sequences. Our method is based on support vector machines (SVMs). We first clarified the definition of carbohydrate-binding proteins and then constructed positive and negative datasets with which the SVMs were trained. By applying the leave-one-out test to these datasets, our method delivered 0.92 of the area under the receiver operating characteristic (ROC) curve. We also examined two amino acid grouping methods that enable effective learning of sequence patterns and evaluated the performance of these methods. When we applied our method in combination with the homology-based prediction method to the annotated human genome database, H-invDB, we found that the true positive rate of prediction was improved.

scholarly journals Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies

1988 ◽  
Vol 256 (2) ◽  
pp. 453-459 ◽  
Author(s):  
O Nakanishi ◽  
Y Homma ◽  
H Kawasaki ◽  
Y Emori ◽  
K Suzuki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phospholipase C ◽  
Rat Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Liver Homogenate ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].

scholarly journals Characterization and Synergistic Interactions of Fibrobacter succinogenes Glycoside Hydrolases

2007 ◽  
Vol 73 (19) ◽  
pp. 6098-6105 ◽  
Author(s):  
Meng Qi ◽  
Hyun-Sik Jun ◽  
Cecil W. Forsberg
Keyword(s):  
Amino Acid ◽  
Synergistic Effect ◽  
Genome Sequence ◽  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolases ◽  
Fibrobacter Succinogenes ◽  
Gene Encoding ◽  
Substrate Specificities ◽  
Synergistic Interactions

ABSTRACT The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. succinogenes, Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.

scholarly journals Novel Bifunctional α-l-Arabinofuranosidase/Xylobiohydrolase (ABF3) from Penicillium purpurogenum

2010 ◽  
Vol 76 (15) ◽  
pp. 5247-5253 ◽  
Author(s):  
Mar�a Cristina Ravanal ◽  
Eduardo Callegari ◽  
Jaime Eyzaguirre
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glycosyl Hydrolase ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Mass Spectrometry Analysis ◽  
Carbohydrate Binding ◽  
Penicillium Purpurogenum ◽  
Spectrometry Analysis ◽  
Amino Terminal

ABSTRACT The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside with a Km of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XlnR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both α-l-arabinofuranosidase and xylobiohydrolase functionalities.

scholarly journals The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

2003 ◽  
Vol 371 (3) ◽  
pp. 1027-1043 ◽  
Author(s):  
Deborah HOGG ◽  
Gavin PELL ◽  
Paul DUPREE ◽  
Florence GOUBET ◽  
Susana M. MARTÍN-ORÚE ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Molecular Architecture ◽  
Catalytic Domains ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

scholarly journals Comparative Analyses of Two Thermophilic Enzymes Exhibiting both β-1,4 Mannosidic and β-1,4 Glucosidic Cleavage Activities from Caldanaerobius polysaccharolyticus

2010 ◽  
Vol 192 (16) ◽  
pp. 4111-4121 ◽  
Author(s):  
Yejun Han ◽  
Dylan Dodd ◽  
Charles W. Hespen ◽  
Samuel Ohene-Adjei ◽  
Charles M. Schroeder ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Biochemical Properties ◽  
Degree Of Polymerization ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Biofuel Industry ◽  
Carbohydrate Binding Modules ◽  
Mannanase Activity ◽  
Hydrolysis Of ◽  
Derivatives Of

ABSTRACT The hydrolysis of polysaccharides containing mannan requires endo-1,4-β-mannanase and 1,4-β-mannosidase activities. In the current report, the biochemical properties of two endo-β-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-β-mannanase and endo-1,4-β-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-β-mannanase activity and little endo-1,4-β-glucanase activity; however, this enzyme also exhibited 1,4-β-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of β-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.

scholarly journals Phenotypic and Enzymatic Comparative Analysis of the Novel KPC Variant KPC-5 and Its Evolutionary Variants, KPC-2 and KPC-4

2008 ◽  
Vol 53 (2) ◽  
pp. 557-562 ◽  
Author(s):  
Daniel J. Wolter ◽  
Philip M. Kurpiel ◽  
Neil Woodford ◽  
Marie-France I. Palepou ◽  
Richard V. Goering ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Upstream Region ◽  
The Novel ◽  
Carbapenem Resistant ◽  
Flanking Regions ◽  
Hydrolysis Of ◽  
Additional Amino Acid

ABSTRACT A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated bla KPC-5, was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of bla KPC-5 showed significant differences from the flanking regions of other bla KPC variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro103→Arg), while KPC-4 contained Pro103→Arg plus an additional amino acid change (Val239→Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus, KPC enzymes may be evolving through stepwise mutations to alter their spectra of activity.

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