scholarly journals S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus

2011 ◽  
Vol 78 (3) ◽  
pp. 768-777 ◽  
Author(s):  
Inci Ozdemir ◽  
Sara E. Blumer-Schuette ◽  
Robert M. Kelly
Keyword(s):  
Cellular Localization ◽  
Catalytic Domain ◽  
Plant Biomass ◽  
Thermophilic Bacteria ◽  
Biochemical Properties ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Content Type

ABSTRACTThe genusCaldicellulosiruptorcontains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes ofCaldicellulosiruptorspecies reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins inCaldicellulosiruptor saccharolyticus(Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequencedCaldicellulosiruptorspecies. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in theC. saccharolyticusgenome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to theC. saccharolyticusS-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in otherCaldicellulosiruptorgenomes may also be important contributors to plant biomass utilization.

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 Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Jordan F. Russell ◽  
Matthew L. Russo ◽  
Xuewen Wang ◽  
Neal Hengge ◽  
Daehwan Chung ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Plant Biomass ◽  
Protein Glycosylation ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Unique Gene ◽  
Glycosyl Transferase ◽  
Caldicellulosiruptor Bescii ◽  
Carbohydrate Binding Modules

ABSTRACT Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases. IMPORTANCE Caldicellulosiruptor has the ability to digest complex plant biomass without pretreatment and have been engineered to convert biomass, a sustainable, carbon neutral substrate, to fuels. Their strategy for deconstructing plant cell walls relies on an interesting class of cellulases consisting of multiple catalytic modules connected by linker regions and carbohydrate binding modules. The best studied of these enzymes, CelA, has a unique deconstruction mechanism. CelA is located in a cluster of genes that likely allows for optimal expression, secretion, and activity. One of the genes in this cluster is a putative isomerase that modifies the CelA protein. In higher eukaryotes, these isomerases are essential for the proper folding of glycoproteins in the endoplasmic reticulum, but little is known about the role of isomerization in cellulase activity. We show that the stability and activity of CelA is dependent on the activity of this isomerase.

scholarly journals Caldicellulosiruptor bescii Adheres to Polysaccharides via a Type IV Pilin-Dependent Mechanism

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Asma M. A. M. Khan ◽  
Valerie J. Hauk ◽  
Mena Ibrahim ◽  
Thomas R. Raffel ◽  
Sara E. Blumer-Schuette
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Elevated Temperatures ◽  
Plant Biomass ◽  
Cell Attachment ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
Type Iv

ABSTRACT Biological hydrolysis of cellulose above 70°C involves microorganisms that secrete free enzymes and deploy separate protein systems to adhere to their substrate. Strongly cellulolytic Caldicellulosiruptor bescii is one such extreme thermophile, which deploys modular, multifunctional carbohydrate-acting enzymes to deconstruct plant biomass. Additionally, C. bescii also encodes noncatalytic carbohydrate binding proteins, which likely evolved as a mechanism to compete against other heterotrophs in carbon-limited biotopes that these bacteria inhabit. Analysis of the Caldicellulosiruptor pangenome identified a type IV pilus (T4P) locus encoded upstream of the tāpirins, that is encoded by all Caldicellulosiruptor species. In this study, we sought to determine if the C. bescii T4P plays a role in attachment to plant polysaccharides. The major C. bescii pilin (CbPilA) was identified by the presence of pilin-like protein domains, paired with transcriptomics and proteomics data. Using immuno-dot blots, we determined that the plant polysaccharide xylan induced production of CbPilA 10- to 14-fold higher than glucomannan or xylose. Furthermore, we are able to demonstrate that recombinant CbPilA directly interacts with xylan and cellulose at elevated temperatures. Localization of CbPilA at the cell surface was confirmed by immunofluorescence microscopy. Lastly, a direct role for CbPilA in cell adhesion was demonstrated using recombinant CbPilA or anti-CbPilA antibodies to reduce C. bescii cell adhesion to xylan and crystalline cellulose up to 4.5- and 2-fold, respectively. Based on these observations, we propose that CbPilA and, by extension, the T4P play a role in Caldicellulosiruptor cell attachment to plant biomass. IMPORTANCE Most microorganisms are capable of attaching to surfaces in order to persist in their environment. Type IV (T4) pili produced by certain mesophilic Firmicutes promote adherence; however, a role for T4 pili encoded by thermophilic members of this phylum has yet to be demonstrated. Prior comparative genomics analyses identified a T4 pilus locus possessed by an extremely thermophilic genus within the Firmicutes. Here, we demonstrate that attachment to plant biomass-related carbohydrates by strongly cellulolytic Caldicellulosiruptor bescii is mediated by T4 pilins. Surprisingly, xylan but not cellulose induced expression of the major T4 pilin. Regardless, the C. bescii T4 pilin interacts with both polysaccharides at high temperatures and is located to the cell surface, where it is directly involved in C. bescii attachment. Adherence to polysaccharides is likely key to survival in environments where carbon sources are limiting, allowing C. bescii to compete against other plant-degrading microorganisms.

scholarly journals Proteomic Dissection of the Cellulolytic Machineries Used by Soil-DwellingBacteroidetes

mSystems ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Marcel Taillefer ◽  
Magnus Ø. Arntzen ◽  
Bernard Henrissat ◽  
Phillip B. Pope ◽  
Johan Larsbrink
Keyword(s):  
Cellular Localization ◽  
Cellulose Degradation ◽  
Biomass Conversion ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Label Free ◽  
Cellulose Conversion ◽  
Content Type ◽  
Highly Efficient

ABSTRACTBacteria of the phylumBacteroidetesare regarded as highly efficient carbohydrate metabolizers, but most species are limited to (semi)soluble glycans. The soilBacteroidetesspeciesCytophaga hutchinsoniiandSporocytophaga myxococcoideshave long been known as efficient cellulose metabolizers, but neither species conforms to known cellulolytic mechanisms. Both species require contact with their substrate but do not encode cellulosomal systems of cell surface-attached enzyme complexes or the polysaccharide utilization loci found in many otherBacteroidetesspecies. Here, we have fractionated the cellular compartments of each species from cultures growing on crystalline cellulose and pectin, respectively, and analyzed them using label-free quantitative proteomics as well as enzymatic activity assays. The combined results enabled us to highlight enzymes likely to be important for cellulose conversion and to infer their cellular localization. The combined proteomes represent a wide array of putative cellulolytic enzymes and indicate specific and yet highly redundant mechanisms for cellulose degradation. Of the putative endoglucanases, especially enzymes of hitherto-unstudied glycoside hydrolase family, 8 were abundant, indicating an overlooked important role during cellulose metabolism. Furthermore, both species generated a large number of abundant hypothetical proteins during cellulose conversion, providing a treasure trove of targets for future enzymology studies.IMPORTANCECellulose is the most abundant renewable polymer on earth, but its recalcitrance limits highly efficient conversion methods for energy-related and material applications. Though microbial cellulose conversion has been studied for decades, recent advances showcased that large knowledge gaps still exist. Bacteria of the phylumBacteroidetesare regarded as highly efficient carbohydrate metabolizers, but most species are limited to (semi)soluble glycans. A few species, including the soil bacteriaC. hutchinsoniiandS. myxococcoides, are regarded as cellulose specialists, but their cellulolytic mechanisms are not understood, as they do not conform to the current models for enzymatic cellulose turnover. By unraveling the proteome setups of these two bacteria during growth on both crystalline cellulose and pectin, we have taken a significant step forward in understanding their idiosyncratic mode of cellulose conversion. This report provides a plethora of new enzyme targets for improved biomass conversion.

scholarly journals Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Jie Zhou ◽  
Zhoukun Li ◽  
Jiale Wu ◽  
Lifeng Li ◽  
Ding Li ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Functional Characterization ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Binding Function ◽  
Catalytic Module

ABSTRACT A novel β-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward β-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel β-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules. IMPORTANCE In this report of a bacterial β-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the β-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-β-(1,3)/(1,6)-glucanase and endo-β-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.

scholarly journals Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

2016 ◽  
Vol 82 (11) ◽  
pp. 3395-3409 ◽  
Author(s):  
Sarah E. Hobdey ◽  
Brandon C. Knott ◽  
Majid Haddad Momeni ◽  
Larry E. Taylor ◽  
Anna S. Borisova ◽  
...  
Keyword(s):  
Active Site ◽  
Phylogenetic Trees ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Cellulolytic Enzymes ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Industrial Enzymes ◽  
Content Type ◽  
Social Amoeba

ABSTRACTGlycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba:Dictyostelium discoideumCel7A (DdiCel7A) andDictyostelium purpureumCel7A (DpuCel7A).DdiCel7A andDpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures ofDdiCel7A andDpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between theDictyosteliumCBHs and the archetypal model GH7 CBH,Trichoderma reeseiCel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity ofTreCel7A, the family 1TreCel7A CBM and linker were added to the C terminus of each of theDictyosteliumenzymes, creatingDdiCel7ACBMandDpuCel7ACBM, which were recombinantly expressed inT. reesei.DdiCel7ACBMandDpuCel7ACBMhydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently asTreCel7A when hydrolysis was compared at their temperature optima. TheKiof cellobiose was significantly higher forDdiCel7ACBMandDpuCel7ACBMthan forTreCel7A: 205, 130, and 29 μM, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.IMPORTANCEGH7 CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungusT. reeseiwhen tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate.

scholarly journals Systematic modification of N-glycosylation sites in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

2020 ◽  
Author(s):  
Bartłomiej Maciej Kołaczkowski ◽  
Kay S. Schaller ◽  
Trine Holst Sørensen ◽  
Günther H. J. Peters ◽  
Kenneth Jensen ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Catalytic Domain ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Site Density ◽  
Steady State Kinetics ◽  
Cellulose Surface

Abstract Background: Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Trichoderma reesei Cel7A (TrCel7A) has served as a model to understand structure-function relationships of processive cellobiohydrolases. The architecture of TrCel7A includes an N-glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible, O-glycosylated linker. Depending on the fungal expression host, glycosylation can vary not only in glycoforms, but also in site occupancy, leading to a complex pattern of glycans, which can affect the enzyme’s stability and kinetics. Results: Two expression hosts, Aspergillus oryzae and Trichoderma reesei, were utilized to successfully express wild-types TrCel7A (WTAo and WTTr) and the triple N-glycosylation site deficient mutants TrCel7A N45Q, N270Q, N384Q (ΔN-glycAo and ΔN-glycTr). Also, we expressed single N-glycosylation site deficient mutants TrCel7A (N45QAo, N270QAo, N384QAo). The TrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (KM) was consistently found to be lowered for the variants with reduced N-glycosylation content, and for the triple deficient mutants, it was less than half of the WTs value on some substrates. The ability of the enzyme to combine productively with sites on the cellulose surface followed a similar pattern on all tested substrates. Thus, site density (number of sites per gram cellulose) was 30-60 % higher for the single deficient variants compared to the WT, and about two-fold larger for the triple deficient enzyme. Molecular dynamic simulation of the N-glycan mutants TrCel7A reveled higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384. Conclusions: The kinetic changes of TrCel7A imposed by removal of N-linked glycans reflected modifications of substrate accessibility. The presence of N-glycans with extended structures increased KM and decreased attack site density of TrCel7A likely due to steric hindrance effect and distance between the enzyme and the cellulose surface, preventing the enzyme from achieving optimal conformation. This knowledge could be applied to modify enzyme glycosylation to engineer enzyme with higher activity on the insoluble substrates.

scholarly journals Systematic modification of N-glycosylation sites in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

2020 ◽  
Author(s):  
Bartłomiej Maciej Kołaczkowski ◽  
Kay S. Schaller ◽  
Trine Holst Sørensen ◽  
Günther H. J. Peters ◽  
Kenneth Jensen ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Catalytic Domain ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Site Density ◽  
Steady State Kinetics ◽  
Cellulose Surface

Abstract Background: Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Trichoderma reesei Cel7A (TrCel7A) has served as a model to understand structure-function relationships of processive cellobiohydrolases. The architecture of TrCel7A includes an N-glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible, O-glycosylated linker. Depending on the fungal expression host, glycosylation can vary not only in glycoforms, but also in site occupancy, leading to a complex pattern of glycans, which can affect the enzyme’s stability and kinetics. Results: Two expression hosts, Aspergillus oryzae and Trichoderma reesei, were utilized to successfully express wild-types TrCel7A (WTAo and WTTr) and the triple N-glycosylation site deficient mutants TrCel7A N45Q, N270Q, N384Q (ΔN-glycAo and ΔN-glycTr). Also, we expressed single N-glycosylation site deficient mutants TrCel7A (N45QAo, N270QAo, N384Ao). The TrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (KM) was consistently found to be lowered for the variants with reduced N-glycosylation content, and for the triple deficient mutants, it was less than half of the WTs value on some substrates. The ability of the enzyme to combine productively with sites on the cellulose surface followed a similar pattern on all tested substrates. Thus, site density (number of sites per gram cellulose) was 30-60 % higher for the single deficient variants compared to the WT, and about two-fold larger for the triple deficient enzyme. Molecular dynamic simulation of the N-glycan mutants TrCel7A reveled higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384. Conclusions: We propose that the marked kinetic changes did not rely on alterations in the overall fold of Cel7A but reflected modifications of substrate accessibility. The presence of N-glycans with extended structures increased KM and decreased attack site density of TrCel7A likely due to steric hindrance effect and distance between the enzyme and the cellulose surface, preventing the enzyme from achieving optimal conformation. This knowledge could be applied to modify enzyme glycosylation to engineer enzyme with higher activity on the insoluble substrates.

scholarly journals A New Group of Modular Xylanases in Glycoside Hydrolase Family 8 from Marine Bacteria

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Xiu-Lan Chen ◽  
Fang Zhao ◽  
Yong-Sheng Yue ◽  
Xi-Ying Zhang ◽  
Yu-Zhong Zhang ◽  
...  
Keyword(s):  
Marine Bacteria ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Domain Architecture ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Binding Ability ◽  
Content Type ◽  
Hydrolase Family

ABSTRACT Xylanases play a crucial role in the degradation of xylan in both terrestrial and marine environments. The endoxylanase XynB from the marine bacterium Glaciecola mesophila KMM 241 is a modular enzyme comprising a long N-terminal domain (NTD) (E44 to T562) with xylan-binding ability and a catalytic domain (CD) (T563 to E912) of glycoside hydrolase family 8 (GH8). In this study, the long NTD is confirmed to contain three different functional regions, which are NTD1 (E44 to D136), NTD2 (Y137 to A193), and NTD3 (L194 to T562). NTD1, mainly composed of eight β-strands, functions as a new type of carbohydrate-binding module (CBM), which has xylan-binding ability but no sequence similarity to any known CBM. NTD2, mainly forming two α-helices, contains one of the α-helices of the catalytic domain's (α/α)6 barrel and therefore is essential for the activity of XynB, although it is far away from the catalytic domain in sequence. NTD3, next to the catalytic domain in sequence, is shown to be helpful in maintaining the thermostability of XynB. Thus, XynB represents a kind of xylanase with a new domain architecture. There are four other predicted glycoside hydrolase sequences with the same domain architecture and high sequence identity (≥80%) with XynB, all of which are from marine bacteria. Phylogenetic analysis shows that XynB and these homologs form a new group in GH8, representing a new class of marine bacterial xylanases. Our results shed light on xylanases, especially marine xylanases. IMPORTANCE Xylanases play a crucial role in natural xylan degradation and have been extensively used in industries such as food processing, animal feed, and kraft pulp biobleaching. Some marine bacteria have been found to secrete xylanases. Characterization of novel xylanases from marine bacteria has significance for both the clarification of xylan degradation mechanisms in the sea and the development of new enzymes for industrial application. With G. mesophila XynB as a representative, this study reveals a new group of the GH8 xylanases from marine bacteria, which have a distinct domain architecture and contain a novel carbohydrate-binding module. Thus, this study offers new knowledge on marine xylanases.

scholarly journals Processivity and Enzymatic Mode of a Glycoside Hydrolase Family 5 Endoglucanase from Volvariella volvacea

2012 ◽  
Vol 79 (3) ◽  
pp. 989-996 ◽  
Author(s):  
Fei Zheng ◽  
Shaojun Ding
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Volvariella Volvacea ◽  
Reaction Conditions ◽  
Content Type ◽  
C Terminus ◽  
Hydrolase Family

ABSTRACTEG1 is a modular glycoside hydrolase family 5 endoglucanase fromVolvariella volvaceaconsisting of an N-terminal carbohydrate-binding module (CBM1) and a catalytic domain (CD). The ratios of soluble to insoluble reducing sugar produced from filter paper after 8 and 24 h of exposure to EG1 were 6.66 and 8.56, respectively, suggesting that it is a processive endoglucanase. Three derivatives of EG1 containing a core domain only or additional CBMs were constructed in order to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1 under stationary and agitated conditions. All four enzymatic forms exhibited the same mode of action on both soluble and insoluble cellulosic substrates with cellobiose as a main end product. An additional CBM fused at either the N or C terminus reduced specific activity toward soluble and insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantly decreased enzyme processivity. Insertion of an additional CBM also resulted in a dramatic decrease in processivity in enzyme-substrate reaction mixtures incubated for 0.5 h, but this effect was reversed when reactions were allowed to proceed for longer periods (24 h). Further significant differences were observed in the substrate adsorption/desorption patterns of EG1 and enzyme derivatives equipped with an additional CBM under agitated reaction conditions. An additional family 1 CBM improved EG1 processivity on insoluble cellulose under highly agitated conditions. Our data indicate a strong link between high adsorption levels and low desorption levels in the processivity of EG1 and possibly other processive endoglucanses.

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