Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

ABSTRACTThe genome of the coprophilic ascomycetePodospora anserinaencodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserinaGH61A [PaGH61A] andPaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced inPichia pastoris. Synergistic cooperation betweenPaGH61A orPaGH61B with the cellobiose dehydrogenase (CDH) ofPycnoporus cinnabarinuson cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference betweenPaGH61A andPaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination ofPaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties ofPaGH61A andPaGH61B and their effect on the interaction with CDH are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

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Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

Applied and Environmental Microbiology ◽

10.1128/aem.01503-12 ◽

2012 ◽

Vol 78 (17) ◽

pp. 6161-6171 ◽

Cited By ~ 80

Author(s):

Christoph Sygmund ◽

Daniel Kracher ◽

Stefan Scheiblbrandner ◽

Kawah Zahma ◽

Alfons K. G. Felice ◽

...

Keyword(s):

Neurospora Crassa ◽

Enzyme System ◽

Cellulose Degradation ◽

Cellulose Hydrolysis ◽

Carbohydrate Binding ◽

Ph Optimum ◽

Content Type ◽

Polysaccharide Monooxygenase

ABSTRACTThe genome ofNeurospora crassaencodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome ofN. crassaand preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced inPichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochromec, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (kcatandKmvalues) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the hemebcofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) fromN. crassawas expressed inP. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

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The Pyrroloquinoline-Quinone-Dependent Pyranose Dehydrogenase from Coprinopsis cinerea Drives Lytic Polysaccharide Monooxygenase Action

Applied and Environmental Microbiology ◽

10.1128/aem.00156-18 ◽

2018 ◽

Vol 84 (11) ◽

Cited By ~ 29

Author(s):

Anikó Várnai ◽

Kiwamu Umezawa ◽

Makoto Yoshida ◽

Vincent G. H. Eijsink

Keyword(s):

Neurospora Crassa ◽

Pyrroloquinoline Quinone ◽

Carbohydrate Binding ◽

Coprinopsis Cinerea ◽

Content Type ◽

Strongly Coupled ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases ◽

Pyranose Dehydrogenase ◽

External Electron Donor

ABSTRACT Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs catalyze the oxidative cleavage of glycosidic bonds after activation by an external electron donor. So far, only flavin-dependent oxidoreductases (from the auxiliary activity [AA] family AA3) have been shown to activate LPMOs. Here, we present LPMO activation by a pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase (PDH) from Coprinopsis cinerea , Cc PDH, the founding member of the recently discovered auxiliary activity family AA12. Cc PDH contains a C-terminal family 1 carbohydrate binding module (CBM1), an N-terminal family AA8 cytochrome domain, and a central AA12 dehydrogenase domain. We have studied the ability of full-length Cc PDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs. The results show that Cc PDH indeed can activate the C-1-oxidizing N. crassa LPMO 9F ( Nc LPMO9F) and the C-4-oxidizing Neurospora crassa LPMO 9C ( Nc LPMO9C), that this activation depends on the cytochrome domain, and that the dehydrogenase and the LPMO reactions are strongly coupled. The two tested Cc PDH-LPMO systems showed quite different efficiencies, and this difference disappeared upon the addition of free PQQ acting as a diphenol/quinone redox mediator, showing that LPMOs differ when it comes to their direct interactions with the cytochrome domain. Surprisingly, removal of the CBM domain from Cc PDH had a considerable negative impact on the efficiency of the Cc PDH-LPMO systems, suggesting that electron transfer in the vicinity of the substrate is beneficial. Cc PDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool for studying cellulose-oxidizing LPMOs. IMPORTANCE Lytic polysaccharide monooxygenases (LPMOs) are currently receiving increasing attention because of their importance in degrading recalcitrant polysaccharides and their potential roles in biological processes, such as bacterial virulence. LPMO action requires an external electron donor, and fungi growing on biomass secrete various so-called glucose-methanol-choline (GMC) oxidoreductases, including cellobiose dehydrogenase, which can donate electrons to LPMOs. This paper describes how an enzyme not belonging to the GMC oxidoreductase family, Cc PDH, can activate LPMOs, and it provides new insights into the activation process by (i) describing the roles of individual Cc PDH domains (a dehydrogenase, a cytochrome, and a carbohydrate-binding domain), (ii) showing that the PDH and LPMO enzyme reactions are strongly coupled, (iii) demonstrating that LPMOs differ in terms of their efficiencies of activation by the same activator, and (iv) providing indications that electron transferring close to the substrate surface is beneficial for the overall efficiency of the Cc PDH-LPMO system.

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Characterization of a Broad-Specificity β-Glucanase Acting on β-(1,3)-, β-(1,4)-, and β-(1,6)-Glucans That Defines a New Glycoside Hydrolase Family

Applied and Environmental Microbiology ◽

10.1128/aem.02572-12 ◽

2012 ◽

Vol 78 (24) ◽

pp. 8540-8546 ◽

Cited By ~ 29

Author(s):

Mickael Lafond ◽

David Navarro ◽

Mireille Haon ◽

Marie Couturier ◽

Jean-Guy Berrin

Keyword(s):

Podospora Anserina ◽

Full Length ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

High Sequence Identity ◽

Content Type ◽

New Family ◽

Domain Of Unknown Function ◽

Type Mode

ABSTRACTHere we report the cloning of thePa_3_10940gene from the coprophilic fungusPodospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeastPichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of β-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this β-glucanase is an exo-acting enzyme on β-(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan substrates. Hydrolysis of short β-(1,3), β-(1,4), and β-(1,3)/β-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-β-(1,3)/(1,6) and endo-β-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.

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Multifunctional Nutrient-Binding Proteins Adapt Human Symbiotic Bacteria for Glycan Competition in the Gut by Separately Promoting Enhanced Sensing and Catalysis

mBio ◽

10.1128/mbio.01441-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 35

Author(s):

Elizabeth A. Cameron ◽

Kurt J. Kwiatkowski ◽

Byung-Hoo Lee ◽

Bruce R. Hamaker ◽

Nicole M. Koropatkin ◽

...

Keyword(s):

Binding Sites ◽

Binding Proteins ◽

Human Microbiome ◽

Gut Bacteria ◽

Nutrient Acquisition ◽

Carbohydrate Binding ◽

Content Type ◽

Carbohydrate Binding Proteins ◽

Gnotobiotic Mice

ABSTRACT To compete for the dynamic stream of nutrients flowing into their ecosystem, colonic bacteria must respond rapidly to new resources and then catabolize them efficiently once they are detected. The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model for nutrient acquisition by symbiotic gut bacteria, which harbor thousands of related Sus-like systems. Structural investigation of the four Sus outer membrane proteins (SusD, -E, -F, and -G) revealed that they contain a total of eight starch-binding sites that we demonstrated, using genetic and biochemical approaches, to play distinct roles in starch metabolism in vitro and in vivo in gnotobiotic mice. SusD, whose homologs are abundant in the human microbiome, is critical for the initial sensing of available starch, allowing sus transcriptional activation at much lower concentrations than without this function. In contrast, seven additional binding sites across SusE, -F, and -G are dispensable for sus activation. However, they optimize the rate of growth on starch in a manner dependent on the expression of the bacterial polysaccharide capsule, suggesting that they have evolved to offset the diffusion barrier created by this structure. These findings demonstrate how proteins with similar biochemical behavior can serve orthogonal functions during different stages of cellular adaptation to nutrients. Finally, we demonstrated in gnotobiotic mice fed a starch-rich diet that the Sus binding sites confer a competitive advantage to B. thetaiotaomicron in vivo in a manner that is dependent on other colonizing microbes. This study reveals how numerically dominant families of carbohydrate-binding proteins in the human microbiome fulfill separate and sometimes cooperative roles to optimize gut commensal bacteria for nutrient acquisition. IMPORTANCE Our intestinal tract harbors trillions of symbiotic microbes. A critical function contributed by this microbial community is the ability to degrade most of the complex carbohydrates in our diet, which not only change from meal to meal but also cannot be digested by our own bodies. A numerically abundant group of gut bacteria called the Bacteroidetes plays a prominent role in carbohydrate digestion in humans and other animals. Currently, the mechanisms that allow this bacterial group to rapidly respond to available carbohydrates and then digest them efficiently are unclear. Here, we present novel functions for four carbohydrate-binding proteins present in one member of the Bacteroidetes, revealing that these proteins serve unique and separable roles in either initial nutrient sensing or subsequent digestion. Because the protein families investigated are numerous in other gut bacteria colonizing nearly all humans and animals, our findings are fundamentally important to understanding how symbiotic microbes assist human digestion.

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Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii

Applied and Environmental Microbiology ◽

10.1128/aem.02009-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 7048-7059 ◽

Cited By ~ 26

Author(s):

Libin Ye ◽

Xiaoyun Su ◽

George E. Schmitz ◽

Young Hwan Moon ◽

Jing Zhang ◽

...

Keyword(s):

Specific Activity ◽

Cellulose Hydrolysis ◽

Thermophilic Bacterium ◽

Carbohydrate Binding ◽

Content Type ◽

Caldicellulosiruptor Bescii ◽

C Terminus ◽

Carbohydrate Binding Modules ◽

Β Sheets ◽

Biochemical Analyses

ABSTRACTA large polypeptide encoded in the genome of the thermophilic bacteriumCaldicellulosiruptor besciiwas determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol.78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases fromC. besciiled to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization byC. bescii.

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Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization

Applied and Environmental Microbiology ◽

10.1128/aem.01408-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 14

Author(s):

Silvia Hüttner ◽

Anikó Várnai ◽

Dejan M. Petrović ◽

Cao Xuan Bach ◽

Dang Thi Kim Anh ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Functional Characterization ◽

Cellulose Microfibrils ◽

Cell Wall Polysaccharide ◽

Poor Growth ◽

Content Type ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

ABSTRACT The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers’ physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall. IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.

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

Applied and Environmental Microbiology ◽

10.1128/aem.07031-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 768-777 ◽

Cited By ~ 43

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.

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Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

Applied and Environmental Microbiology ◽

10.1128/aem.02716-16 ◽

2016 ◽

Vol 83 (2) ◽

Cited By ~ 9

Author(s):

Narumon Tangthirasunun ◽

David Navarro ◽

Sona Garajova ◽

Didier Chevret ◽

Laetitia Chan Ho Tong ◽

...

Keyword(s):

Plant Biomass ◽

Cellulose Degradation ◽

Degradation Mechanism ◽

Podospora Anserina ◽

Cellulosic Biomass ◽

Striking Difference ◽

Crystalline Cellulose ◽

Minor Role ◽

Wild Type ◽

Content Type

ABSTRACT Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

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Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Applied and Environmental Microbiology ◽

10.1128/aem.06814-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2230-2240 ◽

Cited By ~ 40

Author(s):

Xiaoyun Su ◽

Roderick I. Mackie ◽

Isaac K. O. Cann

Keyword(s):

Catalytic Efficiency ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Wild Type ◽

Ph Optimum ◽

Content Type ◽

Caldicellulosiruptor Bescii ◽

Type Protein ◽

Wild Type Protein ◽

Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

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Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

Applied and Environmental Microbiology ◽

10.1128/aem.02118-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 133-140 ◽

Cited By ~ 22

Author(s):

Evelina Kulcinskaja ◽

Anna Rosengren ◽

Romany Ibrahim ◽

Katarína Kolenová ◽

Henrik Stålbrand

Keyword(s):

Signal Peptide ◽

High Performance ◽

Guar Gum ◽

Transmembrane Helix ◽

Exchange Chromatography ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Bifidobacterium Adolescentis ◽

Content Type ◽

Carbohydrate Binding Modules

ABSTRACTThe gene encoding β-mannanase (EC 3.2.1.78) BaMan26A from the bacteriumBifidobacterium adolescentis(living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed inEscherichia coliwithout the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 μM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K,kcatwas determined to be 444 s−1andKm21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.

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