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

Ye, LiBin; Su, Xiaoyun; Schmitz, George E.; Moon, Young Hwan; Zhang, Jing; Mackie, Roderick I.; Cann, Isaac

doi:10.1128/aem.02009-12

Cited by 35 publications

(34 citation statements)

References 42 publications

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“…3). Thus, the two GH domains probably work synergistically, as has been shown for other Caldicellulosiruptor enzymes without SLH domains but with two catalytic domains (76,79,80). Various truncation mutants of Calkro_ 0111 (Fig.…”

Section: Discussionsupporting

confidence: 54%

“…Considering the full-length enzyme, coordination between the GH16 and GH55 domains is expected, such that the GH16 cuts new chain ends for the GH55 to digest, thereby liberating glucose. This synergism is not uncommon in freely secreted Caldicellulosiruptor enzymes with two catalytic domains (76,79,80), but synergism had yet to be demonstrated in any CAZyme SLH domain protein. This is primarily due to the fact that very few SLH domain proteins have two catalytic CAZyme domains, further highlighting the uniqueness of Calkro_0111.…”

Section: Kronotskyensis Produces An S-layer and Slh Enzymes Calkromentioning

confidence: 99%

See 1 more Smart Citation

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

Conway¹,

Pierce

et al. 2016

Journal of Biological Chemistry

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The genome of the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensis encodes 19 surface layer (S-layer) homology (SLH) domain-containing proteins, the most in any Caldicellulosiruptor species genome sequenced to date. These SLH proteins include five glycoside hydrolases (GHs) and one polysaccharide lyase, the genes for which were transcribed at high levels during growth on plant biomass. The largest GH identified so far in this genus, Calkro_0111 (2,435 amino acids), is completely unique to C. kronotskyensis and contains SLH domains. Calkro_0111 was produced recombinantly in Escherichia coli as two pieces, containing the GH16 and GH55 domains, respectively, as well as putative binding and spacer domains. These displayed endo-and exoglucanase activity on the ␤-1,3-1,6-glucan laminarin. A series of additional truncation mutants of Calkro_0111 revealed the essential architectural features required for catalytic function. Calkro_0402, another of the SLH domain GHs in C. kronotskyensis, when produced in E. coli, was active on a variety of xylans and ␤-glucans. Unlike Calkro_0111, Calkro_0402 is highly conserved in the genus Caldicellulosiruptor and among other biomass-degrading Firmicutes but missing from Caldicellulosiruptor bescii. As such, the gene encoding Calkro_0402 was inserted into the C. bescii genome, creating a mutant strain with its S-layer extensively decorated with Calkro_0402. This strain consequently degraded xylans more extensively than wild-type C. bescii. The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptor species.Many bacteria (1, 2) and archaea (3) produce a two-dimensional, para-crystalline array of protein that covers the outside of the cell, referred to as a surface layer (S-layer).5 In bacteria, the majority of S-layer proteins are non-covalently associated with the bacterial cell surface via specialized domains at their N or C terminus, typically from one of three distinct but analogous domain categories: surface layer homology (SLH) (4, 5), CWB2 (pfam04122) (6), or NCAD (previously SLAP, pfam03217) (7,8). In archaea, S-layer proteins are most often attached to the cell membrane. S-layer proteins can be anchored to the membrane via a C-terminal transmembrane helix domain (3, 9) or covalently attached to cell membrane lipid via an archaeosortase, as shown in Haloferax volcanii (10,11). In most microorganisms, the S-layer is composed entirely of one or two proteins (SLPs), which self-assemble. In addition to these SLPs, some Firmicutes produce a family of proteins that also contain SLH, CWB2, or NCAD domains and, as such, are predicted to be associated with the S-layer (12). SLPs and S-layer associated proteins can also contain domains with specialized functions allowing the S-layer to play a role in adherence and biofilm formation (13-16), biogenesis of the cell envelope (17), cell division (18...

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Section: Discussionsupporting

confidence: 54%

Section: Kronotskyensis Produces An S-layer and Slh Enzymes Calkromentioning

confidence: 99%

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

Conway¹,

Pierce

et al. 2016

Journal of Biological Chemistry

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“…The diverse enzyme system is shown in Figure 5. Ye et al [82] used genetic engineering such as cloning and expression to produce a bifunctional enzyme from hyperthermophilic bacterium Caldicellulosiruptor bescii. This enzyme is able to hydrolyze both cellulose and mannan oligosaccharides as well as show a modest synergistic effect with other endoglucanases.…”

Section: Enzymatic Hydrolysismentioning

confidence: 99%

Biomass processing into ethanol: pretreatment, enzymatic hydrolysis, fermentation, rheology, and mixing

Volynets

Ein‐Mozaffari

Dahman

2016

Green Processing and Synthesis

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Alternate energy resources need to be developed to amend for depleting fossil fuel reserves. Lignocellulosic biomass is a globally available renewable feedstock that contains a rich sugar platform that can be converted into bioethanol through appropriate processing. The key steps of the process, pretreatment, enzymatic hydrolysis, and fermentation, have undergone considerable amount of research and development over the past decades nearing the process to commercialization. In order for the commercialization to be successful, the process needs to be operated at high dry matter content of biomass, especially in the enzymatic hydrolysis stage that influences ethanol concentration in the final fermentation broth. Biomass becomes a thick paste with challenging rheology for mixing to be effective. As the biomass consistency increases, yield stress increases which limits efficiency of mixing with conventional stirred tanks. The purpose of this review is to provide features and perspectives on processing of biomass into ethanol. Emphasis is placed on rheology and mixing of biomass in the enzymatic hydrolysis step as one of the forefront issues in the field.

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“…The major enzymes are composed of multidomain structures, two GHs, and one to three CBMs (13,14,16,17). Several multidomain enzymes have been biochemically characterized (18)(19)(20)(21)(22)(23). Nevertheless, the function and biochemical properties of noncatalytic proteins secreted by Caldicellulosiruptor species are unknown.…”

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confidence: 99%

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

et al. 2014

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Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (K a ) values of 5.2 ؋ 10 6 to 44 ؋ 10 6 M ؊1 among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.

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

Cited by 35 publications

References 42 publications

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

Biomass processing into ethanol: pretreatment, enzymatic hydrolysis, fermentation, rheology, and mixing

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

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