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1.
J Sci Food Agric ; 102(1): 85-94, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031874

RESUMO

BACKGROUND: Epilactose, a potential prebiotics, was derived from lactose through enzymatic catalysis. However, production and purification of epilactose are currently difficult due to powerless enzymes and inefficient downstream processing steps. RESULTS: The encoding gene of cellobiose 2-epimerase (CE) from Caldicellulosiruptor sp. Rt8.B8 was cloned and expressed in Escherichia coli BL21(DE3). The enzyme was purified and it was suitable for industrial production of epilactose from lactose without by-products, because of high kcat (197.6 s-1 ) and preferable thermostability. The Rt8-CE gene was further expressed in the Bacillus subtilis strain. We successfully produced epilactose from 700 g L-1 lactose in 30.4% yield by using the recombinant Bacillus subtilis whole cells. By screening of a ß-galactosidase from Bacillus stearothermophilus (BsGal), a process for separating epilactose and lactose was established, which showed a purity of over 95% in a total yield of 69.2%. In addition, a mixed rare sugar syrup composed of epilactose and d-tagatose was successfully produced from lactose through the co-expression of l-arabinose isomerase and ß-galactosidase. CONCLUSION: Our study shed light on the efficient production of epilactose using a food-grade host expressing a novel CE enzyme. Moreover, an efficient and low-cost process was attempted to obtain high purity epilactose. In order to improve the utilization of raw materials, the production process of mixed syrup containing epilactose and d-tagatose with prebiotic properties produced from lactose was also established for the first time. © 2021 Society of Chemical Industry.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Caldicellulosiruptor/enzimologia , Celobiose/metabolismo , Dissacarídeos/biossíntese , Racemases e Epimerases/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caldicellulosiruptor/genética , Estabilidade Enzimática , Expressão Gênica , Temperatura Alta , Lactose/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-34542397

RESUMO

A novel nitrogen-fixing fermentative bacterium, designated as YA01T, was isolated from Nakabusa hot springs in Japan. The short-rod cells of strain YA01T were Gram-positive and non-sporulating. Phylogenetic trees of the 16S rRNA gene sequence and concatenated sequences of 40 single-copy ribosomal genes revealed that strain YA01T belonged to the genus Caldicellulosiruptor and was closely related to Caldicellulosiruptor hydrothermalis 108T, Caldicellulosiruptor bescii DSM 6725T and Caldicellulosiruptor kronotskyensis 2002T. The 16S rRNA gene sequence of strain YA01T shares less than 98.1 % identity to the known Caldicellulosiruptor species. The G+C content of the genomic DNA was 34.8 mol%. Strain YA01T shares low genome-wide average nucleotide identity (90.31-91.10 %), average amino acid identity (91.45-92.10 %) and <70 % digital DNA-DNA hybridization value (41.8-44.2 %) with the three related species of the genus Caldicellulosiruptor. Strain YA01T grew at 50-78 °C (optimum, 70 °C) and at pH 5.0-9.5 (optimum, pH 6.5). Strain YA01T mainly produced acetate by consuming d(+)-glucose as a carbon source. The main cellular fatty acids were iso-C17 : 0 (35.7 %), C16 : 0 (33.3 %), DMA16 : 0 (6.6 %) and iso-C15 : 0 (5.9 %). Based on its distinct phylogenetic position, biochemical and physiological characteristics, and the major cellular fatty acids, strain YA01T is considered to represent a novel species of the genus Caldicellulosiruptor for which the name Caldicellulosiruptor diazotrophicus sp. nov. is proposed (type strain YA01T=DSM 112098T=JCM 34253T).


Assuntos
Fontes Termais , Técnicas de Tipagem Bacteriana , Composição de Bases , Caldicellulosiruptor , DNA Bacteriano/genética , Ácidos Graxos/química , Japão , Nitrogênio , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
3.
Artigo em Inglês | MEDLINE | ID: mdl-34424833

RESUMO

The present study was carried out to re-clarify the taxonomic relationship of Caldicellulosiruptor acetigenus, Caldicellulosiruptor lactoaceticus and Caldicellulosiruptor kristjanssonii. The 16S rRNA sequence similarities between these species of the genus Caldicellulosiruptor were above the threshold values (98.65%) for bacterial species delineation. Similarly, the digital DNA-DNA hybridization and average nucleotide and amino acid identity values were greater than the thresholds for bacterial species delineation. In phylogenetic (based on 16S rRNA gene sequences) and phylogenomic trees Caldicellulosiruptor acetigenus, Caldicellulosiruptor lactoaceticus and Caldicellulosiruptor kristjanssonii clade together. The results of our analysis indicated that these three taxa are conspecific. Therefore, Caldicellulosiruptor lactoaceticus Mladenovska et al. 1997 and Caldicellulosiruptor kristjanssonii Bredholt et al. 1999 should be reclassified as later heterotypic synonyms of Caldicellulosiruptor acetigenus (Nielsen et al. 1994) Onyenwoke et al. 2006.


Assuntos
Caldicellulosiruptor , Filogenia , Técnicas de Tipagem Bacteriana , Caldicellulosiruptor/classificação , DNA Bacteriano/genética , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
4.
Biochemistry ; 60(27): 2206-2220, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34180241

RESUMO

The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1-CBM22.2-Xyn10C construct, indicating a compact arrangement at room temperature.


Assuntos
Proteínas de Bactérias/química , Caldicellulosiruptor/enzimologia , Esterases/química , Xilosidases/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Caldicellulosiruptor/química , Caldicellulosiruptor/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Esterases/metabolismo , Modelos Moleculares , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Conformação Proteica , Temperatura , Xilosidases/metabolismo
5.
Microbes Environ ; 36(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34108360

RESUMO

Fermentative nitrogen-fixing bacteria have not yet been examined in detail in thermal environments. In the present study, we isolated the thermophilic fermentative bacterium, strain YA01 from a hot spring. This strain grew at temperatures up to 78°C. A phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain YA01 belonged to the genus Caldicellulosiruptor, which are fermentative bacteria in the phylum Firmicutes, with 97.7-98.0% sequence identity to its closest relatives. Strain YA01 clearly exhibited N2-dependent growth at 70°C. We also confirmed N2-dependent growth in the relatives of strain YA01, Caldicellulosiruptor hydrothermalis 108 and Caldicellulosiruptor kronotskyensis 2002. The nitrogenase activities of these three strains were examined using the acetylene reduction assay. Similar activities were detected for all tested strains, and were slightly suppressed by the addition of ammonium. A genome analysis revealed that strain YA01, as well as other Caldicellulosiruptor, possessed a gene set for nitrogen fixation, but lacked the nifN gene, which encodes a nitrogenase iron-molybdenum cofactor biosynthesis protein that is commonly detected in nitrogen-fixing bacteria. The amino acid sequences of nitrogenase encoded by nifH, nifD, and nifK shared 92-98% similarity in Caldicellulosiruptor. A phylogenetic tree of concatenated NifHDK sequences showed that NifHDK of Caldicellulosiruptor was in the deepest clade. To the best of our knowledge, this is the first study to demonstrate the nitrogen-fixing ability of fermentative bacteria at 70°C. Caldicellulosiruptor may have retained an ancient nitrogen-fixing enzyme system.


Assuntos
Caldicellulosiruptor/isolamento & purificação , Caldicellulosiruptor/fisiologia , Fixação de Nitrogênio , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caldicellulosiruptor/classificação , Caldicellulosiruptor/genética , Fermentação , Genoma Bacteriano , Fontes Termais/química , Fontes Termais/microbiologia , Temperatura Alta , Nitrogênio/metabolismo , Nitrogenase/química , Nitrogenase/genética , Nitrogenase/metabolismo , Filogenia
6.
Appl Environ Microbiol ; 87(14): e0052421, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33990300

RESUMO

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous ß-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. ß-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and ß-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular ß-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable ß-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCE Caldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.


Assuntos
Actinobacteria/enzimologia , Caldicellulosiruptor/metabolismo , Proteoma/metabolismo , Thermotoga maritima/enzimologia , Xilanos/metabolismo , Xilosidases/metabolismo , Actinobacteria/genética , Celobiose/metabolismo , Escherichia coli/genética , Thermotoga maritima/genética , Xilosidases/genética
7.
J Ind Microbiol Biotechnol ; 48(5-6)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33956122

RESUMO

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.


Assuntos
Celulase/biossíntese , Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Via Secretória , Partícula de Reconhecimento de Sinal/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caldicellulosiruptor/enzimologia , Caldicellulosiruptor/genética , Carboxipeptidases/genética , Celulase/genética , Celulose/metabolismo , DNA Bacteriano , Escherichia coli/genética , Fermentação , Glicosídeo Hidrolases , Microbiologia Industrial , Mutação , Peptidoglicano/genética , Domínios Proteicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes/biossíntese
8.
J Agric Food Chem ; 69(6): 1907-1915, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33541071

RESUMO

Cellobiose 2-epimerase (CE) offers a promising enzymatic approach to produce lactulose. However, its application is limited by the unsatisfactory isomerization activity and thermostability. Our study attempted to optimize the catalytic performances of CEs by flexible loop exchange, for which four mutants were constructed using CsCE (CE from Caldicellulosiruptor saccharolyticus) as a template. As a result, all mutants maintained the same catalytic directions as the templates. Mutant RmC displayed a 2.2- and 1.34-fold increase in the isomerization activity and catalytic efficiency, respectively. According to the results of molecular dynamics (MD) simulations, it was revealed that the loop exchange in RmC enlarged the entrance of the active site for substrate binding and benefited proton transfer involved in the isomerization process. Besides, the t1/2 of mutant StC at 70 °C was increased from 29.07 to 38.29 h, owing to the abundance of rigid residues (proline) within the flexible loop of StC. Our work demonstrated that the isomerization activity and thermostability of CEs were closely related to the flexible loop surrounding the active site, which provides a new perspective to engineer CEs for higher lactulose production.


Assuntos
Caldicellulosiruptor , Celobiose , Estabilidade Enzimática , Isomerismo , Lactulose , Racemases e Epimerases/genética
9.
Acta Crystallogr D Struct Biol ; 76(Pt 11): 1104-1113, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33135681

RESUMO

Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Šresolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148-181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Caldicellulosiruptor/enzimologia , Carboidratos Epimerases/química , Spirochaeta/enzimologia , Proteínas de Bactérias/genética , Biocatálise , Carboidratos Epimerases/genética , Domínio Catalítico , Isomerismo , Mutagênese Sítio-Dirigida , Especificidade por Substrato
10.
J Ind Microbiol Biotechnol ; 47(8): 585-597, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32783103

RESUMO

Caldicellulosiruptor bescii is the most thermophilic cellulolytic organism yet identified (Topt 78 °C). It grows on untreated plant biomass and has an established genetic system thereby making it a promising microbial platform for lignocellulose conversion to bio-products. Here, we investigated the ability of engineered C. bescii to generate alcohols from carboxylic acids. Expression of aldehyde ferredoxin oxidoreductase (aor from Pyrococcus furiosus) and alcohol dehydrogenase (adhA from Thermoanaerobacter sp. X514) enabled C. bescii to generate ethanol from crystalline cellulose and from biomass by reducing the acetate produced by fermentation. Deletion of lactate dehydrogenase in a strain expressing the AOR-Adh pathway increased ethanol production. Engineered strains also converted exogenously supplied organic acids (isobutyrate and n-caproate) to the corresponding alcohol (isobutanol and hexanol) using both crystalline cellulose and switchgrass as sources of reductant for alcohol production. This is the first instance of an acid to alcohol conversion pathway in a cellulolytic microbe.


Assuntos
Caldicellulosiruptor/genética , Ácidos Carboxílicos/metabolismo , Etanol/metabolismo , Lignina/metabolismo , Microrganismos Geneticamente Modificados , Panicum/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Biocombustíveis/análise , Biomassa , Fermentação , Oxirredução , Panicum/microbiologia , Pyrococcus furiosus/enzimologia , Thermoanaerobacter/enzimologia
11.
Appl Environ Microbiol ; 86(20)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32769195

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Caldicellulosiruptor/genética , Celulose/metabolismo , Peptidilprolil Isomerase/genética , Proteínas de Bactérias/metabolismo , Caldicellulosiruptor/metabolismo , Deleção de Genes , Glicosilação , Peptidilprolil Isomerase/metabolismo , Especificidade por Substrato
12.
Appl Environ Microbiol ; 86(16)2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32532871

RESUMO

Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode pectinolytic enzymes. Pectate lyases that cleave the α-1,4-galacturonosidic linkage of pectin are widely used in industries such as papermaking and fruit softening. However, there are few reports on pectate lyases with good thermostability. Here, two pectate lyases (CbPL3 and CbPL9) from a hyperthermophilic bacterium, Caldicellulosiruptor bescii, belonging to family 3 and family 9 polysaccharide lyases, respectively, were investigated. The biochemical properties of the two CbPLs were shown to be similar under optimized conditions of 80°C to 85°C and pH 8 to 9. However, the degradation products from pectin and polygalacturonic acids (pGAs) were different. A family 66 carbohydrate-binding module (CbCBM66) located in the N terminus of the two CbPLs shares 100% amino acid identity. A CbCBM66-truncated mutant of CbPL9 showed lower activities than the wild type, whereas CbPL3 with a CbCBM66 knockout portion was reported to have enhanced activities, thereby revealing the different effect of CbCBM66. Prediction by the I-TASSER server revealed that CbCBM66 is structurally close to BsCBM66 from Bacillus subtilis; however, the COFACTOR and COACH programs indicated that the substrate-binding sites between CbCBM66 and BsCBM66 are different. Furthermore, a substrate-binding assay indicated that the catalytic domains in the two CbPLs had strong affinities for pectate-related substrates, but CbCBM66 showed a weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscopy (SEM) analysis and a total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for the degradation of plant biomass.IMPORTANCE Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium, Caldicellulosiruptor bescii The two enzymes share a high optimum temperature, a low optimum pH, and good thermostability at the evaluated temperature. A family 66 carbohydrate-binding module (CbCBM66) was identified in the two CbPLs, sharing 100% amino acid identity. The deletion of CbCBM66 dramatically decreased the activity of CbPL9 but increased the activity and thermostability of CbPL3, suggesting different roles of CbCBM66 in the two enzymes. Moreover, the degradation products of the two CbPLs were different. These results revealed that these enzymes could represent potential pectate lyases for applications in the paper and textile industries.


Assuntos
Proteínas de Bactérias/genética , Firmicutes/genética , Pectinas/metabolismo , Polissacarídeo-Liases/genética , Proteínas de Bactérias/metabolismo , Biomassa , Caldicellulosiruptor , Escherichia coli/enzimologia , Escherichia coli/genética , Firmicutes/enzimologia , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Polissacarídeo-Liases/metabolismo
13.
Biomed Res Int ; 2020: 1871934, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32351984

RESUMO

Glucose isomerase (GI) that catalyzes the conversion of D-glucose to D-fructose is one of the most important industrial enzymes for the production of high-fructose corn syrup (HFCS). In this study, a novel GI (CbGI) was cloned from Caldicellulosiruptor bescii and expressed in Escherichia coli. The purified recombinant CbGI (rCbGI) showed neutral and thermophilic properties. It had optimal activities at pH 7.0 and 80°C and retained stability at 85°C. In comparison with other reported GIs, rCbGI exhibited higher substrate affinity (Km = 42.61 mM) and greater conversion efficiency (up to 57.3% with 3M D-glucose as the substrate). The high catalytic efficiency and affinity of this CbGI is much valuable for the cost-effective production of HFCS.


Assuntos
Aldose-Cetose Isomerases/química , Proteínas de Bactérias/química , Caldicellulosiruptor/enzimologia , Xarope de Milho Rico em Frutose/química , Zea mays/química
14.
Protein J ; 39(2): 174-181, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32140970

RESUMO

In eukaryotes, chromosome ends (telomeres) are tethered to the inner nuclear membrane. During the early stages of meiosis, telomeres move along the nuclear membrane and gather near the spindle-pole body, resulting in a bouquet-like arrangement of chromosomes. This chromosomal configuration appears to be widely conserved among eukaryotes, and is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In fission yeast, the Bqt1-Bqt2 protein complex plays a key role in tethering the telomere to the inner nuclear membrane. However, the structural details of the complex required to clarify how telomeres are gathered near the spindle-pole body remain enigmatic. Previously, we devised a preparation procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, in which a SUMO tag was fused to the N-terminus of the Bqt1 protein. This allowed us to purify the Bqt1-Bqt2 complex from the soluble fraction. In the present study, we found that a maltose-binding protein homolog, Athe_0614, served as a better fusion partner than the SUMO protein, resulting in the marked increase in the solubility of the Bqt1-Bqt2 complex. The Athe_0614 fusion partner may open up new avenues for X-ray crystallographic analyses of the structure of the Bqt1-Bqt2 complex.


Assuntos
Proteínas de Bactérias/metabolismo , Firmicutes/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Caldicellulosiruptor , Meiose , Proteínas Recombinantes de Fusão/metabolismo , Telômero/metabolismo
15.
FEBS J ; 287(20): 4370-4388, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32064769

RESUMO

Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.


Assuntos
Caldicellulosiruptor/metabolismo , Celulases/metabolismo , Celulose/metabolismo , Celulossomas/metabolismo , Temperatura , Glicosilação , Hidrólise
16.
Appl Environ Microbiol ; 86(9)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32086304

RESUMO

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 tapirins, 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.


Assuntos
Aderência Bacteriana , Proteínas de Fímbrias/metabolismo , Firmicutes/fisiologia , Polissacarídeos Bacterianos/metabolismo , Caldicellulosiruptor , Firmicutes/metabolismo
17.
Appl Environ Microbiol ; 86(7)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980431

RESUMO

Renewable fuels have gained importance as the world moves toward diversifying its energy portfolio. A critical step in the biomass-to-bioenergy initiative is deconstruction of plant cell wall polysaccharides to their unit sugars for subsequent fermentation to fuels. To acquire carbon and energy for their metabolic processes, diverse microorganisms have evolved genes encoding enzymes that depolymerize polysaccharides to their carbon/energy-rich building blocks. The microbial enzymes mostly target the energy present in cellulose, hemicellulose, and pectin, three major forms of energy storage in plants. In the effort to develop bioenergy as an alternative to fossil fuel, a common strategy is to harness microbial enzymes to hydrolyze cellulose to glucose for fermentation to fuels. However, the conversion of plant biomass to renewable fuels will require both cellulose and hemicellulose, the two largest components of the plant cell wall, as feedstock to improve economic feasibility. Here, we explore the enzymes and strategies evolved by two well-studied bacteria to depolymerize the hemicelluloses xylan/arabinoxylan and mannan. The sets of enzymes, in addition to their applications in biofuels and value-added chemical production, have utility in animal feed enzymes, a rapidly developing industry with potential to minimize adverse impacts of animal agriculture on the environment.


Assuntos
Biocombustíveis/análise , Firmicutes/metabolismo , Temperatura Alta , Mananas/metabolismo , Xilanos/metabolismo , Caldicellulosiruptor
18.
Sci Rep ; 9(1): 15924, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685873

RESUMO

Carbohydrate active enzymes are classified in databases based on sequence and structural similarity. However, their function can vary considerably within a similarity-based enzyme family, which makes biochemical characterisation indispensable to unravel their physiological role and to arrive at a meaningful annotation of the corresponding genes. In this study, we biochemically characterised the four related enzymes Tm_Ram106B, Tn_Ram106B, Cb_Ram106B and Ts_Ram106B from the thermophilic bacteria Thermotoga maritima MSB8, Thermotoga neapolitana Z2706-MC24, Caldicellulosiruptor bescii DSM 6725 and Thermoclostridium stercorarium DSM 8532, respectively, as α-L-rhamnosidases. Cobalt, nickel, manganese and magnesium ions stimulated while EDTA and EGTA inhibited all four enzymes. The kinetic parameters such as Km, Vmax and kcat were about average compared to other rhamnosidases. The enzymes were inhibited by rhamnose, with half-maximal inhibitory concentrations (IC50) between 5 mM and 8 mM. The α-L-rhamnosidases removed the terminal rhamnose moiety from the rutinoside in naringin, a natural flavonone glycoside. The Thermotoga sp. enzymes displayed the highest optimum temperatures and thermostabilities of all rhamnosidases reported to date. The four thermophilic and divalent ion-dependent rhamnosidases are the first biochemically characterised orthologous enzymes recently assigned to glycoside hydrolase family 106.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridiales/enzimologia , Firmicutes/enzimologia , Glicosídeo Hidrolases/metabolismo , Thermotoga maritima/enzimologia , Thermotoga neapolitana/enzimologia , Proteínas de Bactérias/química , Caldicellulosiruptor , Clonagem Molecular , Cobalto/química , Cobalto/metabolismo , Ácido Edético/química , Ácido Edético/metabolismo , Flavanonas/metabolismo , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ramnose/metabolismo , Especificidade por Substrato , Temperatura
19.
Int J Mol Sci ; 20(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394870

RESUMO

Platycodin D (PD), a major saponin (platycoside) in Platycodi radix (balloon flower root), has higher pharmacological activity than the other major platycosides; however, its content in the plant root is only approximately 10% (w/w) and the productivities of PD by several enzymes are still too low for industrial applications. To rapidly increase the total PD content, the ß-glucosidase from Caldicellulosiruptor bescii was used for the deglucosylation of the PD precursors platycoside E (PE) and platycodin D3 (PD3) in the root extract into PD. Under the optimized reaction conditions, the enzyme completely converted the PD precursors into PD with the highest productivity reported so far, increasing the total PD content to 48% (w/w). In the biotransformation process, the platycosides in Platycodi radix were hydrolyzed by four pathways: deapiosylated (deapi)-PE → deapi-PD3 → deapi-PD, PE → PD3 → PD, polygalacin D3 → polygalacin D, and 3″-O-acetyl polygalacin D3 → 3″-O-acetyl polygalacin D.


Assuntos
Biotransformação , Firmicutes/metabolismo , Raízes de Plantas/metabolismo , Platycodon/metabolismo , Saponinas/metabolismo , Triterpenos/metabolismo , beta-Glucosidase/metabolismo , Caldicellulosiruptor , Hidrólise , Redes e Vias Metabólicas , Estrutura Molecular , Saponinas/química , Especificidade por Substrato , Triterpenos/química , beta-Glucosidase/química
20.
J Biol Chem ; 294(25): 9995-10005, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31097544

RESUMO

Caldicellulosiruptor bescii is an extremely thermophilic, cellulolytic bacterium with a growth optimum at 78 °C and is the most thermophilic cellulose degrader known. It is an attractive target for biotechnological applications, but metabolic engineering will require an in-depth understanding of its primary pathways. A previous analysis of its genome uncovered evidence that C. bescii may have a completely uncharacterized aspect to its redox metabolism, involving a tungsten-containing oxidoreductase of unknown function. Herein, we purified and characterized this new member of the aldehyde ferredoxin oxidoreductase family of tungstoenzymes. We show that it is a heterodimeric glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GOR) present not only in all known Caldicellulosiruptor species, but also in 44 mostly anaerobic bacterial genera. GOR is phylogenetically distinct from the monomeric GAP-oxidizing enzyme found previously in several Archaea. We found that its large subunit (GOR-L) contains a single tungstopterin site and one iron-sulfur [4Fe-4S] cluster, that the small subunit (GOR-S) contains four [4Fe-4S] clusters, and that GOR uses ferredoxin as an electron acceptor. Deletion of either subunit resulted in a distinct growth phenotype on both C5 and C6 sugars, with an increased lag phase, but higher cell densities. Using metabolomics and kinetic analyses, we show that GOR functions in parallel with the conventional GAP dehydrogenase, providing an alternative ferredoxin-dependent glycolytic pathway. These two pathways likely facilitate the recycling of reduced redox carriers (NADH and ferredoxin) in response to environmental H2 concentrations. This metabolic flexibility has important implications for the future engineering of this and related species.


Assuntos
Biomassa , Firmicutes/metabolismo , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Gliceraldeído 3-Fosfato/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Caldicellulosiruptor , Firmicutes/crescimento & desenvolvimento , Gliceraldeído 3-Fosfato/metabolismo , Metaboloma , Oxirredução , Filogenia
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