Paenibacillus Barcinonensis Research Articles

Flax biorefining for paper production

AbstractIn this work, we assessed the potential of Cel6D, a recently reported exocellulase from Paenibacillus barcinonensis, as a biorefining agent for flax pulp. Pulp fibers were treated with this enzyme, Cel9B (an endocellulase previously shown to possess biorefining action) and the two in combination. Samples of biorefined flax pulp were mechanically refined to obtain handsheets. All three biorefining treatments decreased air permeance in the handsheets. Cel9B increased tensile index and folding endurance, and the Cel6D − Cel9B combination increased tensile index and burst index but also resulted in markedly decreased tear index and folding endurance. On the other hand, Cel6D increased tensile index and burst index; also, more importantly, it increased tear resistance, albeit slightly, relative to the other two treatments. The results of this work can be useful to understand the differential effects of exocellulases and endocellulases as biorefining tools and open up new avenues for exploring their use in other biotechnological applications.

Isolation, Identification and Antimicrobial Activities of Four Bacteria Associated with Entomopathogenic Nematodes, Steinernema spp.

Recently, the search for novel products derived from natural sources has become necessary due to the decreasing effectiveness of current antibiotics in treating bacteria that are antibiotic-resistant. In this context, it is well known that entomopathogenic bacteria (EPBs) produce a broad range of secondary metabolites with antibacterial activities. Therefore, an in-vitro trial was conducted to isolate and identify non-symbiotic bacteria associated with entomopathogenic nematodes, Steinernema spp. and evaluate the antibacterial activity against four antibiotic-resistant bacteria. Four bacterial isolates, i.e. Pseudomonas alcaligenes ST-1, Paenibacillus barcinonensis ST-2, Bacillus mojavensis ST-3, and Bacillus megaterium ST-4 were non-symbiotically isolated from the heamolymph of dead Steinernema-infected Galleria mellonella larvae and molecularly characterized. The bacterial cells and filtrates from P. alcaligenes ST-1 strongly inhibited the growth of Staphylococcus aureus through disk diffusion (43 mm), minimum inhibitory concentration (2.5 µL/mL), and minimal bactericidal concentration (5 µL/mL) assay. Conclusively, the direct application of endogenous Steinernema-associated EPB as an antibacterial agent for antibiotic-resistant bacteria looked promising.

Profiling of the viable bacterial and fungal microbiota in fermented feeds using single-molecule real-time sequencing.

Fermented concentrated feed has been widely recognized as an ideal feed in the animal industry. In this study, we used a powerful method, coupling propidium monoazide (PMA) pretreatment with single-molecule real-time (SMRT) sequencing technology to compare the bacterial and fungal composition of feeds before and after fermentation with four added lactic acid bacteria (LAB) inoculants (one Lactobacillus casei strain and three L. plantarum strains). Five feed samples consisting of corn, soybean meal, and wheat bran were fermented with LAB additives for 3 d. Following anaerobic fermentation, the pH rapidly decreased, and the mean numbers of LAB increased from 106 to 109 colony-forming units (cfu)/g fresh matter. SMRT sequencing results showed that the abundance and diversity of bacteria and fungi in the feed were significantly higher before fermentation than after fermentation. Fifteen bacterial species and eight fungal genera were significantly altered following fermentation, and L. plantarum was the dominant species (relative abundance 88.94%) in the post-fermentation group. PMA treatment revealed that the bacteria Bacillus cereus, B. circulans, Alkaliphilus oremlandii, Cronobacter sakazakii, Paenibacillus barcinonensis, and P. amylolyticus (relative abundance >1%) were viable in the raw feed. After fermentation, their relative abundances decreased sharply to <0.2%; however, viable L. plantarum was still the dominant species post fermentation. We inferred that our LAB additives grew rapidly and inhibited harmful microorganisms and further improved feed quality. In addition, coupling PMA treatment with the Pacific Biosciences SMRT sequencing technology was a powerful tool for providing accurate live microbiota profiling data in this study.

Biological control of the root-knot nematode Meloidogyne incognita on tomatoes and carrots by plant growth-promoting rhizobacteria

In the current study 27 plant growth-promoting rhizobacteria (PGPR) strains were screened for biocontrol activity against Meloidogyne incognita on 6-week old carrot seedlings that were inoculated with a suspension of J2 juveniles five days after treatment with the bacteria. Five of the PGPR strains, namely Bacillus firmus T11, Bacillus aryabhattai A08, Paenibacillus barcinonensis A10, Paenibacillus alvei T30, and Bacillus cereus N10w, caused 86.0, 85.2, 84.6, 81.5 and 82.1% reduction in gall numbers, respectively. In a subsequent greenhouse experiment on carrots, treatment with strain T30 caused a significant reduction in gall index and egg mass index compared to the control. The reduction in gall numbers caused by strain T30 was not statistically different from that obtained with CropGuard (furfural). Treatment with B. aryabhattai A08 gave the best results on tomato, significantly reducing gall index and egg mass index compared to the control. In vitro experiments with all five abovementioned strains resulted in second-stage juvenile (J2) paralyses. Induction of resistance was not observed in a split-root experiment conducted in the greenhouse, suggesting that secondary metabolites produced by the bacterial strains are responsible for the biocontrol activity. It is concluded that the bacterial strains P. alvei T30 and B. aryabhattai A08 have potential as biological control agents of M. incognita on carrots and tomatoes respectively.

Taxonomical and functional characterization of Bacillus strains isolated from tomato plants and their biocontrol activity against races 1, 2 and 3 of Fusarium oxysporum f. sp. Lycopersici

Fusarium wilt is an economically important disease of tomato crops (Solanum lycopersicum L.) grown in Brazil that can reduce tomato yield. Among the alternative approaches for controlling this fungal disease, application of antagonistic bacteria has recently been increasing. This study aimed to select and identify phylogenetically bacteria belonging to the genus Bacillus that have antagonistic activity toward three races of the fungus Fusarium oxysporum f. sp. lycopersici (Fol) that colonizes tomato plants grown in infested areas and cause fusarium wilt. The 16S rRNA subunit sequences of 32 bacterial strains isolated from 20 healthy tomato plants showed high diversity and many had high similarity to species of the genus Bacillus: B. toyonensis, B. cereus, B. aryabhattai, B. megaterium, B. aerius, B. stratosphericus and Paenibacillus barcinonensis. In vitro antagonism tests showed that some bacterial strains reduced mycelial growth of the three fungal races in a dual method. Among the four tomato genotypes tested, the Perinha Água Branca variety was the most susceptible to all three fungal races (highest percentage of vascular infection) and was used for a greenhouse experiment to evaluate the antagonistic role of Bacillus strains against fungal pathogens. The B. cereus strain controlled 100% of the damage caused by the race 1 isolate, whereas strains from species B. megaterium controlled 100% the damage caused by the race 2 isolate. B. toyonensis controlled 100% of disease caused by race 2 and race 3 isolates. Among the bacterial strains tested, 82% and 94% had proteolytic and cellulolytic activity, respectively. Together the results suggest that some Bacillus strains could control fusarium wilt in tomatoes caused by Fol race 1, 2 and 3. In addition, these strains have functional activities that may improve growth of tomato plants.

Paenibacillus polysaccharolyticus sp. nov., a xylanolytic and cellulolytic bacteria isolated from leaves of Bamboo Phyllostachys aureosulcata.

A novel xylanolytic and cellulolytic strain, BL9T, was isolated from leaves of the Bamboo plants maintained at the Tamil Nadu Agricultural University Campus, Coimbatore, India. On the basis of the results of 16S rRNA gene analysis, it was determined to be phylogenetically close to the type strains of Paenibacillus amylolyticus NRRL NRS-290T (98.3 %), Paenibacillus barcinonensis BP-23T (98.1 %), Paenibacillus tundrae A10bT (98.0 %) and Paenibacillus xylanexedens B22aT (97.6 %). The strain stained Gram variable and was aerobic, motile and catalase- and oxidase-positive, with rod-shaped cells. Based upon the genome sequence, the average nucleotide identity with the related species ranged from 66 % to 72 %, and the digital DNA-DNA hybridization value ranged from 13 % to 27 %. The DNA G+C content was 45.6 mol%, meso-diaminopimelic acid was identified as the predominant component of the cell wall, and MK-7 was the only menaquinone in cell membranes. The whole-cell fatty acid profile included C16 : 0, iso-C14 : 0, anteiso-C15 : 0 and iso-C16 : 0. The polar lipids identified were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and diphosphtidylglycerol. On the basis of these polyphasic taxonomic traits, BL9T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus polysaccharolyticus sp. nov. is proposed. The type strain is BL9T (=NBRC 105191T=ICMP 17623T).

Paenibacillus silvae sp. nov., isolated from a tropical rainforest soil.

Two Gram-stain-positive, facultatively aerobic, endospore-forming and rod-shaped bacteria, designated DB13031T and DB13311, were isolated from the soil of the Jiaxi Nature Reserve in Hainan, PR China. 16S rRNA gene analysis of strains DB13031T and DB13311 showed that they fell within the Paenibacillus cluster, with highest similarities to Paenibacillus cucumis AP-115T (98.4 and 98.3 %, respectively), Paenibacillus barcinonensis BP-23T (98.3 and 98.2 %, respectively) and Paenibacillus oceanisediminis L10T (97.7 and 97.7 %, respectively). The DNA-DNA hybridization values between strain DB13031T and the type strains of its closest related species were 48.2, 38.1 and 43.5 %. Strain DB13031T contained menaquinone-7 (MK-7) as the predominant isoprenoid quinone and anteiso-C15 : 0, iso-C16 : 0 and C16 : 0 as the major cellular fatty acids. The cell-wall peptidoglycan was of the A1γ type and the major polar lipid profiles were diphosphatidylglycerol, phosphatidylethanolamine, four unknown aminophospholipids and four unknown phospholipids. Based on the phenotypic and genotypic data, it is proposed that the two isolates represent a novel species of the genus Paenibacillus, for which the name Paenibacillus silvae sp. nov. is proposed. The type strain is DB13031T (=CGMCC 1.12770T=DSM 28013T).

Cellulase- and Xylanase-Producing Bacterial Isolates with the Ability to Saccharify Wheat Straw and Their Potential Use in the Production of Pharmaceuticals and Chemicals from Lignocellulosic Materials

The efficient use of lignocellulosic materials for pharmaceutical and chemical production relies on the hydrolysis of their components into their building blocks (e.g. hexoses and pentoses), which can be converted later into chemicals of interest. This study aimed to isolate cellulase- and xylanase-producing bacteria for the bioconversion of lignocellulosic materials into their respective monomeric sugars. Bacterial isolates were screened using CMC- and Xylan–Trypan blue agar, and then cellulase and xylanase activities were evaluated by the 3,5-dinitro-salicylic acid (DNS) method. Furthermore, bacterial ability to saccharify wheat straw was tested. Ten bacterial isolates were found to have the ability to saccharify wheat straw, and to produce cellulase and xylanase enzymes simultaneously. The bacterial isolates were identified at molecular level using 16S rRNA gene sequencing and phylogenetic analysis. Bacterial isolates were identified as Cellulomonas sp. CX4, Cellulomonas sp. CX5, Bacillus sp. CX6, Bacillus sp. CX10, Paenibacillus illinoisensis CX11, Paenibacillus sp. CX14, Bacillus cereus CX15, and Bacillus sp. CX16, Paenibacillus barcinonensis CX17 and Cellulomonas sp. CX20. Among all the isolates, Bacillus sp. CX6 showed the highest ability to produce total reducing sugar (6.03 and 6.16 mg/ml), while the lowest ability to saccharify wheat straw was found with Cellulomonas sp. CX5 (2.01 and 2.12 mg/ml). This study presents cellulase- and xylanase- producing bacterial isolates for their potential to saccharify lignocellulosic materials for possible use in the production of pharmaceuticals and chemicals.

A bacterial GH6 cellobiohydrolase with a novel modular structure.

Cel6D from Paenibacillus barcinonensis is a modular cellobiohydrolase with a novel molecular architecture among glycosyl hydrolases of family 6. It contains an N-terminal catalytic domain (family 6 of glycosyl hydrolases (GH6)), followed by a fibronectin III-like domain repeat (Fn31,2) and a C-terminal family 3b cellulose-binding domain (CBM3b). The enzyme has been identified and purified showing catalytic activity on cellulosic substrates and cellodextrins, with a marked preference for phosphoric acid swollen cellulose (PASC). Analysis of mode of action of Cel6D shows that it releases cellobiose as the only hydrolysis product from cellulose. Kinetic parameters were determined on PASC showing a K m of 68.73mg/ml and a V max of 1.73U/mg. A series of truncated derivatives of Cel6D have been constructed and characterized. Deletion of CBM3b caused a notable reduction in hydrolytic activity, while deletion of the Fn3 domain abolished activity, as the isolated GH6 domain was not active on any of the substrates tested. Mutant enzymes Cel6D-D146A and Cel6D-D97A were constructed in the residues corresponding to the putative acid catalyst and to the network for the nucleophilic attack. The lack of activity of the mutant enzymes indicates the important role of these residues in catalysis. Analysis of cooperative activity of Cel6D with cellulases from the same producing P. barcinonensis strain reveals high synergistic activity with processive endoglucanase Cel9B on hydrolysis of crystalline substrates. The characterized cellobiohydrolase can be a good contribution for depolymerization of cellulosic substrates and for the deconstruction of native cellulose.

Paenibacillus cucumis sp. nov., isolated from a cucumber plant.

A Gram-positive-staining, aerobic, endospore-forming bacterial strain, isolated from the stem of a cucumber plant, was studied in detail for its taxonomic position. Based on 16S rRNA gene sequence similarity comparisons, strain AP-115T was grouped into the genus Paenibacillus, most closely related to Paenibacillus amylolyticus (98.8 %), Paenibacillus tundrae and Paenibacillus barcinonensis (both 98.4 %). The 16S rRNA gene sequence similarity to other species of the genus Paenibacillus was ≤98.4 %. Chemotaxonomic characterization supported allocation of the strain to the genus Paenibacillus. The quinone system contained exclusively menaquinone MK-7, and in the polar lipid profile diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylserine were predominating. The major component in the polyamine pattern was spermidine, and the diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The major fatty acids were iso- and anteiso-branched fatty acids. The results of physiological and biochemical tests allowed phenotypic differentiation of strain AP-115T from closely related species. Thus, AP-115T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus cucumis sp. nov. is proposed, with AP-115T (=LMG 29222T=CCM 8653T) as the type strain.

Paenibacillus puernese sp. nov., a β-glucosidase-producing bacterium isolated from Pu'er tea.

A Gram-staining-positive, endospore-forming, aerobic, rod-shaped bacterium, designated as DCY97(T), was isolated from ripened Pu'er tea and was identified by using a polyphasic approach. 16S rRNA gene sequence analysis showed that strain DCY97(T) was closely related to Paenibacillus dongdonensis KUDC0114(T) (98.0 %), Paenibacillus oceanisediminis L10(T) (97.7 %), and Paenibacillus barcinonensis BP-23(T) (97.2 %). The phenotypic and chemotaxonomic characteristics of strain DCY97(T) matched with the characteristics of members belonging to the genus Paenibacillus. The major identified polar lipids included phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. The predominant quinone was MK-7. The major fatty acids were anteiso-C15:0 (35.1 %), anteiso-C16:0 (19.0 %), and iso-C16:0 (13.9 %). The peptidoglycan cell wall was composed of meso-diaminopimelic acids, alanine, and D-glutamic acid. The genomic DNA G + C content was determined to be 46.7 mol%. The DNA-DNA relatedness between strain DCY97(T) and Paenibacillus dongdonensis KCTC 33221(T), Paenibacillus oceanisediminis KACC 16023(T), Paenibacillus barcinonensis KCTC 13019(T) were 27, 19, and 10 %, respectively. Based on the genotypic, phenotypic, and chemotaxonomic characteristics, strain DCY97(T) is considered as a novel species of the genus Paenibacillus, for which the name Paenibacillus puernese sp. nov. is proposed. The type strain is DCY97(T) (=KCTC 33596(T) = JCM 140369(T)).

A newly discovered arabinoxylan-specific arabinofuranohydrolase. Synergistic action with xylanases from different glycosyl hydrolase families.

Arabinofuranosidase Abf43A from Bacillussp. BP-7 i s a newly discovered arabinoxylan arabinofuranohydrolase (AXH). It is a modular enzyme comprised of a GH43 catalytic domain and a carbohydrate-binding module of family CBM6. Recombinant Abf43A showed high activity on arabinoxylans, being rye arabinoxylan the preferred substrate on which the purified enzyme exhibited a Km of 10.6 ± 3.3 mg/ml and a Vmax of 29.2 ± 3.4 U/mg. Thin-layer chromatography analysis of hydrolysis products showed arabinose as the only sugar released by the enzyme from its substrates. The GH43 and CBM6 modules of the enzyme were individually cloned and expressed in Escherichia coli. While the isolated catalytic GH43 module did not show hydrolytic activity, the purified CBM6 bound to soluble arabinoxylan in affinity gel electrophoresis analysis. Evaluation of cooperative activity of arabinofuranosidase Abf43A with xylanases from families GH10, GH11, andGH30, (Xyn10A, Xyn11E, and Xyn30D from Paenibacillus barcinonensis) on arabinoxylan depolymerization revealed that the studied enzyme showed synergism with Xyn11E, a 2.54-fold increase in the amount of sugars released. On the contrary, Abf43A did not show synergism with the xylanases of families GH10 or GH30 evaluated. The enzyme characterized contributes to understanding the role of this class of enzymes in the catalytic depolymerization of arabinoxylans and their potential for the production of valuable xylooligosaccharides from these abundant plant polymers.

Exploring Multimodularity in Plant Cell Wall Deconstruction: STRUCTURAL AND FUNCTIONAL ANALYSIS OF Xyn10C CONTAINING THE CBM22-1–CBM22-2 TANDEM

Elucidating the molecular mechanisms regulating multimodularity is a challenging task. Paenibacillus barcinonensis Xyn10C is a 120-kDa modular enzyme that presents the CBM22/GH10/CBM9 architecture found in a subset of large xylanases. We report here the three-dimensional structure of the Xyn10C N-terminal region, containing the xylan-binding CBM22-1-CBM22-2 tandem (Xyn10C-XBD), which represents the first solved crystal structure of two contiguous CBM22 modules. Xyn10C-XBD is folded into two separate CBM22 modules linked by a flexible segment that endows the tandem with extraordinary plasticity. Each isolated domain has been expressed and crystallized, and their binding abilities have been investigated. Both domains contain the R(W/Y)YYE motif required for xylan binding. However, crystallographic analysis of CBM22-2 complexes shows Trp-308 as an additional binding determinant. The long loop containing Trp-308 creates a platform that possibly contributes to the recognition of precise decorations at subsite S2. CBM22-2 may thus define a subset of xylan-binding CBM22 modules directed to particular regions of the polysaccharide. Affinity electrophoresis reveals that Xyn10C-XBD binds arabinoxylans more tightly, which is more apparent when CBM22-2 is tested against highly substituted xylan. The crystal structure of the catalytic domain, also reported, shows the capacity of the active site to accommodate xylan substitutions at almost all subsites. The structural differences found at both Xyn10C-XBD domains are consistent with the isothermal titration calorimetry experiments showing two sites with different affinities in the tandem. On the basis of the distinct characteristics of CBM22, a delivery strategy of Xyn10C mediated by Xyn10C-XBD is proposed.

Comparative evaluation of the action of two different endoglucanases. Part II: On a biobleached acid sulphite pulp

A TCF sulphite pulp, bleached at the laboratory scale with a laccase–violuric acid system and complemented with a pressurized hydrogen peroxide stage, was treated with two endoglucanases, one obtained from Paenibacillus barcinonensis (B) and the other one produced from Cerrena unicolor (F) to improve cellulose reactivity. The treated pulps were evaluated in terms of brightness, viscosity, α-cellulose, water retention value, fibre morphology, Fock solubility, NMR and carbohydrate composition of pulps and liquors. Results revealed that both endoglucanases improved cellulose reactivity, albeit in a different way; thus, B caused no scissions in the cellulose chain and no significant reduction in fibre length, whereas F decreased viscosity and shortened fibre length, leading to lower reactivity value. The liquor composition of soluble carbohydrates released by the enzymatic treatments revealed the B had a processive mode of action since short oligosaccharides, cellobiose and glucose, were obtained. F hydrolysed, from high to low concentration, cellobiose, glucose and cellotriose. Importantly, environmentally friendly dissolving pulp with 90 % Fock solubility was obtained, combining two enzymatic treatments: a laccase–mediator system and then a cellulase from P. barcinonensis (B). In order to improve the quality of final dissolving pulp, a pulp purification step was introduced before the B endoglucanase treatment. The cold caustic extraction lead to reduce the amount of hemicelluloses by 42 % with respect to biobleached pulp, but Fock solubility was also reduced. However, complementing the purification step with F treatment, reduced the amount of hemicelluloses but also improved Fock solubility by 17 %, although some presence of cellulose II was detected by NMR.

New GH16 β‐glucanase from Paenibacillus barcinonensis BP‐23 releases a complex pattern of mixed‐linkage oligomers from barley glucan

The gene coding for a lichenase from Paenibacillus barcinonensis BP-23, a powerful carbohydrate-degrading strain, was obtained using a genome walking strategy and expressed in Escherichia coli for further characterization. The amino acid sequence deduced from lic16A revealed that the lichenase is a single-domain enzyme belonging to the GH16 family. Purified recombinant Lic16A showed exclusive activity on β-1,3-1,4-glucans, showing a Km of 16.88 mg/mL and a Vmax of 266.09 U/mg using lichenan as a substrate. Lic16A was stable at 55°C for at least 3 H in moderate pH conditions. Thin-layer chromatography analysis showed that the enzyme released a complex mixture of hydrolysis products, which consisted of different length oligosaccharides of intermediate mobility among cellooligomers. The health benefits of β-glucans's consumption and the increased interest for the use of their oligomers as prebiotics add interest to the study of Lic16A for the production of β-glucan-derived oligosaccharides and the evaluation of their biotechnological potential. This is the first report on β-1,3-1,4-glucanase produced by P. barcinonensis.

Comparative evaluation of the action of two different endoglucanases. Part I: On a fully bleached, commercial acid sulfite dissolving pulp

A fully bleached commercial acid dissolving pulp was treated with two endoglucanases, one obtained from Paenibacillus barcinonensis (B) and the other one produced from Cerrena unicolor (F) with the intention to improve cellulose reactivity and processability in the viscose process. B cellulase was tested under 120 U/g oven dry pulp (odp) and the F cellulase under two conditions, 12 and 60 U/g odp. In addition, a purification stage, consisting in a cold caustic extraction (CCE) of 9 % w/v NaOH, was applied before or after the enzymatic treatment in order to reduce the amount of hemicellulose and improve the action of enzymes. The treated pulps were evaluated in terms of brightness, viscosity, water retention value, fibre morphology, carbohydrate composition, Fock solubility and NMR. In general, results revealed that both endoglucanases improved cellulose reactivity, albeit in a different way; thus, B caused no scissions in the cellulose chain and no significant reduction in fibre length, whereas F strongly decreased viscosity, shortened fibre length and increased considerably the amount of fines. The result of applying two different doses of F cellulase was reflected on Fock solubility and fibre morphology. F60 treatment was found to give the highest value of Fock solubility and the biggest reduction of fibre length. The effect of both endoglucanases on Fock solubility was increased by introducing an earlier CCE stage. Finally, a CCE_B120 pulp with 3 % of hemicellulose and 69 % of Fock solubility was obtained.

Isolation and characterization of a novel thermostable β-glucosidase from Bacillus subtilis SU40.

A new bacterial strain SU40 with ability to produce thermostable β-glucosidase was isolated from soil located at Puducherry (India) and identified as Bacillus subtilis. The conditions like temperature, pH, carbon and nitrogen sources were optimized conditions for the maximum production of β-glucosidase. The enzyme was purified using ammonium sulphate precipitation and BioSep and HPLC chromatography. Purified enzyme was found to be a monomeric protein with an apparent molecular mass of 38.3 kDa. The enzyme was stable over a wide range of pH and temperatures and exhibited maximal activity at 75°C and pH 12.0. The enzyme was found to retain approximately 60% of maximal activity for 1 h at 70°C and hydrolyzed a wide range of aryl-β-D-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and p-nitrophenyl-β-D-glucopyranoside. The Km and Vmax values of the enzyme were 0.21 μM and 9.38 μmoles mg(-1) min(-1), respectively. The enzyme demonstrated stability in the presence of 0.5% surfactants and was completely inhibited after addition of EDTA and Hg2+ ions. Synergistic action of β-glucosidase from B. subtilis SU40 with endoglucanase from Paenibacillus barcinonensis MG7 completely degraded the rice straw.

Unusual carboxylesterase bearing a GGG(A)X-type oxyanion hole discovered in Paenibacillus barcinonensis BP-23.

Strain Paenibacillus barcinonensis BP-23, previously isolated from Ebro's river delta (Spain), bears a complex hydrolytic system showing the presence of at least two enzymes with activity on lipidic substrates. EstA, a cell-bound B-type carboxylesterase from the strain was previously isolated and characterized. The gene coding for a second putative lipase, located upstream cellulase Cel5A, was obtained using a genome walking strategy and cloned in Escherichia coli for further characterization. The recombinant clone obtained displayed high activity on medium/short-chain fatty acid-derivative substrates. The enzyme, named Est23, was purified and characterized, showing maximum activity on pNP-caprylate (C8:0) or MUF-heptanoate (C7:0) under conditions of moderate temperature and pH. Although Est23 displays a GGG(A)X-type oxyanion hole, described as an important motif for tertiary alcohol ester resolution, neither conversion nor enantiomeric resolution of tertiary alcohols could be detected. Amino acid sequence alignment of Est23 with those of known bacterial lipase families and with closely related proteins suggests that the cloned enzyme does not belong to any of the described bacterial lipase families. A phylogenetic tree including Est23 and similar amino acid sequences showed that the enzyme belongs to a differentiated sequence cluster which probably constitutes a new family of bacterial lipolytic enzymes.

Xyn11E from Paenibacillus barcinonensis BP-23: a LppX-chaperone-dependent xylanase with potential for upgrading paper pulps

A new xylanase from Paenibacillus barcinonensis BP-23, Xyn11E, has been identified and characterized. Xyn11E has been cloned and heterologously expressed in Escherichia coli. It is a single-domain xylanase belonging to the family 11 of glycosyl hydrolases (GH11) with a predicted molecular weight of 20.652kDa and an isoelectric point (pI) of 8.7. Substrate specificity, kinetic properties, and mode of action of the purified xylanase were characterized. Xyn11E exhibited high activity toward branched xylans, being beechwood xylan the preferred substrate. The optimum pH and temperature of the purified enzyme were 6.5 and 50°C, respectively. Catalytic constants were determined on beechwood xylan, on which Xyn11E showed a Km of 12.98mg/ml and a Vmax of 3,023U/mg. The enzyme hydrolyzed long xylooligosaccharides, while oligomers shorter than xylotetraose were not degraded. Products released from glucuronoxylans were shorter than those liberated from cereal arabinoxylans. The xylanase was dependent on P. barcinonensis BP-23 LppX for its expression in an active form. Coexpression of Xyn11E with E. coli chaperones could not replace the need of LppX, which seems to act as a specific chaperone for Xyn11E correct folding. Activity of the enzyme on bleached pulps was evaluated. Xyn11E liberated reducing sugars from ECF and TCF pulps from eucalyptus, sisal, and flax, which makes it a good candidate for the enzymatic-assisted production of high-cellulose-content pulps from paper-grade pulps.

The influence of supramolecular structure of cellulose allomorphs on the interactions with cellulose-binding domain, CBD3b from Paenibacillus barcinonensis

The interaction of recombinant cellulose-binding domains (CBDs) of endoglucanase Cel9B from Paenibacillus barcinonensis with different cotton cellulose allomorphs (I, II and III) has been investigated, in order to bring new insights regarding the CBD adsorption and desorption processes. The highest CBD adsorption capacity was recorded for cellulose I, confirming the affinity of proteins to the most crystalline substrate. The weakening and splitting of the hydrogen bonds within cellulose structure after CBD adsorption, as well as a decrease of the crystallinity degree were identified by ATR-FTIR spectroscopy and XRD. The CBD's adsorption kinetic was shown to be rendered by properties as, specific surface area and porosity, being confirmed by dynamic vapor sorption measurements. An important influence of temperature (25, 37 and 50°C) and/or pH medium (4, 5.5, 7 and 10) on the CBD desorption capacity was confirmed, being related to the hydrophobic interactions formed between the CBD and the cellulose allomorphs.