Endoglucanase Gene Research Articles

Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers' rumen.

ObjectiveThis study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism.MethodsTo isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21.ResultsThe maximum activity of recombinant Cel5A (rCel5A) was observed at 50°C and pH 4.0. The enzyme was constant at the temperature range of 20°C to 40°C but also, at the pH range of 3 to 9. The metal ions including Ca2+, K+, Ni2+, Mg2+, and Fe2+ increased the endoglucanase activity but the addition of Mn2+, Cu2+, and Zn2+ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and 45.66 μmol/min/mg. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was 96.69 (s−1) and 6.88 (mL/mg/s), respectively.ConclusionOur results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

Diversity, Pathogenicity, and Current Occurrence of Bacterial Wilt Bacterium Ralstonia solanacearum in Peru

The current bacterial wilt infestation level in the potato fields in the Peruvian Andes was investigated by collecting stem samples from wilted plants and detecting Ralstonia solanacearum. In total 39 farmers’ fields located in the central and northern Peru between the altitudes 2111 and 3742 m above sea level were sampled. R. solanacearum was detected in 19 fields, and in 153 out of the 358 samples analyzed. Phylogenetic analysis using the partial sequence of the endoglucanase gene on strains collected in Peru between 1966 and 2016 from potato, pepper, tomato, plantain or soil, divided the strains in phylotypes I, IIA, and IIB. The Phylotype IIB isolates formed seven sequevar groups including the previously identified sequevars 1, 2, 3, 4, and 25. In addition to this, three new sequevars of phylotype IIB were identified. Phylotype IIA isolates from Peru clustered together with reference strains previously assigned to sequevars 5, 39, 41, and 50, and additionally one new sequevar was identified. The Phylotype I strain was similar to the sequevar 18. Most of the Peruvian R. solanacearum isolates were IIB-1 strains. In the old collection sampled between 1966 and 2013, 72% were IIB-1 and in the new collection at 2016 no other strains were found. The pathogenicity of 25 isolates representing the IIA and IIB sequevar groups was tested on potato, tomato, eggplant and tobacco. All were highly aggressive on potato, but differed in pathogenicity on the other hosts, especially on tobacco. All IIA strains caused latent infection on tobacco and some strains also caused wilting, while IIB strains caused only few latent infections on this species. In conclusion, high molecular diversity was found among the R. solanacearum strains in Peru. Most of the variability was found in areas that are no longer used for potato cultivation and thus these strains do not pose a real threat for potato production in the country. Compared to the previous data from the 1990s, the incidence of bacterial wilt has decreased in Peru. The epidemics are likely caused by infected seed tubers carrying the clonal brown rot strain IIB-1.

Recombinant thermo-alkali-stable endoglucanase of Myceliopthora thermophila BJA (rMt-egl): Biochemical characteristics and applicability in enzymatic saccharification of agro-residues

Codon adaptation index (CAI) of a 1263bp long endoglucanase encoding gene from the thermophilic mould Myceliopthora thermophile BJA has been improved from 0.44 to 0.76 by in vitro gene synthesis. The codon optimized endoglucanase gene (Mt-egl) has been constitutively expressed in Pichia pastoris under the regulation of GAP promoter. Recombinant endoglucanase (rMt-egl), purified by size exclusion chromatography, has been confirmed to be a monomeric protein of ∼47kDa. rMt-egl is optimally active at pH 10 and 50°C, displaying stability in broad pH and temperature ranges, with a t1/2 of 60 and 15min at 90 and 100°C, respectively. This retained ∼70% of activity after 3h incubation at pH 5–12. The Km, Vmax, kcat and kcat/Km of rMt-egl were 5mgmL−1, 20μmolesmin−1mg−1, 1.02×103s−1 and 204s−1mg−1mL−1, respectively. Homology modeling and bioinformatics analysis confirmed catalytically important role of glutamate 234 and 344. rMt-egl released high amounts of reducing sugars from wheat bran and corn cobs (421 and 382mgg−1), thus making it a useful biocatalyst for producing bioethanol and fine chemicals from agro-residues.

Deletion Analysis of GH7 Endoglucanase Gene (cel7B) Promoter Region in a Talaromyces cellulolyticus ligD-Disrupted Strain.

Talaromyces cellulolyticus is expected to become an industrial cellulase producer. In this study, we performed deletion analysis of the promoter region of the GH7 endoglucanase gene (cel7B), which encodes one of the major cellulases, using a β-glucuronidase reporter system. To obtain strains that harbor each gene cassette at the same locus, we had to improve the homologous recombination frequency. Hence, the ligD gene, encoding DNA ligase IV, was disrupted by homologous recombination. After that, the introduced pyrF marker gene, encoding orotate phosphoribosyl transferase, was deleted by a marker recycling system. The resultant strain, YDLP, exhibits high homologous recombination frequency. These data suggest that this approach will drastically improve the genetic modification tools of T. cellulolyticus. We obtained 7 strains for reporter analysis using YDLP as the host strain. Reporter analysis revealed that the promoter region between -812 and -612 is important for expression of cel7B. These results imply a relationship between this region and novel transcriptional factors.

Genetic diversity of Ralstonia solanacearum strains causing bacterial wilt of solanaceous crops in Myanmar

In 2013 and 2014, an extensive survey of bacterial wilt in Myanmar was performed, and 70 strains of Ralstonia solanacearum (Rs) were collected from wilting plants of tomato, potato, chili and eggplant. Myanmar Rs strains were characterized by traditional and molecular methods. Polymerase chain reaction (PCR) test using Rs-specific primer set amplified one specific band (281-bp) from template DNA of all strains. Pathogenicity tests on the four solanaceous plants differentiated the strains into six pathogenic groups. Biovar determination tests showed that biovar 3 strains predominated (63%) among all Rs strains. Biovar 4 strains (7%) were obtained from both tomato and chili strains, whereas biovar 2 (30%) strains were isolated only from potato. Multiplex-PCR analysis indicated that tomato, eggplant and chili strains belonged to phylotype I, whereas potato strains comprised phylotype I and phylotype II. Strains in phylotype I, which was suggested to have originated from Asia, were the most prevalent in all surveyed areas. Phylogenetic analysis based on the endoglucanase (egl) gene sequences revealed that Myanmar strains partitioned into two major clusters that corresponded to phylotype I and II. Strains in phylotype I were further divided into seven subclusters, each corresponding to a distinct sequevar (15, 17, 46, 47, 48, unknown 1 or unknown 2). All strains in phylotype II belonged to sequevar 1. This is the first comprehensive report of the presence of diverse Rs strains in Myanmar.

Cloning and characterization of thermo-alkalistable and surfactant stable endoglucanase from Puga hot spring metagenome of Ladakh (J&K)

A thermo-alkalistable and surfactant stable endoglucanase (PHS) gene consisting of 554 amino acids was identified from metagenomic library of Puga hot spring using functional screening. PHS gene was overexpressed and purified to homogeneity using affinity chromatography The purified PHS protein presented a single band of 60kDa on the SDS-PAGE gel and zymogram. The recombinant PHS exhibited activity over a broad range of pH and temperature with optima at pH 8.0 and 65°C, respectively and having optimum stability at 60°C and pH 8.0, respectively. The recombinant PHS showed highest substrate specificity using CMC (218.4U/mg) as compared with Barley β-glucan (89.2U/mg) and Avicel (0.8U/mg). The Km and Vmax of recombinant PHS for CMC were 3.85mg/ml and 370.37μmolmin−1mg−1, respectively. The activity of the recombinant PHS was enhanced by treatment with 10mM non-ionic detergents such as Tween 20, Tween 40, Tween 80, Triton X- 100 and PEG and was inhibited by CTAB, SDS. Its functionality was stable in the presence of Fe3+ but inhibited by Cu2+, Hg2+, Mn2+ and Zn2+. These properties make PHS endoglucanase a potential candidate for use in laundry, textile,paper and pulp industries

Bacterial Wilt caused by Ralstonia solanacearum in Ethiopia: StatusaAnd Management Approaches: A Review

Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating plant diseases of economically important crops mainly Solanaceous family such as tomato, potato, pepper and eggplant. These crops play a significant role primarily as sources of income and food security for the small scale farming community in Ethiopia. The occurrence of bacterial wilt disease in Ethiopia was reported in 1956 and is known to cause significant yield loss on different Solanaceous crops in different parts of the country. On the basis of conventional characterization and classification, the strains of R. solanacearum found in Ethiopia have been identified as biovar 1 and 2. Recent characterization of R. solanacearum strains based on phylotype grouping using multiplex PCR and partial endoglucanase gene sequencing identified the occurrences of phylotype II and III. The association of biovar and phylotyping schemes indicated that phylotype II comprises only biovar 2, and phylotype III comprises strains of biovar 1 and biovar 2. The importance of the disease on Solanaceous crop is increasing from time to time specially in potato producing areas of the country. Apart from Solanaceous crops, the disease has also been posing a catastrophic damage to ginger production. Latently infected ginger rhizomes and potato seed tuber and decreasing of land holdings that limit crop rotation have contributed to the wider spread of the disease. In this review attempt has been made to summarize relevant scientific studies on this economically important disease in Ethiopia as well as its different disease management options, challenges and future considerations. Because, there is no single effective control measure against the target pathogen so far, a well-coordinated effort is required to develop an integrated disease management program that will help to minimize the damage and yield loss caused by the disease.

Efficient yeast cell-surface display of an endoglucanase of Aspergillus flavus and functional characterization of the whole-cell enzyme.

The endoglucanase gene endo753 from Aspergillus flavus NRRL3357 strains was cloned, and the recombinant Endo753 was displayed on the cell surface of Saccharomyces cerevisiae EBY100 strain by the C-terminal fusion using Aga2p protein as anchor attachment tag. The results of indirect immunofluorescence and Western blot confirmed the expression and localization of Endo753 on the yeast cell surface. The hydrolytic activity test of the whole-cell enzyme revealed that Endo753 immobilized on the yeast cell surface had high endoglucanase activity. The functional characterization of the whole-cell enzyme was investigated, and the whole-cell enzyme displayed the maximum activity at pH 8 and 50°C. The enzyme was stable in a pH range of 7.0-10.0. Furthermore, the whole-cell enzyme displayed high thermostability below 50°C and moderate stability between 50 and 70°C. These properties make endo753 a good candidate in bioethanol production from lignocellulosic materials after displaying on the yeast cell surface.

Expression, purification and molecular characterization of a novel endoglucanase protein from Bacillus subtilis SB13

Bacillus subtilis strain SB13 which is isolated in our previous work was confirmed to produce endoglucanase. In this study, a novel endoglucanase gene (accession number: KX576676) was identified and cloned from SB13. Compared with other consensus sequence of reported endoglucanase genes in the GenBank database, this gene displays five differences (including T740C,A874G,A983G, T1210G and T1301C), which leading to five amino acid changes. Homology modeling has indicated that these five changes were located in the α-helix and random coil regions of the glycosyl hydrolase family 5 (GH5) domain, the random coil and β-sandwich of the type 3 carbohydrate-binding module (CBM3) domain, and the random coil domain. Aprokaryotic expression vector pET30a-endoglucanase was constructed and the endoglucanase was induced to express. The expressed endoglucanase was confirmed by liquid chromatography–tandem mass spectrometry (LC–MSMS) and detected via reaction with carboxymethyl cellulose. In order to obtain the highest expression level of endoglucanase, the expression conditions including IPTG concentration, temperature and pH were optimized. The recombinant endoglucanase protein was purified using a Ni-NTA column, and the 6 × His-tag was removed with thrombin. The results showed that both the modified and unmodified purified endoglucanase had high activity (7.65 ± 0.35 U and 15.05 ± 1.81 U, respectively), thus demonstrating the potential use of this enzyme in various industrial applications. The substitutions of L247P,N292D, F404V and L434P might contribute to the activity of the endoglucanase, and the insertion of a 6 × His-tag at the N-terminal of the endoglucanase might also affect its activity.

Escherichia coli expressing endoglucanase gene from Thai higher termite bacteria for enzymatic and microbial hydrolysis of cellulosic materials

Background: Endoglucanase plays a major role in initiating cellulose hydrolysis. Various wild-type strains were searched to produce this enzyme but mostly low extracellular enzyme activities were obtained. To improve extracellular enzyme production for potential industrial applications, endoglucanase gene of Bacillus subtilis M015, isolated from Thai higher termite, was expressed in a periplasmic-leaky Escherichia coli . Then, the use of crude recombinant endoglucanase (EglS) with a commercial cellulase (Cel) for hydrolyzing celluloses and microbial hydrolysis using whole bacterial cells was evaluated. Results: E. coli Glu5 expressing endoglucanase at high levels was successfully constructed. It produced EglS (55 kDa) with extracellular activity of 18.56 U/mg total protein at optimal hydrolytic conditions (pH 4.8 and 50oC). EglS was highly stable (over 80% activity retained) at 40-50oC after 100 h. The addition of EglS significantly improved the initial sugar production rates of Cel on the hydrolysis of carboxymethyl cellulose (CMC), microcrystalline cellulose and corncob about 5.2, 1.7, and 4.0 folds, respectively, as compared to those with Cel alone. E. coli Glu5 could secrete EglS with high activity in the presence of glucose (1% w/v) and Tween 80 (5% w/v) with low glucose consumption. Microbial hydrolysis of CMC using E. coli Glu5 yielded 26 mg reducing sugar/g CMC at pH 7.0 and 37oC after 48 h. Conclusions: The recombinant endoglucanase activity was improved by 17 times compared with that of the native strain, and could greatly enhance the enzymatic hydrolysis of all studied celluloses when combined with a commercial cellulase. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Times New Roman,serif;}

Presence and transcriptional activity of anaerobic fungi in agricultural biogas plants

Bioaugmentation with anaerobic fungi (AF) is promising for improved biogas generation from lignocelluloses-rich substrates. However, before implementing AF into biogas processes it is necessary to investigate their natural occurrence, community structure and transcriptional activity in agricultural biogas plants. Thus, AF were detected with three specific PCR based methods: (i) Copies of their 18S genes were found in 7 of 10 biogas plants. (ii) Transcripts of a GH5 endoglucanase gene were present at low level in two digesters, indicating transcriptional cellulolytic activity of AF. (iii) Phylogeny of the AF-community was inferred with the 28S gene. A new Piromyces species was isolated from a PCR-positive digester.Evidence for AF was only found in biogas plants operated with high proportions of animal feces. Thus, AF were most likely transferred into digesters with animal derived substrates. Additionally, high process temperatures in combination with long retention times seemed to impede AF survival and activity.

Molecular characterization of Ralstonia solanacearum strains from Ethiopia and tracing potential source of bacterial wilt disease outbreak in seed potatoes

Bacterial wilt, caused by Ralstonia solanacearum, is emerging as a major threat to potato production in Ethiopia, reaching epidemic proportions in the Chencha district recently, with a prevalence of 97% of potato fields in 2015. The recent disease outbreak in the district coincided with a significant introduction of seed potatoes. This research was therefore initiated to genetically characterize the pathogen so as to trace its source, identify its relationship with outbreaks in the rest of the country, and make intervention recommendations. Ralstonia solanacearum isolates were sampled both from seed and ware potato fields in Chencha and from seed potato fields in production regions suspected of being potential sources of the pathogen. Multiplex PCR and phylogenetic analysis of partial endoglucanase gene sequences identified all of the isolates as phylotype IIB sequevar 1. VNTR sequence analysis distinguished 11 different haplotypes, nine of which were unique to the Chencha district. However, one of the haplotypes was common to all seed potato producer regions of Ethiopia except for the Shashemene area. The unique and diverse VNTR haplotypes of the pathogen in Chencha indicates that it is well established in the district. When a geographical map of the VNTR haplotypes was superimposed with the main cross‐regional seed potato distribution pattern of the country, it became evident that the pathogen was being disseminated via latently infected seed from the Holeta‐Jeldu area in the Central Highlands of Ethiopia. Identification of largely uninfected highland districts and multiplication of high‐grade seed potato exclusively in those districts should be given priority.

Expression and Characteristics of an Endoglucanase from Trichoderma atroviride (TaEGII) in Saccharomyces cerevisiae.

Endoglucanase secreted by the fungus Trichoderma atroviride is a kind of cellulase. An endoglucanase gene egII was cloned from T. atroviride AS3.3013 and expressed in Saccharomyces cerevisiae INVScI. The open reading frame of the egII gene was composed of 1257bp, encoding 418 amino acids with a molecular weight of 44.23kDa plus a signal peptide of 21 amino acids. Based on sequence similarity, TaEGII belonged to the glycosyl hydrolase family 5. Expression of the egII gene in T. atroviride AS3.3013 can be induced by microcrystalline cellulose (MCC), bran, carboxymethyl cellulose (CMC), rice straw, and corn stalk but is inhibited by glucose. A highly efficient integrated expression vector (pYPIGH-B includes a sequence of the α-mating factor signal peptide (MF-α)) was constructed. The enzymatic activity of the supernatant of recombinant yeast YPIGH-B3 was 1.29 times higher than that of YES2-egII, demonstrating that the MF-α can significantly improve the expression of the recombinant EGII in S. cerevisiae. The recombinant endoglucanase TaEGII produced by S. cerevisiae showed maximum activity at pH 5.0 and temperature 60°C. Under these conditions, the Km and Kcat values for Avicel and raffinose hydrolysis were 1.22×10-2mgml-1, 9.09×10-2s-1 and 1.06×10-2mgml-1 , 9.18×10-2s-1, respectively. The enzymatic activity of recombinant TaEGII was stable when incubated from 40 to 60°C for 1h. It was stable in a wide range of pH (4.0-7.0) and sensitive to various metal ions. Transgenic yeast strain YPIGH-B3 might be applied to cellulosic ethanol production.

Efficient screening of potential cellulases and hemicellulases produced by Bosea sp. FBZP-16 using the combination of enzyme assays and genome analysis.

Identification of bacteria that produce carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. Twenty lignocellulose-degrading bacterial isolates from Algerian compost and different soils were screened for their potential to produce different enzymes involved in biomass deconstruction. Based on 16S rRNA gene sequencing, the isolates belonged to Proteobacteria and Actinobacteria. Differences among species were reflected both as the presence/absence of enzymes or at the level of enzyme activity. Among the most active species, Bosea sp. FBZP-16 demonstrated cellulolytic activity on both amorphous cellulose (CMC) and complex lignocellulose (wheat straw) and was selected for whole-genomic sequencing. The genome sequencing revealed the presence of a complex enzymatic machinery required for organic matter decomposition. Analysis of the enzyme-encoding genes indicated that multiple genes for endoglucanase, xylanase, β-glucosidase and β-mannosidase are present in the genome with enzyme activities displayed by the bacterium, while other enzymes, such as certain cellobiohydrolases, were not detected at the genomic level. This indicates that a combination of functional screening of bacterial cultures with the use of genome-derived information is important for the prediction of potential enzyme production. These results provide insight into their possible exploitation for the production of fuels and chemicals derived from plant biomass.

Production and characteristics of the recombinant extracellular bifunctional endoglucanase of the polyextremophilic bacterium Bacillus halodurans and its applicability in saccharifying agro-residues.

The recombinant alkalistable and moderately thermostable bifunctional endoglucanase gene (BhCell-Xyl) of polyextremophilic bacterium Bacillus halodurans TSLV1 has been expressed in Pichia pastoris under constitutive GAP as well as inducible AOX promoters. A higher titre of recombinant BhCell-Xyl was attained after induction (4.8UmL-1) as compared to that of the constitutive production (2.1UmL-1). The recombinant P. pastoris strains integrated two copies of BhCell-Xyl under AOX and GAP promoters. The pure recombinant BhCell-Xyl is a glycoprotein of 66kDa, which is optimally active at 60°C and pH 6.0 and 8.0. Glycosylated BhCell-Xyl exhibits higher thermostability than that of the native enzyme. The analysis of amino acids of BhCell-Xyl revealed that multiple factors are responsible for its thermostability. Kinetics and in silico analysis of the enzyme suggested that BhCell-Xyl has one active site for both endocellulase and endoxylanase activities. The BhCell-Xyl possesses a carbohydrate binding domain and saccharifies lignocellulosic agro-residues to xylo-oligosaccharides and cello-oligosaccharides, suggesting its potential application in generating fermentable sugars from renewable agro-residues for biofuel and fine chemical industries.

First Report of Ralstonia pseudosolanacearum Phylotype I Causing Bacterial Wilt on Rodrigues Island, Indian Ocean

The Ralstonia solanacearum species complex (Rssc) encompasses strains that are highly destructive on a wide host range, and some are listed as quarantine organisms in Europe, the United States, and many other countries. This soil-borne bacterial plant pathogen invades the roots and colonizes the xylem vessels, causing a bacterial wilt (BW) disease. Strains are distributed into four phylogenetic groups (phylotypes) linked to their geographical origin of evolution: phylotype I (Asia), phylotype II (America), phylotype III (Africa), and phylotype IV (Australia - Indonesia - Japan). Phylotypes are subdivided into sequevars, encompassing strains that share high homology on the endoglucanase gene partial sequence (750 bp) (Fegan and Prior 2005). Rodrigues is part of the Mascarene Islands in the Southwest Indian Ocean. In February 2016, a survey was conducted to identify the causal agent of BW, encountered for years on this island. Typical BW disease symptoms, including leaf epinasty and browning vessels, were observed on tomato (Solanum lycopersicum L.) in Terre Rouge and on chili pepper (Capsicum frutescens L.) in Nouvelle Decouverte and Montagne Malgache. Seventy-eight stem sections were collected, surface disinfected with 70% ethanol, and crushed into 2 ml of Tris-buffer, then processed for bacterial isolation by plating 50 µl on SM-1 medium (Granada and Sequeira 1983). Among all of the collected stems, typical Ralstonia pseudosolanacearum colonies grew on the semiselective medium from 51 of the 78 samples. Corresponding strains were subcultured and typed as R. pseudosolanacearum phylotype I (Safni et al. 2014), based on the PMX-PCR (Fegan and Prior 2005). A subset of six strains, two from each location, was selected for sequevar identification (Fegan et al. 1998). Strains CFBP8472 and CFBP8465 (Terre Rouge) were identified as sequevar I-31 (GenBank KX242311 and KX242312), but strains CFBP8467 and CFBP8468 (Montagne Malgache) along with CFBP8469 and CFBP8466 (Nouvelle Decouverte) were identified as sequevar I-33 (KX242313 to KX242316). This subset of strains was inoculated on five plants per strain by soil drenching on the susceptible tomato cultivar L390 (Lebeau et al. 2011) with 5 ml of a calibrated suspension (108 CFU/ml in Tris-buffer). Plants were grown in a fully controlled environment at day/night temperatures of 30/25°C ± 1°C under high relative humidity (∼90%). Sterile Tris-buffer inoculated negative controls were grown with the same parameters. Typical BW symptoms were observed within 6 days postinoculation and strains were reisolated and characterized using the same protocol as for the strains that were inoculated, fulfilling Koch's postulates. No symptoms were observed and no Rssc strains could be isolated from negative controls. To our knowledge, this is the first report of R. pseudosolanacearum phylotype I strains causing bacterial wilt on tomato and pepper on Rodrigues. Further studies need to be conducted on the prevalence and diversity of the Rssc strains causing the BW disease, in order to implement adapted control measures, such as the use of local and adapted resistant cultivars. Further surveys on other Southwest Indian Ocean islands such as Mauritius, Reunion, and Seychelles should be achieved to determine the potential origin of the R. pseudosolanacearum phylotype I strains isolated on Rodrigues island. (Texte integral)

Characterization of endoglucanase from Paenibacillus sp. M33, a novel isolate from a freshwater swamp forest.

The newly isolated Paenibacillus sp. M33 from freshwater swamp forest soil in Thailand demonstrated its potential as a cellulose degrader. One of its endoglucanase genes from Paenibacillus sp., celP, was cloned to study the molecular characteristics of its gene product. The celP gene was recognized firstly by degenerate primer designed from Paenibacillus endoglucanase gene, and subsequently identified flanking region by inverse PCR technique. The celP gene consists of an open reading frame of 1707 bp encoding for 569 amino acids including 33-amino acids signal sequence. CelP is a member of glycoside hydrolase family 5 appended with a family 46 carbohydrate-binding module. CelP from recombinant Escherichia coli was purified by affinity chromatography. SDS-PAGE analysis of purified CelP showed a protein band at about 60 kDa. The purified enzyme gave a specific CMCase activity of 0.03 μmol min-1 mg-1 . It had higher activities on lichenan (0.19 μmol min-1 mg-1 ) and barley β-glucan (0.14 μmol min-1 mg-1 ). Maximum activity on lichenan was obtained at 50 °C, pH 5.0. CelP was stable over a pH range of 3.0-10.0 and retained 80% activity when incubated at 50 °C for 1 h. The properties of its CelP endoglucanase, especially substrate specificity, will make it useful in various biotechnological applications including biomass hydrolysis.

A novel pH-stable, endoglucanase (JqCel5A) isolated from a salt-lake microorganism, Jonesia quinghaiensis

Background: Endoglucanase, one of three type cellulases, can randomly cleave internal β-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene ( JqCel5A ) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance. Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Times New Roman,serif;} Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Times New Roman,serif;}

Phylotype and sequevar variability of Ralstonia solanacearum in Brazil, an ancient centre of diversity of the pathogen

The β‐proteobacterium Ralstonia solanacearum causes bacterial wilt of many plant species. Knowledge of phylotype and sequevar variability in populations of this microorganism is useful for implementing control measures, particularly host resistance. To this end, 301 isolates of R. solanacearum were collected from different geographic regions and hosts in Brazil. Their phylotype and sequevar characterization was used to determine the amount and distribution of phenetic and phylogenetic variability. Isolates were classified into phylotypes I (n = 48), clade 1; and phylotype II, clades 2–5. Phylotype II was divided into subclusters IIA (n = 112) and IIB (n = 141). Phylotype II was widely distributed, whereas phylotype I isolates were found in Central, Northern, and Northeastern regions of Brazil. There were 108 haplotypes identified among endoglucanase (egl) gene sequences from 301 isolates and 32 haplotypes among DNA repair (mutS) gene regions from 176 isolates. The egl and mutS sequence analyses identified eight known (1, 4, 7, 18, 27, 28, 41 and 50) and four new (54, 55, 56 and 57) sequevars. Phylotype IIB showed high diversity in sequevars and host range. Multiplex PCR, using primers specific to the Moko ecotype, characterized banana and long pepper isolates as sequevar 4 and 4/NPB, respectively. This constitutes the first report of the emergent ecotype IIB/4NPB in a new host, long pepper. The majority of sequevars were associated with geographic regions. This high variability of R. solanacearum in Brazil suggests use of host resistance to control bacterial wilt should be mainly focused by region.

Two Ralstonia species associated with bacterial wilt of Eucalyptus

Ralstonia solanacearum sensu lato causes bacterial wilt in many economically important agronomic crops and tree species. Eucalyptus is one of the recorded hosts but, despite the losses to forestry, little is known regarding the disease. The aim of this study was to identify the Ralstonia species that are associated with bacterial wilt of Eucalyptus trees growing in different parts of the world. Phylotyping, sequevar and multilocus sequence analysis were used to identify and determine the genetic diversity of the available isolates. The isolates obtained from Africa and Asia grouped in phylotype I, and those isolates from South America in phylotype II. Following the recent reclassification of R. solanacearum sensu lato, these results show that there are two Ralstonia species associated with bacterial wilt of Eucalyptus, namely, R. solanacearum and R. pseudosolanacearum. The isolates from South America represented R. solanacearum, while those from Africa and Asia were R. pseudosolanacearum. Analysis of the endoglucanase (egl), transcriptional regulator (hrpB) and DNA mismatch repair (mutS) gene regions supported this finding. In addition, one previously unidentified sequevar was identified using partial endoglucanase (egl) gene sequence analysis of those isolates obtained from Colombia. This study raises questions regarding the aetiology of the two Ralstonia species found on Eucalyptus, their importance relating to quarantine, potential impact and management of the disease with which they are associated.