Biodegradation of Deoxynivalenol by Nocardioides sp. ZHH-013: 3-keto-Deoxynivalenol and 3-epi-Deoxynivalenol as Intermediate Products.

Deoxynivalenol (DON) is one of the most devastating and notorious contaminants in food and animal feed worldwide. A novel DON-degrading strain, Nocardioides sp. ZHH-013, which exhibited complete mineralization of DON, was isolated from soil samples. The intermediate products of DON generated by this strain were identified by high-performance liquid chromatography and ultra-performance liquid chromatography tandem mass spectrometry analyses. It was shown that, on an experimental level, 3-keto-DON was a necessary intermediate product during the conversion from DON to 3-epi-DON. Furthermore, the ZHH-013 strain could also utilize 3-epi-DON. This DON degradation pathway is a safety concern for food and feed. The mechanism of DON and 3-epi-DON elimination will be further studied, so that new enzymes for DON degradation can be identified.

A Swollenin From Talaromyces leycettanus JCM12802 Enhances Cellulase Hydrolysis Toward Various Substrates.

Swollenins exist within some fungal species and are candidate accessory proteins for the biodegradation of cellulosic substrates. Here, we describe the identification of a swollenin gene, Tlswo, in Talaromyces leycettanus JCM12802. Tlswo was successfully expressed in both Trichoderma reesei and Pichia pastoris. Assay results indicate that TlSWO is capable of releasing reducing sugars from lichenan, barley ß-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin. The specific activity of TlSWO toward lichenan, barley ß-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin is 9.0 ± 0.100, 8.9 ± 0.100, 2.3 ± 0.002 and 0.79 ± 0.002 U/mg, respectively. Additionally, TlSWO had disruptive activity on Avicel and a synergistic effect with cellobiohydrolases, increasing the activity on pretreated corn stover by up to 72.2%. The functional diversity of TlSWO broadens its applicability in experimental settings, and indicating that it may be a promising candidate for future industrial applications.

Phenotypic Plasticity of Staphylococcus aureus in Liquid Medium Containing Vancomycin.

Phenotypic plasticity enables individuals to develop different phenotypes in a changing environment and promotes adaptive evolution. Genome-wide association study (GWAS) facilitates the study of the genetic basis of bacterial phenotypes, and provides a new opportunity for bacterial phenotypic plasticity research. To investigate the relationship between growth plasticity and genotype in bacteria, 41 Staphylococcus aureus strains, including 29 vancomycin-intermediate S. aureus (VISA) strains, were inoculated in the absence or presence of vancomycin for 48 h. Growth curves and maximum growth rates revealed that strains with the same minimum inhibitory concentration (MIC) showed different levels of plasticity in response to vancomycin. A bivariate GWAS was performed to map single-nucleotide polymorphisms (SNPs) associated with growth plasticity. In total, 227 SNPs were identified from 14 time points, while 15 high-frequency SNPs were mapped to different annotated genes. The P-values and growth variations between the two cultures suggest that non-coding region (SNP 738836), ebh (SNP 1394043), drug transporter (SNP 264897), and pepV (SNP 1775112) play important roles in the growth plasticity of S. aureus. Our study provides an alternative strategy for dissecting the adaptive growth of S. aureus in vancomycin and highlights the feasibility of bivariate GWAS in bacterial phenotypic plasticity research.

Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents.

Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (ßα)8-barrel structure, where each (ßα) module is essential for the enzyme's stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A from Talaromyces emersonii (TeEgl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from Stegonsporium opalus (SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity (T50) (15 and 19°C), and the melting temperature (Tm ) (16.5 and 22.9°C) and extended half-lives (t1/2) (∼240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 from Aspergillus niger (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the SoCel5 and TeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2 and α3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent SoCel5.IMPORTANCE Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure, SoCel5 and TeEgl5A, with different thermal properties, were hybridized to study the roles of different (ßα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

[A novel bifunctional xylanase/cellulase TcXyn10A from Thermoascus crustaceus JCM12803].

Efficient utilization of cellulose and xylan is of importance in the bioethanol industry. In this study, a novel bifunctional xylanase/cellulase gene, Tcxyn10a, was cloned from Thermoascus crustaceus JCM12803, and the gene product was successfully overexpressed in Pichia pastoris GS115. The recombinant protein was then purified and characterized. The pH and temperature optima of TcXyn10A were determined to be 5.0 and 65-70 °C, respectively. The enzyme retained stable under acid to alkaline conditions (pH 3.0-11.0) or after 1-h treatment at 60 °C. The specific activities of TcXyn10A towards beechwood xylan, wheat arabinoxylan, sodium carboxymethyl cellulose and lichenan were (1 480±26) U/mg, (2 055±28) U/mg, (7.4±0.2) U/mg and (10.9±0.4) U/mg, respectively. Homologous modeling and molecular docking analyses indicated that the bifunctional TcXyn10A has a single catalytic domain, in which the substrate xylan and cellulose shared the same binding cleft. This study provides a valuable material for the study of structure and function relationship of bifunctional enzymes.

Enhancing the catalytic activity of a novel GH5 cellulase GtCel5 from Gloeophyllum trabeum CBS 900.73 by site-directed mutagenesis on loop 6.

BACKGROUND: Cellulases of glycosyl hydrolase (GH) family 5 share a (ß/α)8 TIM-barrel fold structure with eight ßα loops surrounding the catalytic pocket. These loops exposed on the surface play a vital role in protein functions, primarily due to the interactions of some key amino acids with solvent and ligand molecules. It has been reported that motions of these loops facilitate substrate access and product release, and loops 6 and 7 located at the substrate entrance of the binding pocket promote proton transfer reaction at the catalytic site motions. However, the role of these flexible loops in catalysis of GH5 cellulase remains to be explored. RESULTS: In the present study, an acidic, mesophilic GH5 cellulase (with optimal activity at pH 4.0 and 70 °C), GtCel5, was identified in Gloeophyllum trabeum CBS 900.73. The specific activities of GtCel5 toward CMC-Na, barley ß-glucan, and lichenan were 1117 ± 43, 6257 ± 26 and 5318 ± 54 U/mg, respectively. Multiple sequence alignment indicates that one amino acid residue at position 233 on the loop 6 shows semi-conservativeness and might contribute to the great catalytic performance. Saturation mutagenesis at position 233 was then conducted to reveal the vital roles of this position in enzyme properties. In comparison to the wild type, variants N233A and N233G showed decreased optimal temperature (- 10 °C) but increased activities (27 and 70%) and catalytic efficiencies (kcat/Km; 45 and 52%), respectively. The similar roles of position 233 in catalytic performance were also verified in the other two GH5 homologs, TeEgl5A and PoCel5, by reverse mutation. Further molecular dynamics simulations suggested that the substitution of asparagine with alanine or glycine may introduce more hydrogen bonds, increase the flexibility of loop 6, enhance the interactions between enzyme and substrate, and thus improve the substrate affinity and catalytic efficiency. CONCLUSION: This study proposed a novel cellulase with potentials for industrial application. A specific position was identified to play key roles in cellulase-substrate interactions and enzyme catalysis. It is of great importance for understanding the binding mechanism of GH5 cellulases, and provides an effective strategy to improve the catalytic performance of cellulases.

[A highly active GH11 xylanase from Penicillium sp. L1 with potential applications in xylo-oligosaccharide production].

Xylanase is a high-profile glycoside hydrolase with applications in brewing, feed, pharmacy and bioenergy industries, but most of xylanases are in active below 30 ℃. In order to obtain low temperature active xylanase, a xylanase gene, XYN11A, was cloned from Penicillium sp. L1 and expressed in Pichia pastoris GS115. After purification and enzyme assay, optimal pH and temperature were determined to be 3.5 to 4.0 and 55 ℃. This enzyme was stable at acid and neutral condition (pH 1.0 to 7.0) or under the treatment of 40 ℃ for 1 hour. This xylanase displayed strong resistance to all tested ions and chemicals. Noteworthily, XYN11A maintained a higher activity of 6 700 U/mg than a lot of GH11 xylanase, and demonstrated higher activity (24% to 58%) at lower temperature from 20 to 40 ℃. After beechwood xylan hydrolysis for 16 h, the hydrolysates consisted mainly of xylobiose, xylotriose and xylotetraose and barely of xylose, thus XYN11A could be used for the production of prebiotic xylooligosaccharide. Possessing the features of acidophilic, highly active at lower temperature and oligosaccharide production, XYN11A demonstrated great potential in food and feed industrials.

Improvement of the catalytic efficiency of a hyperthermophilic xylanase from Bispora sp. MEY-1.

Extremophilic xylanases have attracted great scientific and industrial interest. In this study, a GH10 xylanase-encoding gene, Xyl10E, was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris GS115. Deduced Xyl10E shares the highest identities of 62% and 57% with characterized family GH10 xylanases from Talaromyces leycettanus and Penicillium canescens (structure 4F8X), respectively. Xyl10E was most active at 93 to 95°C and pH 4.0, retained more than 75% or 48% of the initial activity when heated at 80°C or 90°C for 30 min, respectively, and hardly lost activity at pH 1.0 to 7.0, but was completely inhibited by SDS. Two residues, A160 and A161, located on loop 4, were identified to play roles in catalysis. Mutants A160D/E demonstrated higher affinity to substrate with lower Km values, while mutants A161D/E mainly displayed elevated Vmax values. All of these mutants had significantly improved catalytic efficiency. According to the molecular dynamics simulation, the mutation of A160E was able to affect the important substrate binding site Y204 and then improve the substrate affinity, and the mutation of A161D was capable of forming a hydrogen bond with the substrate to promote the substrate binding or accelerate the product release. This study introduces a highly thermophilic fungal xylanase and reveals the importance of loop 4 for catalytic efficiency.

Thermostability improvement of a Talaromyces leycettanus xylanase by rational protein engineering.

Thermophilic xylanases with high catalytic efficiency are of great interest in the biofuel, food and feed industries. This study identified a GH11 xylanase gene, Tlxyn11B, in Talaromyces leycettanus JCM12802. Recombinant TlXyn11B produced in Pichia pastoris is distinguished by high specific activity (8259 ± 32 U/mg with beechwood xylan as substrate) and excellent pH stability (from 1.0 to 10.5). The beechwood xylan hydrolysates consisted mainly of xylobiose, xylotriose and xylotetraose, thus TlXyn11B could be used for the production of prebiotic xylooligosaccharide. By using the structure-based rational approach, the N-terminal sequence of TlXyn11B was modified for thermostability improvement. Mutants S3F and S3F/D35V/I/Q/M had elevated T m values of 60.01 to 67.84 °C, with S3F/D35I the greatest. Homology modeling and molecular dynamics (MD) simulation analysis revealed that the substituted F3 and I35 formed a sandwich structure with S45 and T47, which may enhance the overall structure rigidity with lowered RMSD values. This study verifies the efficiency of rational approach in thermostability improvement and provides a xylanase candidate of GH11 with great commercialization potential.

Overexpressing key component genes of the secretion pathway for enhanced secretion of an Aspergillus niger glucose oxidase in Trichoderma reesei.

Vast interest exists in developing T. reesei for production of heterologous proteins. Although rich genomic and transcriptomic information has been uncovered for the T. reesei secretion pathway, little is known about whether engineering its key components could enhance expression of a heterologous gene. In this study, snc1, a v-SNARE gene, was first selected for overexpression in T. reesei. In engineered T. reesei with additional copies of snc1, the Aspergillus niger glucose oxidase (AnGOD) was produced to a significantly higher level (2.2-fold of the parental strain). hac1 and bip1, two more component genes in the secretion pathway, were further tested for overexpression and found to be also beneficial for AnGOD secretion. The overexpression of one component gene more or less affected the expression of the other two genes, suggesting a complex regulating mechanism. Our study demonstrates the potential of engineering the secretion pathway for enhancing heterologous gene production in T. reesei.

Improvement of the catalytic performance of a hyperthermostable GH10 xylanase from Talaromyces leycettanus JCM12802.

A xylanase gene of GH 10, Tlxyn10A, was cloned from Talaromyces leycettanus JCM12802 and expressed in Pichia pastoris. Purified recombinant TlXyn10A was acidic and hyperthermophilic, and retained stable over the pH range of 2.0-6.0 and at 90°C. Sequence analysis of TlXyn10A identified seven residues probably involved in substrate contacting. Three mutants (TlXyn10A_P, _N and _C) were then constructed by substituting some or all of the residues with corresponding ones of hyperthermal Xyl10C from Bispora sp. MEY-1. TlXyn10A_P with mutations at subsites +2 to +4 exhibited improved specific activity (by 0.44-fold) and pH stability (2.0-10.0). Molecular dynamics simulation analysis indicated that mutations E229I and F232E probably weaken the substrate affinity at subsites +3 to +4, and G149D may introduce a new hydrogen bond. These modifications altogether account for the improved performance of TlXyn10A_P. Moreover, TlXyn10A_P was able to hydrolyze wheat straw persistently, and has the application potentials in various industries.

Improvement of the catalytic performance of a Bispora antennata cellulase by replacing the N-terminal semi-barrel structure.

The aim of this work was to study the contribution of the N-terminal structure to cellulase catalytic performance. A wild-type cellulase (BaCel5) of glycosyl hydrolase (GH) family 5 from Bispora antennata and two hybrid enzymes (BaCel5(127) and BaCel5(167)) with replacement of the N-terminal (ßα)3 (127 residues) or (ßα)4 (167 residues)-barrel with the corresponding sequences of TeEgl5A from Talaromyces emersonii were produced in Pichia pastoris and biochemically characterized. BaCel5 exhibited optimal activity at pH 5.0 and 50°C but had low catalytic efficiency (25.4±0.8mLs(-1)mg(-1)). In contrast, BaCel5(127) and BaCel5(167) showed similar enzymatic properties but improved catalytic performance. When using CMC-Na, barley ß-glucan, lichenan, and cellooligosaccharides as substrates, BaCel5(127) and BaCel5(167) had increased specific activities and catalytic efficiencies by ∼1.8-6.7-fold and ∼1.0-4.7-fold, respectively. The catalytic efficiency of BaCel5(167) was even higher than that of parental proteins. The underlying mechanism was analyzed by molecular docking and molecular dynamic simulation.

A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry.

A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-ß-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5-5.0 and 75°C, retained stability over the pH range of 2.0-7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries.

Novel Mutation Sites in the Development of Vancomycin- Intermediate Resistance in Staphylococcus aureus.

Increased use of vancomycin has led to the emergence of vancomycin-intermediate Staphylococcus aureus (VISA). To investigate the mechanism of VISA development, 39 methicillin-susceptible strains and 3 MRSA strains were treated with vancomycin to induce non-susceptibility, and mutations in six genes were analyzed. All the strains were treated with vancomycin in vitro for 60 days. MICs were determined by the agar dilution and E-test methods. Vancomycin was then removed to assess the stability of VISA strains and mutations. Following 60 days of vancomycin treatment in vitro, 29/42 VISA strains were generated. The complete sequences of rpoB, vraS, graR, graS, walK, and walR were compared with those in the parental strains. Seven missense mutations including four novel mutations (L466S in rpoB, R232K in graS, I594M in walk, and A111T in walR) were detected frequently in strains with vancomycin MIC ≥ 12 µg/mL. Jonckheere-Terpstra trend test indicated these mutations might play an important role during VISA evolution. After the vancomycin treatment, strains were passaged to vancomycin-free medium for another 60 days, and the MICs of all strains decreased. Our results suggest that rpoB, graS, walk, and walR are more important than vraS and graR in VISA development.

Implication of a galactomannan-binding GH2 ß-mannosidase in mannan utilization by Caldicellulosiruptor bescii.

Many glycoside hydrolases involved in deconstruction of cellulose and xylan from the excellent plant cell wall polysaccharides-degrader Caldicellulosiruptor bescii have been cloned and analyzed. However, far less is known about the enzymatic breakdown of mannan, an important component of hemicellulose. We herein cloned, expressed and purified the first ß-mannosidase CbMan2A from C. bescii. CbMan2A is thermophilic, with an optimal temperature of 80 °C. CbMan2A hydrolyzes mannooligosaccharides with degrees of polymerization from 2 to 6 mainly into mannose and shows strong synergy with CbMan5A, an endo-mannanase from the same bacterium, in releasing mannose from ß-1,4-mannan. Thus CbMan2A forms the missing link in enzymatic conversion of mannan into the ready-to-use mannose by C. bescii. Based on these observations, a model illustrating how CbMan2A may assist C. bescii in mannan utilization is presented. In addition, CbMan2A appeared to bind to insoluble galactomannan in a pH-dependent fashion. Although the relation of this feature to mannan utilization remains elusive, CbMan2A represents an excellent model for investigation of the binding of GH2 ß-mannosidases to galactomannan.

A Novel GH7 Endo-ß-1,4-Glucanase from Neosartorya fischeri P1 with Good Thermostability, Broad Substrate Specificity and Potential Application in the Brewing Industry.

An endo-ß-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1-20, a catalytic domain of glycoside hydrolase family 7 (GH7), a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1). The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0-6.0) and excellent stability at pH3.0-8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley ß-glucan (2020 ± 9 U mg-1), moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 µg) reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.

Application of a novel alkali-tolerant thermostable DyP-type peroxidase from Saccharomonospora viridis DSM 43017 in biobleaching of eucalyptus kraft pulp.

Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50-80°C. It also showed broad pH adaptability (>35% activity at pH 4.0-9.0) and alkali-tolerance (>80% activity after incubation at pH 5-10 for 1 h at 37°C), and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0). SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.

Analysis of miRNAs and their targets during adventitious shoot organogenesis of Acacia crassicarpa.

Organogenesis is an important process for plant regeneration by tissue or cell mass differentiation to regenerate a complete plant. MicroRNAs (miRNAs) play an essential role in regulating plant development by mediating target genes at transcriptional and post-transcriptional levels, but the diversity of miRNAs and their potential roles in organogenesis of Acacia crassicarpa have rarely been investigated. In this study, approximately 10 million sequence reads were obtained from a small RNA library, from which 189 conserved miRNAs from 57 miRNA families, and 7 novel miRNAs from 5 families, were identified from A. crassicarpa organogenetic tissues. Target prediction for these miRNAs yielded 237 potentially unique genes, of which 207 received target Gene Ontology annotations. On the basis of a bioinformatic analysis, one novel and 13 conserved miRNAs were selected to investigate their possible roles in A. crassicarpa organogenesis by qRT-PCR. The stage-specific expression patterns of the miRNAs provided information on their possible regulatory functions, including shoot bud formation, modulated function after transfer of the culture to light, and regulatory roles during induction of organogenesis. This study is the first to investigate miRNAs associated with A. crassicarpa organogenesis. The results provide a foundation for further characterization of miRNA expression profiles and roles in the regulation of diverse physiological pathways during adventitious shoot organogenesis of A. crassicarpa.

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection.

The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF- α , and IFN- γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products.

A novel, alkali-tolerant thermostable xylanase from Saccharomonospora viridis: direct gene cloning, expression and enzyme characterization.

A xylanase gene, designated Svixyn10A, was cloned from actinomycetes Saccharomonospora viridis and the gene product was characterized. Gene Svixyn10A contains 1,374 bp and encodes a polypeptide of 457 amino acids composed of a glycoside hydrolase family 10 catalytic domain with a putative signal peptide, a short Gly-rich linker and a family 2 carbohydrate-binding module (CBM). The deduced amino acid sequence of SviXyn10A shared the highest identity (57 %) with a hypothetical xylanase from Streptomyces lividans TK24 (ZP_05528201). A recombinant His-tagged xylanase, SviXyn10A was expressed in Escherichia coli BL21 and purified. The optimum pH and temperature for SviXyn10A is 8.0 and 60 °C. Compared with thermophilic and mesophilic counterparts, SviXyn10A was more active at high temperatures, retaining >63 % of its maximum activity at 65-70 °C and ~40 % even at 80 °C. It had broad pH adaptability (>35 % activity at pH 5.0-11.0) and alkali-tolerance (>70 % activity after incubation at pH 8.0-11.0 for 1 h at 37 °C), and was highly thermostable (>75 % activity after incubation at 70 °C for 3 h at pH 8.0). It may be the first alkali-tolerant thermostable xylanase reported from Saccharomonospora. These favorable properties make SviXyn10A a good candidate for application in pulp and paper industries.