Cloning, Characterization, and Functional Expression of a Thermostable Type B Feruloyl Esterase from Thermophilic Thielavia Terrestris.

Feruloyl esterases (FAEs) have great potential applications in paper and breeding industry. A new thermo-stable feruloyl esterase gene, TtfaeB was identified from the thermophilic fungus Thielavia terrestris h408. Deduced protein sequence shares the identity of 67% with FAEB from Neurospora crassa. The expression vector pPIC9K-TtfaeB was successfully constructed and electro-transformed into GS115 strain of Pichia pastoris. One transformant with high feruloyl esterase yield was obtained through plate screening and named TtFAEB1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TtFAEB1 showed a distinct protein band appearing at the position of about 35-kDa, indicating that TtfaeB gene has been successfully expressed in P. pastoris. The recombinant TtFAEB was purified by affinity chromatography and the specific activity of purified TtFAEB was 6.06 ± 0.72 U/mg. The optimal temperature and pH for purified recombinant TtFAEB was 60 °C and 7.0, respectively. TtFAEB was thermostable, retaining 96.89 and 84.16% of the maximum activity after being treated for 1 h at 50 °C and 60 °C, respectively. Additionally, the enzyme was stable in the pH range 4.5-8.0. The homology model of TtFAEB showed that it consists of a single domain adopting a typical α/ß-hydrolase fold and contains a catalytic triad formed by Ser117, Asp201, and His260. TtFAEB in association with xylanase from Trichoderma reesei could release 77.1% of FA from destarched wheat bran. The present results indicated that the recombinant TtFAEB with excellent enzymatic properties is a promising candidate for potential applications in biomass deconstruction and biorefinery.

Effects of endophytic fungi in Mongolian pine on the selection behavior of woodwasp (Sirex noctilio) and the growth of its fungal symbiont.

BACKGROUND: The European woodwasp, Sirex noctilio, is a global invasive pest, attacking a wide variety of pine species by inoculating spores of a symbiotic fungus (Amylostereum areolatum) at oviposition. The woodwasp larvae depend on the growth of the symbiotic fungus to feed. The relationship among host endophytic fungi, symbiotic fungus and woodwasp remain elusive. Here, the effects of endophytes in Mongolian pine on the growth of Amylostereum areolatum and the selection behavior of female woodwasp were investigated by quantifying the mycelium growth rates and olfactometry assays. RESULTS: The endophytic plant fungi, Trichoderma harzianum, Phlebiopsis gigantea, T. viride and T. atroviride, completely killed the mycelia of Amylostereum areolatum. Mycelium fermentation broth of Chaetomium globosum inhibited the growth of the symbiont. Moreover, we observed that volatiles of Ophiostoma minus and Aspergillus niger (acetophenone, acetylacetone, hexadecane, phenylethyl alcohol, and isopropyl myristate) had repellent effects on adult female woodwasp. While volatiles of Amylostereum areolatum ((-)-globulol, 2-hexene, cycloprop[e]indene-1a,2(1H)-dicarboxaldehyde, terpene and cyclopentanone) had a significant attractiveness to adult female woodwasp. CONCLUSIONS: Some species of the host endophytic fungi had a significant negative effect on the growth and development of woodwasps, which could be useful in the monitoring and effective management of woodwasps. © 2018 Society of Chemical Industry.

[Screening and identification of proteins interacting with p38 MAP kinase via T7 phage-display screening system].

OBJECTIVE: To screen and identify proteins that interact with p38 mitogen-activated protein (MAP) kinases by means of T7 phage-display screening system. METHOD: His-tagged fusion protein of p38 MAP kinase was used to coat a 96-well ELISA plate and Ni-NTA resin, which served as the media for screening human lung and liver T7 phage cDNA libraries. RESULTS: After 4 rounds of biopanning, 86 independent plaques were selected and processed by EDTA. The inserted gene fragments from these plaques were amplified by PCR, the products purified by a gel recovery method. The sequences of the insertions were identified and analyzed with BLAST program in GenBank. Forty-six clones were found to encode proteins. CONCLUSION: T7 phage-display screening system is convenient, rapid and effective for screening the P38 MAP kinase-binding proteins.

[Construction of vector of his-tagged cytoplasmic fragment of human toll like receptor 4 and its expression in E.coli].

OBJECTIVE: To construction of vector of his-tagged cytoplasmic fragment of human Toll like receptor 4 (hTLR4) and its expression in E.coli. METHODS: hTLR4 cytoplasmic cDNA codon domain was amplified by polymerase chain reaction (PCR) and cloned into pET-DsbA2.0 plasmid expressing His-DsbA fusion protein. After being identified by the assay of restrictional enzyme and sequencing, His-Dsb A fusion proteins were induced with isopropy-beta-D-thiogalactoside (IPTG) and further purified. RESULTS: A fusion protein with molecular weight of 42 kd was obtained. CONCLUSION: hTLR4 which was constructed and expressed successfully under nondenaturing conditions provides a tool for further studies.

Regulation of inducible nitricoxide synthase gene transcription by p38 mitogen-activated protein kinase in human embryonic kidney 293 cells.

OBJECTIVE: To study the transcriptional regulation of inducible nitric oxide synthase (iNOS) gene by p38 mitogen-activated protein kinase (MAPK). METHODS: With human embryonic kidney (HEK) 293 cells as the target and the assistance of lipofectamine-mediated co-transfection techniques and luciferase reporter gene systems, FLAG-tagged p38 isoforms (namely FLAG-p38 alpha, FLAG-p38 beta;, FLAG-p38 gamma and FLAG-p38 phi;) in pcDNA3, pcDNA3, piNOS-Luc and pCMV-beta; were transfected into HEK 293 cells, and the relative activity of luciferase was subsequently tested. RESULTS: Highest luciferase activity occurred only in p38 alpha group compared with the other three isoform groups under no stimulation. Under the stimulation by lipopolysaccharide (LPS), the luciferase activity of each group was obviously increased and the highest activity occurred in p38 beta group. CONCLUSION: LPS can induce transcription and activation of iNOS gene, and p38 MAPK is involved in the transcription regulation of iNOS gene in HEK 293 cells.

Role of p38 mitogen-activated protein kinase in lipopolysaccharide-induced expression of inducible nitric oxide synthase in human endothelial cells.

OBJECTIVE: To understand the role of p38 mitogen-activated protein kinase (p38MAPK) in the expression of inducible nitric oxide synthase (iNOS) and NO production in human endothelial cells under the stimulation by lipopolysaccharide (LPS). METHODS: NO level in the supernatant of the cell culture media was measured with Griess method, and iNOS protein and mRNA expressions by the cells were detected with immunofluorescence analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. Immunoprecipitation assay was employed to examine p38 MAPK activity. RESULTS: It was shown that in comparison with the basal level of iNOS expression in cultured endothelial cells line ECV304, iNOS mRNA and protein expressions were significantly increased in the cells after LPS stimulation. In response to LPS treatment, obvious enhancement of p38 MAPK activity in ECV304 took place after the stimulation, with the peak level occurring at 15 min that maintained for approximately 45 min before gradual decline. When treated with SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole], a highly specific inhibitor of p38 MAPK, significant inhibition of LPS-induced iNOS protein and mRNA expressions was observed. CONCLUSIONS: p38 MAPK plays an important role in iNOS expression and NO production in ECV304 cells, and, inhibition of the signal transduction pathway may consequently be an effective approach to reduce the production of iNOS and other cytokines for the treatment of septic shock.

[Construction and expression of red fluorescent protein fusion vector incorporating p38 mitogen-activated protein kinase].

OBJECTIVE: To construct the vector that expresses the fusion protein of p38 mitogen-activated protein kinase (MAPK) and red fluorescent protein (RFP) in mammalian cells. METHODS: FLAG-tagged p38 MAPK in pcDNA3 vector was subcloned into RFP vector pDsRed1-N1, the construct of which was then transfected into HeLa cells and observed with fluorescence microscope. RESULTS: The recombinant plasmid was verified by enzyme digestion, PCR and sequence analysis, and p38 MAPK-RFP fusion protein was highly expressed in HeLa cells. Fluorescence microscope found the red fluorescence distributed all over the cytoplasm and in the nuclei as well. CONCLUSION: The expression vector for p38 MAPK-RFP fusion protein is successfully constructed and effective expression of this fusion protein is achieved, which might be instrumental in the study of intracellular localization of p38 MAPK.