Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere

Abstract Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30 °C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.

Potential application of β-1, 3 glucanase from an environmental isolate of Pseudomonas aeruginosa MCCB 123 in fungal DNA extraction.

Pseudomonas aeruginosa MCCB 123 was grown in a synthetic medium for β-1,3 glucanase production. From the culture filtrate, β-1,3 glucanase was purified with a molecular mass of 45 kDa. The enzyme was a metallozyme as its β-1,3 glucanase activity got inhibited by the metal chelator EDTA. Optimum pH and temperature for β-1,3 glucanase activity on laminarin was found to be 7 and 50 °C respectively. The MCCB 123 β-1,3 glucanase was found to have good lytic action on a wide range of fungal isolates, and hence its application in fungal DNA extraction was evaluated. β-1,3 glucanase purified from the culture supernatant of P. aeruginosa MCCB 123 could be used for the extraction of fungal DNA without the addition of any other reagents generally used. Optimum pH and temperature of enzyme for fungal DNA extraction was found to be 7 and 65 °C respectively. This is the first report on β-1,3 glucanase employed in fungal DNA extraction.

Stem Blight Control of Schizonepeta tenuifolia Caused by Phytophthora nicotianae Using Trichoderma spp / 中草药·英文版

Objective To control stem blight disease of Schizonepeta tenuifolia caused by Phytophthora nicotianae.Methods The antagonist effect of 13 Trichoderma strains(including T.viride and T.hamianum)was evaluated upon mycelia growth of P.nicotianae.Trichoderma strains with high antagonistic activities against the pathogen were used to control stem blight of S.tenuifolia in the field.Results Of 13 Trichoderma strains tested,T.viride strain M3 showed maximum mycelia growth inhibition(83.2%)to the pathogen,followed by T.viride strain Tv04-2(78.2%)and then T.harziamum strain ThB(65.0%),in vitro.Fungal cell wall degrading enzymes,protease,and β-1,3-glueanase were analyzed qualitatively and quantitatively in further study.T.viride strains M3,Tv04-2,and T.harzianum strain ThB efficiently against P.nicotianae were used to control stem blight of S.tenuifolia in the field,and T.viride strain M3 showed the best biocontrol potential.Conclusion Trichoderma spp.can be used as alternatives of pesticides to control stem blight,one of the serious soilhome diseases of S.tenuifolia caused by P.nicotianae.However,though T.viride strains Tv04-2 aad T.harzianum strain ThB are also highly against P.nicotianae in vitro,the controlling efficacy of them on stem blight disease is not as excellent as T.viride strains M3 in the field.

The Identification of Antagonistic Bacteria B-04 against Botrytis cinerea and Improving Biocontrol Effect through Genetic Modification with ?-1,3-glucanase Gene / 中国生物工程杂志

Bacillus cereus B-04 antagonist to Botrytis cinerea were isolated from samples of tomato soil infected by Botrytis cinerea in Zibo, which are identified through a series of morphological and biochemical characteristics and the sequence of 16SrDNA. Aiming at enhancing the inhibitory effect of this strain, a 4.1kb DNA fragment containing ?-1,3-glucanases gene from pUC1940 was inserted into vector pBE2 and pHY300PLK to construct recombination plasmids, PBE2-glu and pHY300PLK-glu, which were transferred into Bacillus cereus B-04, resulting in a new strain named B-04-glu. Restriction enzyme digestion and ?-1,3-glucanases plate culture confirmed that B-04-glu contained a functional ?-1,3-glucanases gene. Compared to the wild strain B-04, B-04-glu had an increased inhibitory effect against Botrytis cinerea on tomato.

Transgenic Atractylodes macrocephala with double defense genes exhibiting resistanceto Rhizoctonia solani / 中草药

Objective The transgenic Atractylodes macrocephala resistance to Rhizoctonia solani was obtained by gene engineering. Methods On the base of the efficient regeneration system of Baizhu via shoot organogenesis, the rice chitinase gene (RCH10) and the alfalfa ?-1, 3-glucanase gene (AGLU) were tandem-inserted into the transformation vector pB101, which was transformed into A. macrocephala with gene gun. Transformants were confirmed by PCR, GUS assay, and disease resistant. Results Twenty-five independent transformants possessed desired genes were observed by PCR detection, and among them five transformants exhibiting resistance to Rhizoctonia solani. Conclusion The disease resistant variety has been obtained by transformation, which provides a shorten avenue for the direct introduction of novel traits into A. macrocephala through genetic engineering without the need for numerous back-crossing in breeding programs that slow down cultivar improvement, meanwhile it improves disease resistant gene pool.