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1.
The efficacy of Chaetomium globosum as a biocontrol agent against the late blight pathogen Phytophthora infestans was evaluated in potato plants. Among eight Chaetomium isolates evaluated C. globosum isolate Cg-6 showed greater inhibition to mycelial growth of P. infestans in vitro. TLC studies showed that isolate Cg-6 produced an antibiotic called ‘Chaetomin’. Isolate Cg-6 showed greater exo- and endo-glucanase enzyme activity when compared to other isolates. PCR amplification of the ITS region and sequencing of the PCR product confirmed that isolate Cg-6 belongs to the C. globosum group. C. globosum Cg-6 was formulated as a liquid and applied as a tuber, soil and foliar treatment either individually or in combination against Phytophthora infection in potato plants. Among different treatments, combined application of C. globosum as a tuber treatment @ 1 ml/kg of tubers, as a soil application @ 1 ml/kg of Farm Yard Manure (FYM) and foliar spray @ 0.7% resulted in significantly less late blight infection (72%) compared to untreated control (100%) under field conditions. The application of C. globosum resulted in greater tuber yield by reducing late blight infection in two field trials when compared to untreated controls. The study clearly demonstrated the potential use of C. globosum as a biocontrol agent in the management of late blight disease in potato plants. 相似文献
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《中国畜牧杂志》2019,(9)
本实验利用PCR方法扩增1株贝莱斯芽孢杆菌(Bacillus velezensis)源的内切葡聚糖酶基因(CMCase),插入到p ET32a(+)中构建重组表达大肠杆菌系统,对重组内切葡聚糖酶基因进行生物信息学分析以及酶学性质研究。结果表明:内切葡聚糖酶由499个氨基酸组成,预测分子量为55.02 ku,最大开放阅读框ORF约为1 500 bp。经诱导表达优化后,培养上清中可检测到内切葡聚糖酶酶活力为5.81 IU/mL,菌体中为1.61 IU/mL,诱导后原菌液直接超声破碎处理可达7.41 IU/mL,其酶活力为野生菌株的2.3倍。重组内切葡聚糖酶具有典型内切纤维素酶的特征,其最适酶反应温度和pH分别为50℃和6,在60℃条件下放置1 h相对剩余酶活力可保持在70%以上,在pH 6～10范围内可保持90%以上。Na~+、Mg~(2+)可以促进重组内切葡聚糖酶酶活力,而Co~(2+)、Hg~(2+)、Fe~(2+)等金属离子以及表面活性剂SDS则具有较强的抑制作用。由此可见,本实验在大肠杆菌BL21(DE3)中成功表达了内切葡聚糖酶,且该酶主要进行分泌表达,具有一定的耐热性和良好的pH稳定性。 相似文献
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[目的]对热纤梭菌内切葡聚糖酶进行酿酒酵母细胞表面展示研究,探索降低纤维素酶成本和提高酶活的方法。[方法]通过提取产内切葡聚糖酶的热纤梭菌总DNA,根据热纤梭菌内切葡聚糖酶基因序列和酿酒酵母表面展示质粒载体pYD1上的多克隆位点设计引物,克隆内切葡聚糖酶目的基因,将其连接到酵母表面展示质粒载体pYD1上,构建重组质粒pYD1-CelA,并将其转化入酿酒酵母菌株EBY100中,经诱导表达后,测定表达产物的最适温度和pH,并分析影响其活性的金属离子种类和浓度。[结果]PCR扩增获得1 400bp左右的内切葡聚糖酶CelA基因片段,并成功构建了该基因与酵母表面展示质粒载体pYD1的重组质粒pYD1-CelA。重组质粒转化酿酒酵母菌株EBY100后,在鉴定培养基上测定透明圈的大小得出最佳诱导时间为60 h。表达产物性质的研究结果显示其最适反应温度为50℃,在pH 4.6时酶活性相对较高。[结论]该研究结果为后续对纤维素酶的进一步研究、改造、大量生产及应用奠定了一定的基础。 相似文献
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The fungus Ulocladium botrytis was isolated from Scutia buxifolia leaf litter and its growth was evaluated on both liquid and solid medium with sodium-carboxy-methylcellulose (CMC, 0.5%)
as sole C source at a pH range between 4.0 and 10.0 and the synthesis of cellulose-degrading enzymes on litter. Growth on
CMC-agar medium was maximum at pH 6.0, while in liquid CMC cultures, the highest biomass levels were found at pH 8.0 in both
cases after 7 days of incubation. Cellulose-degrading enzyme activities such as β-glucosidase (2.40 U dry leaf g−1), cellobiohydrolase (3.92 10−3 U dry leaf g−1), and endoglucanase (2.01 U dry leaf g−1) activities were detected in water-soluble fractions of inoculated leaves after 30 days of incubation. Endoglucanase activity
was maximum at pH 6.0 and relatively stable as the pH increase, being 100 and 60% stable at pH 7 and 8, respectively. As a
consequence of these enzyme activities, leaf mass was reduced by 5.8%. Our findings suggest that U. botrytis contains a cellulose-degrading enzyme complex that, unlike other cellulolytic systems, can degrade recalcitrant plant litter
under alkaline conditions. 相似文献
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纤维素酶的工业应用一直受酶活性低,成本高的限制,为了提高中性内切葡聚糖酶的活性,本研究利用易错PCR技术对来自枯草芽胞杆菌(Bacillus subtilis )C-36的内切葡聚糖酶基因进行定向进化研究,在酶分子水平上改造内切葡聚糖酶分子.实验获得了最佳突变株b-15和b-28,其内切葡聚糖酶活力比亲本酶分别提高了2.1和3.6倍.序列分析表明:突变体b-15有6个碱基发生了突变,分别是A360G、T402A、A419T、T648A、A1208G和A1397T,导致4个氨基酸突变,分别是N134K、Q140L、N403S和Q466L;b-28只有1个碱基发生了突变,导致了398位Lys突变为Arg.通过SWISS-MODEL服务器模拟酶的三维结构,分析表明,b-15改变的4个氨基酸分别位于催化结构域的第4和第5个α螺旋之间的转角和结合结构域的第5个β折叠及第9和第10个β折叠之间的转角;b-28的突变位于结合结构域的第4个β折叠.同时,对获得的两株突变株b-15和b-28用正交试验优化产酶条件,优化后的酶活分别达到4.542和5.136 U/mL,是优化前的2.2和1.5倍.实验获得了酶活性提高的内切葡聚糖酶菌株,为进一步在分子水平研究内切葡聚糖酶的功能和其应用打下了基础,也为高酶活酶分子在其他高表达系统的表达提供了基础材料. 相似文献
7.
Two strains of Gluconacetobacter diazotrophicus (Pal 5, UAP5541) and the arbuscular mycorrhizal fungus Glomus intraradices increased both the shoot and root dry weight of sorghum 45 days after inoculation, whereas they had no effect on the shoot and root dry weight of maize. Co-inoculation (Gluconacetobacter diazotrophicus plus Glomus mosseae) did not increase the shoot and root dry weight of either plant. There was a synergistic effect of Gluconacetobacter diazotrophicus on root colonization of maize by Glomus intraradices, whereas an antagonistic interaction was observed in the sorghum root where the number of Gluconacetobacter diazotrophicus and the colonization by Glomus intraradices were reduced. Plant roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices, either separately or together, significantly increased root endoglucanase, endopolymethylgalacturonase and endoxyloglucanase activities. The increase varied according to the plant. For example, in comparison with non-inoculated plants, there were higher endoglucanase (+328%), endopolymethylgalacturonase (+180%) and endoxyloglucanase (+125%) activities in 45-day old co-inoculated maize, but not in 45-day old sorghum. The possibility is discussed that hydrolytic enzyme activities were increased as a result of inoculation with Gluconacetobacter diazotrophicus, considering this to be one of the mechanisms by which these bacteria may increase root colonization by AM fungi. 相似文献
8.
Summary Cellulase activity in a silt loam soil was assayed and characterised using a microcrystalline cellulose substrate (Avicel). Activity was maximal between pH 5.3 and pH 6.0. A 64% loss in activity was observed on air-drying the soil. However, the residual activity was stable to storage at 40°C for 7 days and was resistant to the action of added protease. The component endoglucanase and -D-glucosidase activities in field-moist and air-dried soil were also assayed. The proportion of the soil microbial population able to utilise cellulose was investigated and the persistence of two free (soluble) cellulase preparations of microbial origin was determined following their addition to soil. A rapid decline in the endoglucanase activity of a Streptomyces sp. cellulase preparation was observed while 30% of the original activity of a Trichoderma viride cellulase preparation could still be detected after 20 days. From the data obtained in this study it appears that the major portion of the -D-glucosidase activity is bound to and protected by the soil colloids. By contrast, the major portion of the exo- and endoglucanase activity appears to be free in the soil solution, attached to the outer surfaces of cellulolytic microorganisms or associated in enzyme substrate complexes. The low residual activity measured in air-dried soil may owe its stability to an association with soil colloids or with recalcitrant cellulosic material present in soil. 相似文献
9.
Joselito E. Villa Kenichi Tsuchiya Mitsuo Horita Marina Natural Nenita Opina Mitsuro Hyakumachi 《Journal of General Plant Pathology》2005,71(1):39-46
The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050 相似文献
10.
Cristiane S. Farinas Gabriela L. VitcosqueRafael F. Fonseca Victor Bertucci NetoSonia Couri 《Industrial Crops and Products》2011,34(1):1186-1192
Cellulase production is one of the most critical steps in the economics of second generation ethanol. Although solid-state fermentation (SSF) is an attractive process for the production of enzymes, SSF is highly limited by the difficulty in controlling the operating variables which affect microbial growth and metabolites production. In this context, this work evaluates the effects of operational conditions on endoglucanase production by a selected strain of Aspergillus niger cultivated under SSF using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity and substrate initial moisture on endoglucanase production were evaluated using a statistical design methodology. A correlation coefficient of 0.9106 and a calculated value of F, 5.46 folds higher than the listed value (P-value < 0.05) allowed the modeling of endoglucanase production under different process conditions. Higher endoglucanase production (56.1 U/g) was achieved for a selected condition of substrate initial moisture of 72%, air inlet humidity of 70%, and flow rate of 20 mL/min. A significant increase in endoglucanase production was also found to be achieved under forced aeration conditions (50.2 IU/g) compared to static conditions (29.8 IU/g) after 72 h of cultivation. Besides, respirometric analysis revealed that the total amount of CO2 produced was linearly correlated with enzyme production (R2 of 0.988). The bioreactor system used, as well as the methodology employed herein, was very effective in evaluating the influence of operational variables on enzymes production under SSF. 相似文献