泡盛曲霉植酸酶的酶学性质研究

泡盛曲霉植酸酶作为动物饲料添加剂具有广泛的应用前景。以半固体发酵方式培养泡盛曲霉AS3.324(Aspergillus awamori),并得到纯化的植酸酶。对其酶学性质研究表明:其反应最适温度为50～55℃,最适pH为5.5,在37℃下以植酸钠为底物的Km值为1.05nmol/L,Vmax为2.16μmol/(L.min)。EDTA基本不影响植酸酶活性;Ca2 、Mg2 、Mn2 对植酸酶活性有轻微的抑制作用;Fe2 、Zn2 对酶促反应有显著的抑制作用。对该酶的耐热性研究表明,在较高温度条件处理后,仍有较高残余酶活性,与当今商品化的植酸酶相比,有较强的耐热性。     

Biological control of onion neck rot (Botrytis aclada): Protection of wounds made by leaf topping

The efficacy of fungal antagonists in protecting onions from neck rot caused by Botrytis aclada was investigated. Leaf wounds made by topping of onions during harvest, which are considered as important entrance sites for B. aclada, were treated with conidial suspensions (5 × 10⁷ conidia ml^‐1) of antagonists. In field experiments with artificial inoculation with conidia of B. aclada, applications of Trichoderma viride during harvest reduced the incidence of neck rot, assessed after three months’ storage of the onions at 9° C, from 35% to 24% in 1989 and from 28% to 18% in 1990, when onions initially had been stored under favourable conditions for fungal development. Comparable results were obtained with T. hamatum and Gliocladium roseum. A bioassay based on wound treatment of detached onion leaves was developed to select further antagonists. From 40 candidate antagonists tested, 20 strains gave similar or better control than the strain of T. viride applied in the field experiments. Effective antagonists could be found amongst strains of Trichoderma spp. Gliocladium spp. and Penicillium spp. as well as amongst fungi such as Aureobasidium pullulans and yeasts isolated from green leaves of onion or rye.     

Enhancement of operation and storage stability of glucoamylase from Aspergillus awamori by a protease inhibitor preparation

Glucoamylase was produced extracellularly by fermentation of strain Aspergillus awamori, which had been genetically modified to have high-level glucoamylase activity. Initial experiments showed that the enzyme deactivated quickly, with a half-life of less than 6 days even stored at 5°C. A possible reason for the rapid deactivation was the presence of proteases, attacking and degrading the glucoamylase. Therefore a liquid protease inhibitor cocktail (Sigma, USA) was selected and applied to enhance the stability of the enzyme. The activity of the enzyme (stored at 5°C) measured by the Schoorl-method with starch as substrate showed that the cocktail was effective with the enzyme maintaining 95% of its initial storage activity for almost one year. The enzyme preparation has been used for starch hydrolysis in a flat-sheet membrane bioreactor at 60°C to manufacture glucose solution and its operation stability extended by using the cocktail.     

Enhancement of operation and storage stability of glucoamylase from Aspergillus awamori by a protease inhibitor preparation

Glucoamylase was produced extracellularly by fermentation of strain Aspergillus awamori, which had been genetically modified to have high-level glucoamylase activity. Initial experiments showed that the enzyme deactivated quickly, with a half-life of less than 6 days even stored at 5°C. A possible reason for the rapid deactivation was the presence of proteases, attacking and degrading the glucoamylase. Therefore a liquid protease inhibitor cocktail (Sigma, USA) was selected and applied to enhance the stability of the enzyme. The activity of the enzyme (stored at 5°C) measured by the Schoorl-method with starch as substrate showed that the cocktail was effective with the enzyme maintaining 95% of its initial storage activity for almost one year. The enzyme preparation has been used for starch hydrolysis in a flat-sheet membrane bioreactor at 60°C to manufacture glucose solution and its operation stability extended by using the cocktail.     

红海榄根际土壤来源的泡盛曲霉（Aspergillus awamori）F12及其代谢产物的抗菌活性分析

摘要：【目的】鉴定一株来自于红海榄根际土壤并具有分泌抑菌活性代谢产物的真菌菌株F12,并从其发酵液乙酸乙酯浸膏中分离抑菌活性成分。【方法】通过形态学观察以及ITS序列分析方法对菌株F12进行鉴定;利用色谱技术分离发酵液乙酸乙酯浸膏中的次生代谢产物,根据化合物的质谱、氢谱、碳谱以及理化性质确定其结构,并检测它们对细菌生长的抑制作用。【结果】菌株F12被鉴定为Aspergillus awamori strain F12;从其发酵液乙酸乙酯浸膏中分离到3种化合物：1,4-二甲氧基苯（1）、大黄素（2）和3,6-二苯甲基哌嗪-2,5-二酮（3）,其中化合物1属于在本属真菌中首次报道。化合物2对金黄色葡萄球菌和枯草芽孢杆菌的生长具有明显的抑制作用,最低抑菌浓度（MIC）分别为16ng/L和32ng/L,化合物1和3对上述菌株的生长无明显的抑制活性。【结论】首次发现从红海榄根际土壤中分离到的泡盛曲霉（Aspergillus awamori）菌株F12具有合成1,4-二甲氧基苯和大黄素的能力,其中后者对微生物的生长具有明显的抑制作用。     

Isolation and characterisation of Aspergillus awamori BS05, a root-knot-nematode-trapping fungus

Nematode-trapping fungi are important biocontrol agents against parasitic nematodes through adhesive or mechanical hyphal traps. Aspergillus awamori, a root-knot-nematode-trapping fungus from tomato rhizosphere soil, was identified based on morphology and molecular characteristics of internal transcribed spacer DNA sequence. Conidial heads were white to black brown, loosely globose, and 72–127 μm in diameter. Conidiophores usually arose from the foot cell of basal mycelium, straight, and 960–1730 × 10.2–13.4 μm, hyaline to pale brown, not constricted below the vesicles; vesicles hemispherical to elongate, 43–56 μm in diameter, black brown, fertile over the upper half to two-thirds. Aspergilla were biseriate, and metulae were variable, 12–26 × 3.8–4.7 μm; phialides were 8.2–9.4 × 2.5–3 μm. Conidia were globose or subglobose, 3.6–4.8 μm in diameter, rough, grey brown and parallel in chains. A. awamori BS05 showed 44.9% control efficacy against Meloidogyne incogtina in pot experiments which suggests it as a potential biocontrol agent against Meloidogyne. This is the first report on A. awamori as nematode-trapping fungus.     

Production, partial purification and characterization of feruloyl esterase by Aspergillus awamori in submerged fermentation

Microbial feruloyl esterases acting on plant cell wall polymers represent key tools for the degradation of plant cell wall. In this paper, we describe in detail the microbial production, partial purification and characterization of feruloyl esterase from a culture medium of Aspergillus awamori strain IFO4033 obtained from a crude hemicellulose preparation of wheat straw, corncobs and wheat germ. Feruloyl esterase was extracted using centrifugation and dialysis, and then purified by ion exchange chromatography and microfiltration to homogeneity, which was checked by SDSPAGE and isoelectric focusing-PAGE. Protein content and activity of the enzyme were measured in each step of extraction and purification. Biomass was determined by the dry weight method. pH and temperature optima of feruloyl esterase enzyme were also determined. The effects of culturing time, and carbon and nitrogen sources on enzyme production were systematically investigated. Finally, enzyme activities under different storage conditions were examined.     

Production of high-fructose syrup from date by-products in a packed bed bioreactor using a novel thermostable invertase from Aspergillus awamori

Abstract

Dates by-products (discarded dates) from the sucrose-rich variety of ‘Deglet Nour’ were used as starting biomass to produce high-fructose syrup (HFS) based on an immobilized invertase process. A novel extracellular thermostable invertase obtained from Aspergillus awamori cultivated in submerged medium was induced with sucrose at 1% and used for this purpose. A zymogram of the crude extract showed the presence of a unique enzyme form that was optimally produced on the 5^th day. This enzyme preparation was biochemically characterized and immobilized on acetic acid-solubilized chitosan by covalent binding using glutaraldehyde (Yi = 88%, Ya = 54% and 15.53 U/g). When deployed in a packed bed reactor (PBR), HFS was efficiently and continuously produced from sucrose derived from aqueous date extracts. Feeding with an extract initially containing 139.2 g/L total sugar with 78.6 g/L sucrose at a flow rate of 17 ml/h, 50°C and pH 6 resulted in a conversion factor of 0.95 and a final fructose content in the syrup of 69 g/L.     

Competitive ability of the antagonists Ulocladium atrum and Gliocladium roseum at temperatures favourable for Botrytis spp. development

Ulocladium atrum and Gliocladium roseum are fungal antagonists capable of suppressing sporulation of Botrytis spp. on dead plant parts. The effect of temperature (3 to 36 °C) on antagonist conidial germination and mycelial growth was assessed on agar. In addition conidial germination of U. atrum was measured on dead lily leaves. The optimum temperature of both antagonists for both conidial germination and mycelial growth was between 27 and 30 °C. U. atrum was less affected by lower temperatures than G. roseum. At optimum temperature, 50% of conidia of U. atrum and G. roseum germinated within 2.6 and 10.0 hrs, respectively. At low sub-optimal temperatures (6 °C), 50% of conidia germinated within 18 and 96 hours, respectively.In bioassays on dead onion leaves, U. atrum suppressed sporulation of B. cinerea and B. aclada at all temperatures tested (6 to 24 °C) by more than 85%. On dead cyclamen leaves, G. roseum was more efficient than U. atrum at 21 and 24 °C but, in contrast to U. atrum, showed no antagonistic activity at temperatures below 21 °C. On dead hydrangea leaves, U. atrum significantly reduced sporulation of B. cinerea at temperatures as low as 3 and 1 °C. Under Dutch growing conditions, the mean air temperature during leaf wetness periods in onion and lily fields was 15 °C with temperatures only occasionally above 20 °C. In greenhouse crops of cyclamen, the mean temperature during high humidity periods was 17 °C. It is therefore concluded that U. atrum is better adapted than G. roseum to temperatures which occur in the field, in greenhouse crops such as cyclamen, or during cold storage of plant stocks.     

Conversion of dextrinized cassava starch into ethanol using cultures of Aspergillus awamori and Saccharomyces cerevisiae

Aspergillus awamori and Saccharomyces cerevisiae have been used to convert dextrinized cassava root flour into ethanol. A batch culture of the combined microorganisms produced 4.3% alcohol by weight from 15% cassava flour slurry in 39 h. Two-stage continuous fermentation was done using A. awamori in an airlift fermenter and yeast in a tower fermenter. A residence time of 12.5 h for the first stage resulted in 12.5% sugar concentration and a saccharification efficiency of 88%. A residence time of 5.6 h for the second stage gave an alcohol concentration of 5.3% alcohol and a starch-into-ethanol conversion efficiency of 72.5%.     

Semisynthesis and Antifungal Activity of Novel Oxime Ester Derivatives of Carabrone Modified at C(4) against Botrytis cinerea

To continuously improve the potential utility of the natural lead compound of carabrone in agrochemistry, carabrone oxime and 36 novel oxime ester derivatives of carabrone modified at C(4) were synthesized, and evaluated for their antifungal activities against Botrytis cinerea in vitro and in vivo. Of these 36 oxime ester derivatives, some compounds exhibited antifungal activities in vitro or in vivo. It was found that compounds with a pyridinyl residue can either efficiently inhibit spore germination or efficiently inhibit hyphal growth of B. cinerea, and compound 9 exhibited the highest activity in vitro and in vivo with IC₅₀ and EC₅₀ values of 1.17 and 12.9 μg/ml, respectively. Further, the structure? activity relationships are also discussed.     

Ethanol production from starch by immobilized Aspergillus awamori and Saccharomyces pastorianus using cellulose carriers

A simultaneous saccharification and fermentation (SSF) process was investigated to produce ethanol using two kinds of cellulose carriers that were respectively suitable for immobilization of Aspergillus awamori and Saccharomyces pastorianus. The maximum ethanol concentration attained by the batch operation was 25.5 g l⁻¹. Under suitable conditions, both cellulose carriers with immobilized cells could be reused efficiently for three cycles. The total amount of ethanol production was 66.0 g (per 1 l working volume) after the repeated operation. Ethanol productivity mainly depends on a saccharification process. There is a limit in durability in the repeated batch operation, and it is important to maintain high activity of the fungus in order to produce ethanol efficiently. Journal of Industrial Microbiology & Biotechnology (2001) 27, 52–57. Received 11 December 2000/ Accepted in revised form 02 June 2001     

绿僵菌SC0924酚酸类代谢产物及其抗荔枝霜疫霉活性

从绿僵菌SC0924固体发酵物中分离得到8个酚酸类化合物,通过波谱分析,分别鉴定为香草酸(1)、丁香酸(2)、邻氨基苯甲酸(3)、苯乙酸(4)、阿魏酸(5)、二氢阿魏酸(6)、2-羟基-3-苯丙酸(7)和2-羟基-3-对羟基苯丙酸丁酯(8).以滤纸片琼脂扩散法对以上化合物进行抗荔枝霜疫霉活性试验,结果表明除化合物2和6外,其余化合物均有抑菌活性.     

Purification and Characterization of a Feruloylesterase from Aspergillus awamori

A feruloylesterase was purified from the extracellular broth of Aspergillus awamori grown on wheat bran culture. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and isoelectric focusing, with an apparent M _r of 35,000 and a pI of 3.8, respectively. The substrate specificity of the purified enzyme differed obviously from that of acetylesterase of A. awamori. The enzyme bound to microcrystalline cellulose.     

Antifungal activity of Juniperus essential oils against dermatophyte, Aspergillus and Candida strains

AIMS: The increasing resistance to antifungal compounds and the reduced number of available drugs led us to search therapeutic alternatives among aromatic plants and their essential oils, empirically used by antifungal proprieties. In this work the authors report on the antifungal activity of Juniperus essential oils (Juniperus communis ssp. alpina, J. oxycedrus ssp. oxycedrus and J. turbinata). METHODS AND RESULTS: Antifungal activity was evaluated by determination of MIC and MLC values, using a macrodilution method (NCCLS protocols), on clinical and type strains of Candida, Aspergillus and dermatophytes. The composition of the oils was ascertained by GC and GC/MS analysis. All essential oils inhibited test dermatophyte strains. The oil from leaves of J. oxycedrus ssp. oxycedrus is the most active, with MIC and MLC values ranging from 0.08-0.16 microl ml(-1) to 0.08-0.32 microl ml(-1), respectively. This oil is mainly composed of alpha-pinene (65.5%) and delta-3-carene (5.7%). CONCLUSIONS: J. oxycedrus ssp. oxycedrus leaf oil proved to be an emergent alternative as antifungal agent against dermatophyte strains. delta-3-Carene, was shown to be a fundamental compound for this activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Results support that essential oils or some of their constituents may be useful in the clinical management of fungal infections, justifying future clinical trials to validate their use as therapeutic alternatives for dermatophytosis.     

碎米莎草茎总生物碱对植物和病原菌的生物活性

以水稻(Oryza sativa)等5种植物和水稻稻瘟病(Magnaporthe grisea)等6种植物病原真菌为试验对象,测定了碎米莎草(Cyperus iria)茎总生物碱对植物和病原菌的生物活性.结果表明,250～4000 μg mL-1的总生物碱对水稻种子的萌发无显著影响,对千金子(Leptochloa chinensis)幼苗的苗高有显著抑制作用.4000μmL-1的总生物碱对烟草(Nicotiana tabacum)、鳢肠(Eclipta prostrate)和车前草(Plantago asiatica)种子的萌发、烟草幼苗的苗高以及水稻、烟草和鳢肠幼苗的鲜重有显著抑制作用,相对较低浓度总生物碱对千金子种子萌发、千金子和车前草幼苗的鲜重以及5种植物幼苗的根长和苗高(烟草幼苗的苗高除外)有显著抑制作用.25～400 μg mL-1的碎米莎草茎总生物碱对小麦赤霉病菌(Fusarium gramblearum)的生长无显著影响,而对苹果轮纹病菌(Physalospora piricola)的生长有显著抑制作用,100～400μg mL-1的总生物碱对稻瘟病菌、油菜菌核病菌(Sclerotinia sclerotiovum)和番茄早疫病菌(Alternaria solani)的生长有显著抑制作用,200～400 μg mL-1的总生物碱对杨树溃疡病菌(Dothiorella gregaria)的生长有显著抑制作用,400μg mL-1的总生物碱对上述病原真菌的抑菌率分别为8.20%、74.87%、40.93%、60.91%、52.70%和16.28%.     

11种植物精油对6种植物病原真菌的抑菌活性研究

为筛选有效的植物杀菌成分,采用菌丝生长法,测定了香茅油、薰衣草油、菊花油、月桂油、柠檬油、广藿香油、肉桂油、天竺葵油、迷迭香油、茶树油、薄荷油对6种植物病原真菌的抑菌活性。发现在2 g/L的浓度下,上述11种精油对6种供试病菌均有明显的抑制作用,其中香茅油、肉桂油、天竺葵油、月桂油、茶树油和薄荷油对6种病原真菌的抑制率均为100%。剂量效应试验表明,肉桂油对灰葡萄孢（Botrytis cinerea）和禾谷镰孢菌（Fusariumgraminearum）的EC50值分别为29.05μg/mL和42.96μg/mL,而天竺葵油对灰葡萄孢（Botrytis cinerea）和禾谷镰孢菌（Fusarium graminearum）的EC50值分别为34.02μg/mL和68.48μg/mL。     

一株拮抗真菌的蛇足石杉内生细菌分离鉴定及培养条件优化

从蛇足石杉(Huperzia serrata)叶片中分离到一株对小麦赤霉(Fusarium graminearum)等多种植物病原真菌有强拮抗作用的内生细菌H-6。通过形态和培养特性观察、生理生化实验及16S rDNA序列分析, 初步鉴定该菌属于伯克霍尔德属, 命名为Burkholderia sp. H-6。同时还对该菌培养条件进行了优化, 得出马铃薯浸出液中添加2.5%的甘露醇和0.1% NaNO3有利于细胞生长和抑菌活性的产生, 最适培养温度为28℃, 最佳初始pH4.0。