不同银杏内酯对黑曲霉线粒体结构及功能的影响

真菌感染作为威胁人类健康的传染性疾病之一,是普遍的全球性问题。本研究通过在黑曲霉的孢子悬浮液中添加不同浓度的银杏内酯,收集黑曲霉菌丝体,评估黑曲霉线粒体的结构和功能,考察不同银杏内酯B和C对黑曲霉的抗真菌效果。透射电子显微镜扫描发现经银杏内酯B处理后的黑曲霉线粒体表现出明显的空泡化,基质结构被破坏;线粒体三羧酸循环中关键酶琥珀酸脱氢酶和苹果酸脱氢酶活性下降,线粒体膜电位降低,丙二醛和活性氧水平显著升高。研究结果初步揭示了银杏内酯以剂量依赖的方式损坏线粒体膜的完整性,破坏线粒体结构,进一步损害线粒体功能;银杏内酯B的抗黑曲霉效果要显著高于银杏内酯C。     

黑曲霉木质纤维素降解酶基因的可变剪接分析及验证

黑曲霉Aspergillus niger因能够产生大量的木质纤维素降解酶而在木质纤维素资源利用中发挥重要作用。目前,有关黑曲霉基因组中与木质纤维素降解相关的基因是否存在可变剪接的情况尚不清楚。本研究以黑曲霉CBS513.88菌株为研究对象,采用rMATS和ABLas两种方法对黑曲霉在葡萄糖为唯一碳源(G组)和小麦秸秆为唯一碳源(WS组)下的56个木质纤维素降解酶基因的可变剪接事件进行分析,并通过RT-PCR扩增和内含子特异性扩增对3个典型基因的可变剪接体进行了验证。结果表明,ABLas可变剪接分析算法相较于rMATS分析算法更为准确,ABLas分析算法显示G组和WS组共有21个木质纤维素降解酶基因出现了可变剪接,可变剪接类型以内含子保留(IR)为主,占所有可变剪接事件的82.85%。另外,G组和WS组发生可变剪接的木质纤维素降解酶基因也有所不同：G组发生可变剪接的基因为13个,WS组发生可变剪接的基因为14个,两组都发生可变剪接的基因为6个,这表明黑曲霉木质纤维素降解酶基因的可变剪接在不同生长条件下存在差异,另一方面,黑曲霉中众多可变剪接体的存在也为开发新型的木质纤维素降解酶资源提供基础。     

Modelling the effect of temperature and water activity on growth of Aspergillus niger strains and applications for food spoilage moulds

AIMS: To develop a model for the combined effect of water activity (a(w)) and temperature on growth of strains of Aspergillus niger, and comparison with data on food spoilage moulds in the literature. METHODS AND RESULTS: An extended combined model describing the growth of two strains of A. niger, as a function of temperature (25-30 degrees C) and a(w) (0.90-0.99) was developed. The growth rate (micro) was expressed as the increase in colony radial growth per unit of time. This extends the previous square root model showing the relationship between temperature and bacterial growth rate developed by Ratkowsky et al. (1983) and the parabolic relationship between the logarithm of the growth rate and a(w) developed by Gibson et al. (1994). A good correlation between the experimental data and the model predictions was obtained, with regression coefficients (r(2)) > 0.99. In addition, the use of this model allowed predictions of the cardinal a(w) levels: a(w(min)), and a(w(opt)). The estimation of the minimum a(w) levels (a(w(min))) was in accordance with data in the literature for similar and a range of other Aspergillus and related species, regardless of the solutes used for a(w) modification. The estimation of the optimal a(w) (a(w(opt))) and the optimal growth rate (micro(opt)) were in good agreement with the experimental results and data from the literature. CONCLUSIONS: This approach enables accurate prediction of the combined effects of environmental factors on growth of spoilage fungi for rapid prediction of cardinal limits using surface response curves. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a rapid method for predicting optimal and marginal conditions for growth of a wide range of spoilage micro-organisms in relation to interacting environmental conditions and will have applications for improving shelf-life of intermediate moisture foods.     

The relationship between multiple mating by queens, within-colony genetic variability and fitness in the ant Lasius niger

Multiple mating has been suggested to benefit social insect queens because high genetic variation within colonies might decrease the load imposed by sterile diploid males, enhance resistance to parasites and pathogens, and lead to a more effective division of labour and/or a wider range of tolerable environmental conditions. We tested these hypotheses in the ant Lasius niger with three population samples from Switzerland and Sweden. We found no diploid males in young or mature colonies suggesting a lack of diploid male load. Colonies with multiply-mated queens were not larger nor did they produce more sexuals than colonies with singly-mated queens. We did find a significantly lower frequency of multiple mating among newly mated queens than among the queens heading mature colonies in one population sample (Switzerland 1997). However, this result was not repeated in the other study population, or in the following year in the Swiss population.     

Elevated temperature effects on the oxidant/antioxidant balance in submerged batch cultures of the filamentous fungus Aspergillus niger B1-D

In the present study the relationship between oxidative stress and elevated culture temperature was examined in an industrially relevant fungal culture, Aspergillus niger B1-D. For the first time, both the intracellular levels of the main stressor species (superoxide radical [O(2) (.-)]) and activities of cellular defensive enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxide [GPx]) were quantified at varying temperature (25, 30, 35, 40 degrees C) to more fully characterize culture response in different growth phases. Elevated culture temperature led to increased O(2) (.-) levels in various culture phases. In the exponential phase this was due to an enhanced generation of O(2) (.-), whereas in stationary phase a decreased dismutation rate may also have contributed. CAT activities generally increased with culture temperature, whereas GPx activity changed little as temperature rose, indicating that GPx played only a minor role in destroying H(2)O(2) in this A. niger. The combination of elevated temperature (35 degrees C) and increased O(2) supply (50% enrichment) led to decreased levels of O(2) (.-) compared to the cultivation at 35 degrees C gassed with air, probably due to enhanced activity of the alternative fungal respiratory pathway. Our findings indicate that while elevated cultivation temperature does clearly induce oxidative stress events, mechanistically, it does so by a rather more complex route than previous studies indicate. Elevated temperature caused a marked disparity in the activities of SOD and CAT, very distinct from the integrated increase in activity of these enzymes in response to oxidative stress.     

Purification and characterization of a novel carbaryl hydrolase from Aspergillus niger PY168

A fungus capable of using carbaryl as the sole source of carbon and energy was isolated from a soil enrichment, and characterized as Aspergillus niger and designated strain PY168. A novel carbaryl hydrolase from cell extract was purified 262-fold to apparent homogeneity with 13.6% overall recovery. It had a monomeric structure with a molecular mass of 50,000 Da and a pI of 4.6, and the enzyme activity was optimal at 45 degrees C and pH 7.5, The activities were strongly inhibited by Hg(2+), Ag+, rho-chloromercuribenzoate, iodoacetic acid, diisofluorophosphate and phenylmethylsulfonyl fluoride but not EDTA and phenanthroline. The purified enzyme hydrolyzed various N-methylcarbamate insecticides. Carbaryl is the preferred substrate.     

Some properties of eucalyptus kraft pulp treated with xylanase from Aspergillus niger

An Aspergillus niger isolate produced about 2500nkat xylanase/ml when cultivated in a medium containing 3% xylose. Application of the crude xylanolytic preparation to unbleached eucalyptus kraft pulp resulted in a decreased kappa number and increased brightness. Handsheets made from the xylanase-treated pulp after ECF bleaching retained good structural and mechanical characteristics.     

黑曲霉3.795glaA基因及其5'调控区的克隆和序列分析

黑曲霉Ｔ２１是由黑曲霉３．７９５经诱变育种获得的糖化酶高产菌株,为阐明其高产的分子机制,由黑曲霉３．７９５克隆了糖化酶结构基因及其５′旁侧序列,并与黑曲霉Ｔ２１的相应序列进行了比较．由黑曲霉３．７９５菌丝体分离染色体ＤＮＡ,Ｓｏｕｔｈｅｒｎ杂交分析表明,糖化酶结构基因位于～２．５ｋｂ的ＥｃｏＲⅠ－ＥｃｏＲⅤ染色体ＤＮＡ片段上,在此ＥｃｏＲⅠ位点上游约１．０ｋｂ处有一ＳａｌⅠ位点．为构建糖化酶结构基因及其５′旁侧序列的基因组文库,该染色体ＤＮＡ分别用ＥｃｏＲⅠ＋ＥｃｏＲⅤ和ＥｃｏＲ＋ＳａｌⅠ消化,琼脂糖凝胶电泳分离并回收长度在１．０ｋｂ左右和２．５ｋｂ左右的ＤＮＡ片段,分别与ｐＵＣ１９载体连接后转化入Ｅ．ｃｏｌｉＤＨ５．用原位杂交方法筛选到了携带糖化酶基因编码区及其１５０５ｂｐ５′旁侧序列的阳性克隆．对克隆片段的ＤＮＡ序列进行了测定并与黑曲霉Ｔ２１的相应序列进行了比较,结果表明,在糖化酶基因编码区及其１５０ｂｐ３′非编码区内,未发现碱基差异,但在－３４０～－１５０５的５′上游区内发生了９个位置的碱基变化,包括缺失、插入和替换．这些结果表明,黑曲霉Ｔ２１与３．７９５的糖化酶产量的差异与其结构基因无关,但可能与其     

Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems

Production of pectinesterase and polygalacturonase by Aspergillus niger was studied in submerged and solid-state fermentation systems. With pectin as a sole carbon source, pectinesterase and polygalacturonase production were four and six times higher respectively in a solid state system than in a submerged fermentation system and required a shorter time for enzyme production. The addition of glucose increased pectinesterase and polygalacturonase production in the solid state system but in submerged fermentation the production was markedly inhibited. A comparison of enzyme productivities showed that those determined for pectinesterase and polygalacturonase with pectin as a carbon source were three and five times higher by using the solid state rather than the submerged fermentation system. The productivities of the two enzymes were affected by glucose in both fermentation systems. The membranes of cells from the solid state fermentation showed increased levels of C18:1, C16:0 and C18:0 fatty acids. Differences in the regulation of enzyme synthesis by Aspergillus niger depended on the fermentation system, favoring the solid state over the submerged fermentation for pectinase production. Received 12 May 1997/ Accepted in revised form 19 September 1997     

Dynamic and structural properties of glucose oxidase enzyme

The catalytic oxidation of β-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I′ band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH₂ does not change the amount of α-helix present in the protein outer shell, but reorganises a fraction of random coil to β-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism. Received: 27 December 1996 / Accepted: 18 July 1997