酿酒酵母两种不同嗜杀菌株的比较研究

以酿酒酵母两种不同类型的嗜杀菌株SK4（K1型）和ERRI（K2型）为材料,分析了不同嗜杀酵母的嗜杀特性,两株嗜杀酵母具有相互杀死作用,其嗜杀活性与菌体生长有关。SK4和ERRI的嗜杀质粒的比较表明：M1－dsRNA质粒和M2-dsRNA质粒分子量分别为1．7kb和1．5kb,两株菌的L－dsRNA质粒均为4．0kb。用高温和紫外线处理嗜杀酵母,嗜杀活性随之消失,消除菌中的M－dsRNA质粒也相应消失,嗜杀活性的消除率随菌株和消除剂的不同而变化。实验证明两株菌产生的毒素蛋白的最适嗜杀作用条件不同,最适pH和温度分别为4．8、16℃和4．0、22℃,但两种毒素蛋白均对在对数生长期的敏感细胞作用最显著。     

嗜杀酵母的生物学功能及其应用

嗜杀酵母能够分泌毒素蛋白,杀死敏感酵母。嗜杀酵母对自身分泌的嗜杀毒素具有免疫力。嗜杀酵母的嗜杀特性与两种双链线状RNA(dsRNA)有关,即编码产生毒素蛋白的M-dsRNA和编码自身和M-dsRNA外壳蛋白的L-dsRNA。嗜杀毒素破坏细胞跨膜化学质子梯度,造成ATP和钾离子泄漏,导致细胞死亡。应用嗜杀酵母可避免野生型酵母污染,净化发酵体系,改善发酵产物品质;嗜杀毒素也可作为抵制病原酵母和类酵母微生物的抗真菌剂。     

嗜杀酵母毒素蛋白活性的平板检测

本文介绍一种在平板上直接检测嗜杀酵母产生的毒素蛋白活性的方法,此方法操作简便,可分析不同条件下毒素蛋白对敏感酵母细胞的嗜杀作用.     

一种检测酵母嗜杀活性的简便方法及其应用

本文以嗜杀酵母ＥＲＲＩ为材料建立了一种双层平板单菌落嗜杀活性检测法。此法与常规营养缺陷型筛选方法相结合,可直接筛选出具有嗜杀活性的酵母营养缺陷型菌株ＭＫ２—３：Ｋ＋Ｒ＋Ｌｅｕ－,并成功地用于检测理化因子对嗜杀质粒的消除作用。进一步采用此法在直接混合培养中做出了嗜杀酵母对敏感酵母作用的动力学曲线。结果表明,敏感酵母活菌数在混合培养的对数生长后期开始急剧下降。     

酿酒酵母两种不同嗜杀菌的比较研究

以酿酒酵母两种不同类型的嗜杀菌株ＳＫ４（Ｋ１型）和ＥＲＲ１（Ｋ２型）为材料,分析了不同嗜杀酵母的嗜杀特性,两株嗜杀酵母具有相互杀死作用,其嗜杀活性与菌体生长有关。ＳＫ４和ＥＲＲ１的嗜杀质粒的比较表明：Ｍ１－ｄｓＲＮＡ质粒和Ｍ２－ｄｓＲＮＡ质粒分子量分别为１．７ｋｂ和１．５ｋｂ,两株菌的Ｌ－ｄｓＲＮＡ质粒均为４．０ｋｂ。用高温和紫外线处理嗜杀酵母,嗜杀活性随之消失,消除菌中的Ｍ－ｄｓＲＮＡ质粒也相应     

嗜杀酵母KillerYeast的杀伤性质和酵母的病毒假说

嗜杀酵母Killer Yeasf,杀伤酵母或逆戟酵母,是指能释放毒素杀死其同类的酵母。具有杀伤性质的真菌有酵母属(Sac-charomyces),黑粉菌属(Usfflago),吲酵母属(Torulopsfs),德巴里酵母属(Debaromyces),汉逊酵母属(Han-     

利用细胞质导入法选育嗜杀啤酒酵母

利用细胞质导入(Cytoduction)法中的核融合缺陷细胞融合技术,在对酿酒酵母(Saccharomyces cerevisiae)D518菌株不做任何遗传标记,将嗜杀酵母5045菌株的嗜杀质粒转移到受体菌D518中,获得了具有两亲株优良性状的融合子KD102菌株。对融合子分析表明:融合子遗传性状稳定,不仅含有供体菌5045的嗜杀质粒,而且受体菌D518的核基因被原封不动地保留下来,为异质体细胞(Heteroplasmon)。将融合子KD102菌株用于小型、中型及生产性酿酒试验,结果表明,具有与亲株D518同样的酿造特性。在发酵过程中,能抑制野生酵母污染,净化发酵体系。对于保证啤酒纯种酿造及提高成品酒的生物稳定性具有明显效果。     

杀松材线虫细菌的分离、鉴定及其培养条件研究

为了对松萎蔫病进行安全、无污染的生物防治,从青岛大学校园内采集的样品中分离出120株菌。采用浸渍法从中筛选出1株具有较高杀线虫活性的细菌G8-1。杀线虫活性测定结果表明,该菌株发酵上清液48 h后的杀线虫率为90.81%。通过16S rRNA的序列分析,将其鉴定为Flectobacillus rhizosphaerae。通过单因素分析法和正交实验对该菌株的培养条件进行研究,确定最佳培养条件:最适初始pH为8.0,最适培养温度为35℃,最佳接种菌龄为9 h,最佳培养时间为3 d,菌株G8-1发酵上清液的杀线虫率可达到96.31%。对菌株产生的活性物质的稳定性进行初步分析,结果表明该菌株的活性物质都具有较好的热稳定性,并且在弱碱性条件下具有较高的杀线虫活性稳定性。本研究为松材线虫的生物防治提供参考。     

利用细胞质导人法选育嗜杀啤酒酵母

利用细胞质导入(Cytoduction)法中的核融合缺陷细胞融合技术,在对酿酒酵母(Saccharomyces cerevisiae)D518菌株不做任何遗传标记,将嗜杀酵母5045菌株的嗜杀质粒转移到受体菌D518中,获得了具有两亲株优良性状的融合子KDl02菌株。对融合于分析表明：融合子遗传性状稳定,不仅含有供体菌5045的嗜杀质粒,而且受体菌D518的核基因被原封不动地保留下来,为异质体细胞(Heteroplasmon)。将融合子KDl02菌株用于小型、中型及生产性酿酒试验。结果表明．具有与亲株D518同样的酿造特性。在发酵过程中．能抑制野生酵母污染,净化发酵体系。对于保证啤酒纯种酿造及提高成品酒的生物稳定性具有明显效果。     

两株耐碱酵母的pH耐受实验观察

对分离鉴定的两株酵母菌用比浊法和Bradford法进行pH耐受范围检测。Trichosporon asahiiXJU-1的pH耐受范围为2.0~13.0,最适pH值为8.0。Rhodotorula mucilaginosaXJU-1的pH耐受范围为3.0~12.0,最适pH值为8.5。比浊法在pH4.0~10.0测出数值比较可靠,而在pH<4.0和pH≥10.0产生了较大误差。在最适pH时,Bradford法间接测定两酵母的生长量,T.asahii总溶解蛋白为290μg/mL,Rh.mucilaginosa总溶解蛋白为164μg/mL。Bradford法在pH2.0~13.0范围均能较准确地反映出菌株生长状况,数据表明两株酵母有广阔的pH耐受性,它们是耐碱酵母菌的新成员。     

Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins

Influenza virus (strain X-47) was labeled with the triplet probe, eosin 5-isothiocyanate. Most of the label was found to be associated with haemagglutinin, the major glycoprotein of the viral envelope. Rotational diffusion of the glycoprotein was investigated by measuring flash-induced transient dichroism of the eosin probe. The anisotropy decay curves showed that mobility of haemagglutinin measured at pH 7.3 increased considerably with temperature with the greatest change occurring over the range 20-30 degrees C. However, at pH 5.2 no mobility was detectable over the time range of the experiment. The activity of the virus was determined by assaying haemolysis of human erythrocytes. The haemolytic activity showed an optimum at pH 5.2 and increased markedly with temperature, being negligible below 20 degrees C. In addition, inactivation of the virus by incubation at pH 5.2 was also strongly temperature dependent. A 15 min incubation at pH 5.2 inactivated the virus above 30 degrees C but had no effect below 20 degrees C. On the basis of these results, it is proposed that mobility of haemagglutinin is significant for its functional properties. When the pH is reduced from 7.3 to 5.2, the mobility observed at higher temperatures is required for the molecular rearrangements which accompany the fusion event. In the absence of an apposing membrane, these rearrangements result in irreversible aggregation of haemagglutinin in the viral membrane, and hence loss of mobility and activity.     

A yeast assay for high throughput screening of natural anti-viral agents

Over the last decade the yeast Saccharomyces cerevisiae has become a popular organism for studying heterologous gene expression and in vivo protein-protein interactions. Many variations of these basic systems have originated over the years. Besides these vast and varied applications of the yeast expression system, S. cerevisiae has also been used extensively in fundamental research as a model simple eukaryote. We have used the S. cerevisiae system to design a high throughput screen for anti-viral agents from natural sources. The design of the assay rests on the ability of the L-A helper virus and the M(1) satellite virus to detect small variations in -1 ribosomal frameshifting. A minor change in frameshifting efficiencies can be detected and clearly shown phenotypically in terms of zones of clearing on an agar plate. Using such a process, we have initiated a high throughput screening process for natural anti-viral agents.     

PH,pCO2, and temperature effect on R-adenovirus production

The effects of pH, carbon dioxide vapor pressure, pCO(2), and temperature on E1 and E3 deleted recombinant adenovirus vector (rAV) production with HEK293S cells have been studied in the ranges of pH = 6.7-7.7, pCO(2) = 0.05-0.20 atm, and T = 32-39 degrees C, respectively. The experiments were performed in four 500-mL bioreactors in parallel, which make possible the reduction of inter-run variability. Cell concentration and viability, relative oxygen uptake rate (OUR), fluorescence, and viral titer were measured. It was found that, although pH and pCO(2) did not affect significantly cell viability in the range studied, they had an important effect on virus titer. pCO(2) allowed the maximum production of rAV at 0.05 atm, and pH showed a very sharp optimum at 7.2. Temperature had an effect on both cell metabolism and virus titer. Low temperature prolonged cell viability and high OUR. Most of all, a 3-fold increase in virus yield was found at 35 degrees C compared to that at 37 degrees C, while 32 degrees C was not as beneficial (1.5-fold increase). This finding could have an important impact on large-scale production. This phenomenon was modeled using a simple 3-parameter synthesis-decay model. This model shows how the optimum gain in virus production at 35 degrees C is due to a balance between the production and decay processes at that temperature.     

Recombinant luciferase-expressing human cytomegalovirus (CMV) for evaluation of CMV inhibitors

Recombinant Towne CMV expressing luciferase under the control of CMV-DNA polymerase (POL) or the late pp28 (UL99) promoters were evaluated for potential application in high-throughput screening of anti-viral compounds. POL-and pp28-luciferase displayed maximal expression 48 and 72 hours post infection, respectively. The pp28-luciferase virus achieved a wider dynamic range of luciferase expression (6-7 logs) and was selected for testing of inhibition by five anti-viral compounds. Luciferase expression highly correlated with plaque reduction and real-time PCR. The pp28-luciferase reporter system is rapid, reproducible, and highly sensitive. It may be applied to screening of novel anti-CMV compounds.     

Enhancement of the anti-herpetic effect of trichosanthin by acyclovir and interferon

Trichosanthin (TCS) is a type I ribosome-inactivating protein that has a wide range of pharmacological activities. The present study investigated the effectiveness of TCS on herpes simplex virus (HSV-1). The anti-viral activity and toxicity of TCS on Vero cells were measured. Results showed that the ED(50), TD(50) and the therapeutic indices were 38.5, 416.5 and 10.9 microg/ml, respectively. Anti-viral activity of TCS was substantially potentiated when it was used in conjunction with other anti-viral agents. The ED(50) of TCS was reduced 125-fold by acyclovir at a concentration of 0.001 microg/ml, which was practically devoid of significant anti-viral activity. Similarly, the ED(50) of TCS was reduced 100-fold by interferon-alpha2a at a concentration of 100 IU/ml. In conclusion, TCS is effective against HSV-1 and other anti-viral agents such as acyclovir or interferon can potentiate its action substantially.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

The effect of soil acidification on atmospheric methane uptake by a Maine forest soil(1)

Soil from the zone of maximal methanotrophic activity (approximately 5-8 cm depth) in a mixed coniferous-hardwood forest consumed atmospheric methane over a wide pH range (3.5-7.5) with a broad optimum between 4.8 and 6.0. Methane uptake at native soil pH values (4.4-4.8) was only slightly less rapid than rates at optimal pH values. Addition of mineral acids to intact soil cores in pulsed applications decreased atmospheric methane consumption. The extent of inhibition varied with the type, concentration and volume of acid added: nitric acid was more inhibitory than sulfuric acid at an equivalent soil pH, and methane uptake decreased with increasing volumes and concentrations of added acid. Although ammonium chloride at 1 μmol g fresh weight (gfw) soil(-1) inhibited methane uptake, the extent of inhibition did not vary significantly with decreasing soil pH below values of 4.4.     

Acid and alkaline phosphatase activities in the clam Scrobicularia plana: kinetic characteristics and effects of heavy metals

The acid and alkaline phosphatase activities of the clam Scrobicularia plana have been partially characterised in different organs and tissues (digestive gland, gills, foot, siphon and mantle) and the 'in vitro' effect of heavy metals on both types of enzymatic activity have been analysed. The optimal pH ranged between 4.0 and 5.5 for acid phosphatase activity and 8.5 and 9.5 for alkaline phosphatase activity. The apparent optimum temperature was in the 30-60 degrees range for acid phosphatase activity and in the 30-40 degrees C range for alkaline phosphatase activity. The effect of substrate concentration on enzymatic activities in the tissues showed a good fit to the Michaelis-Menten model. For both types of enzymatic activity, the highest values were found in the digestive gland. The effect of heavy metals was dependent on the tissue analysed. Mercury showed the highest inhibition in the organs/tissues and the parameters Km and Vmax were modified when the inhibitor concentration increased, thus indicating a mixed type of inhibition.     

Characterization of alpha-galactosidase from Lactobacillus fermentum

α-Galactosidase activity was studied in Lactobacillus fermentum strains. The optimum temperature was found to be 45°C. The enzyme was inactivated at temperatures higher than 55°C, but remained active during storage at low temperatures (0, -30 and -70°C) for 5 months. Enzyme activity was observed within a 5.0–6.5 pH range, while optimum pH was dependent on the particular strain assayed. The addition of Zn²⁺ to the reaction buffer exerted a slight negative effect upon the activity, while Hg²⁺ and p -chloromercuribenzoate produced a strong inhibition. These results would indicate the presence of -SH groups in the catalytic site of the enzyme.     

Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. Using p-nitrophenyl-β-D-glucoside as substrate, K(m) was 0.54 and 2.11 mM, and k(cat)/K(m) was 1489 and 787 mM(-1) min(-1) for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30°C, while the optimum values for Bgl1E were pH 10.0 and 25°C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl(3) and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5-10.5), making it a potential candidate for industrial applications.