L-干燥法和冷冻真空干燥法保藏放线菌效果的比较

L-干燥法和冷冻真空干燥法保藏不同放线菌效果是不一致的。在低温5℃多数放线菌用L-干燥法保藏,无论是干燥后及保存11个月以后其成活率均比冷冻真空干燥法高,但L-干燥法对温度较敏感,在温度37℃保藏成活率有下降的趋势。而冷冻真空干燥法在短期内保藏受温度影响较小。两种方法使用三种保护剂:10%脱脂牛奶粉,3%谷氨酸钠,1%蛋白胨+10%蔗糖,其中以10%脱脂牛奶粉为最理想。因此,采用L-干燥法以10%脱脂牛奶粉为保护剂,在低温5℃长期保存放线菌是较理想的方法之一。两种方法三种保护剂保存11个月后对葡萄糖异构酶     

L-干燥法和冷冻真空干燥法保藏放线菌效果的比较

L-干燥法和冷冻真空干燥法保藏不同放线菌效果是不一致的。在低温5℃多数放线菌用L-干燥法保藏,无论是干燥后及保存11个月以后其成活率均比冷冻真空干燥法高,但L-干燥法对温度较敏感,在温度37℃保藏成活率有下降的趋势。而冷冻真空干燥法在短期内保藏受温度影响较小。两种方法使用三种保护剂:10%脱脂牛奶粉,3%谷氨酸钠,1%蛋白胨+10%蔗糖,其中以10%脱脂牛奶粉为最理想。因此,采用L-干燥法以10%脱脂牛奶粉为保护剂,在低温5℃长期保存放线菌是较理想的方法之一。两种方法三种保护剂保存11个月后对葡萄糖异构酶     

石蜡油保藏苏云金芽孢杆菌13年的活性检验

石蜡油法保藏苏云金芽孢杆菌 ,一般可以保藏 1～ 5a,保藏研究发现石蜡油法保存 1 3a的苏云金芽孢杆菌经复壮后菌株仍存活 ,且营养特征、生理特征正常。     

双歧杆菌培养和保藏条件的研究

通过培养和保藏ＳＢ３２１方法的比较,得到了一种适合一般微生物学实验室培养和保藏双歧杆菌的简易方法。     

定期转种法和低温冷冻法保藏致病真菌活性的能力比较

定期转种法和低温冷冻保存法是临床实验室最常用的两种真菌保存方法,为比较两种方法保藏致病真菌活性的能力,本研究使用两种保藏方法对实验室689株致病真菌保藏5年后进行检测。定期转种法是将菌落接种于马铃薯斜面培养基并将其储存在4℃冰箱,每6个月转种1次。低温冷冻法是挑取马铃薯斜面培养基上生长良好的菌落于无菌10%甘油中,放置在-80℃储存。保藏5年后,将两种方法保藏的菌株转种复苏,比较菌株的复活率。对于念珠菌属Candida、新生隐球菌Cryptococcus neoformans、毛癣菌属Trichophyton、曲霉属Aspergillus和孢子丝菌属Sporothirix真菌,两种方法的菌株复活率无统计学差异;对于小孢子菌属Microsporum真菌和马尔尼菲蓝状菌Talaromyces marneffei,使用低温冷冻法保藏的菌株复活率高于定期转种法保藏的菌株复活率;对于着色霉属Fonsecaea真菌,低温冷冻法保藏的菌株复活率低于定期转种法保藏的菌株复活率。因此,我们认为对于常见致病真菌的长期保藏,使用10%甘油作为保护剂的低温冷冻法优于定期转种法,但其不适用于着色霉属Fonsecaea真菌的长期保藏。     

海藻糖对猪精子冷冻真空干燥保存效果的影响

猪精子经冷冻干燥后,在光学显微镜和电子显微镜下观察其超微结构,并借助辅助生殖技术将其注入猪卵母细胞后,进一步观察受精卵的发育情况。结果表明：海藻糖组雄原核形成率 (68.52％)、卵裂率 (59.17％) 和囊胚率 (19.16％) 优于EDTA组 (64.59％、56.26％和15.62％) 和对照组 (35.36％、52.33％和8.60％) (P＜0.05);海藻糖组的冷冻真空干燥猪精子分别在4℃下保存60、120、180 d,雄原核形成率、卵裂率和囊胚率均无显著差异 (P＞0.05);海藻糖组的冷冻真空干燥猪精子复水化后孵育1 h和2 h,卵裂率、卵裂率和囊胚率均差异显著 (P＜0.05);海藻糖处理组与EDTA处理组中的冷冻真空干燥猪精子分别在4℃和?20℃下保存后各处理组间精子形态差异不显著 (P＞0.05);海藻糖组中B级冷冻真空干燥精子百分数显著多于EDTA处理组 (P＜0.05)。超微结构分析表明,冷冻真空干燥猪精子的损伤主要表现在顶体和颈部的肿胀与缺损、尾部断裂。     

电脉冲法转化苏云金芽孢杆菌BMB171的研究

研究了用电脉冲法转化苏云金芽孢杆菌受体菌ＢＭＢ１７１的优化条件以及由转化导入的几类ｃｒｙ基因在ＢＭＢ１７１中的表达效果。结果表明,采用ＳＧ溶液作电脉冲缓冲液,用１０．ｏｋＶ／ｃｍ的脉冲场强和１次电脉冲（４．６ｍｓ）以及采用对数前期（ＯＤ_６５０ｎｍ为０．２～０．３）收获的受体菌,可以达到最高转化频率,其中用ｐＨＴ３１０１电转化 ＢＭＢ１７１的最高频率达 ８×１０７转化子／μｇ ＤＮＡ。转化频率随质粒ｐＨＴ３１０１浓度的增加,在５４．６９ｐｇ／ｍＬ至３．５０μｇ／ｍＬ范围内     

真空渗入转化法中农杆菌在植株体内的分布和活力变化

以真空渗入处理后的白菜植株为材料,采用组织化学染色法和细菌平板培养的方法,研究了农杆菌在植株体内的分布特点及其活力变化。结果表明：不同器官中农杆菌的分布量不同,以花器官中分布最多,叶中次之,茎中最少;农杆菌存在于细胞间隙中,维管束及其周围分布较集中,在胚珠中大量分布。处理后植株体内,各器官中农杆菌的生活力及其数量都随时间延长而减少,但是花器官中的农杆菌存活量较大,处理15d后的花蕾中仍然有一定量(约10.3个CFU/g组织)具有活力的农杆菌存在。讨论了这些研究结果在揭示真空渗入转化法的转化过程和提高转化频率中的意义。     

抗体-适配子混合夹心法检测炭疽芽孢杆菌芽孢的研究

通过SELEX(SystemEvolutionofLigandsbyEXponentialenrichment)体外筛选技术寻获的能与炭疽芽孢杆菌芽孢特异结合的DNA适配子(aptamer)考察其作为检测分子的能力。利用抗体-适配子混合夹心法,根据显色反应强度,评价适配子检测炭疽芽孢杆菌芽孢的能力。结果表明,适配子可以作为检测分子检测靶物质的存在。适配子检测范围在2-16μg,芽孢的检测范围在4×104-4×107,当适配子16μg,芽孢量为4×107显色的指示强度最强。说明适配子作为检测用分子,具有潜在的实用价值。     

1株枯草芽胞杆菌的鉴定及其弹性蛋白酶结构研究

鉴定弹性蛋白酶产生菌株EL32,确定该酶的基本结构。采用分子生物学、形态学及生理生化性质对菌株EL32进行鉴定;用硫酸铵沉淀、离子交换层析和分子筛层析纯化酶蛋白;借助肽指纹图谱及酶基因克隆技术研究该酶的一级结构;利用同源建模的方法研究该酶的空间结构。菌株EL32的16S rDNA与枯草芽胞杆菌16S rDNA的同源性达到99%,其菌落呈乳白色,其细胞革兰染色阳性,具有芽胞;发酵葡萄糖试验产酸不产气,明胶水解试验呈阳性,能够水解淀粉,V-P反应呈阳性,鉴定为枯草芽胞杆菌。从菌株BL32发酵液中纯化得到了弹性蛋白酶,SDS-PAGE分析显示其分子量为31 ku。用LTQ-MS测定肽指纹图谱表明该弹性蛋白酶是枯草芽胞杆菌蛋白酶subtilisin,该酶的基因和蛋白质序列与枯草芽胞杆菌蛋白酶subtilisin的同源性都高达99%。菌株EL32弹性蛋白酶的三维结构含有6个α-螺旋,7个扭曲的平行β-折叠以及2个反平行的β-折叠,His、Asp和Ser是其活性中心的关键基团。鉴定了1株产弹性蛋白酶的枯草芽胞杆菌,确定了其弹性蛋白酶是蛋白酶subtilisin,为该枯草芽胞杆菌蛋白酶的应用提供了基础。     

Growth conditions and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosyntheis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5–1%, gelatin at concentrations of 0.5–1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V–VII stages of sporulation).     

枯草芽孢杆菌在系统与合成生物技术中研究进展及工业应用

枯草芽孢杆菌Bacillus subtilis是微生物生理生化机理研究的模式菌株,也是工业应用生产小分子化合物、大宗化学品、工业酶、药物及保健品等生物制剂的良好底盘细胞.近些年,研究枯草芽孢杆菌的合成生物技术和代谢工程方法日新月异,为利用其作为底盘细胞生产目标产品提供了良好的工具和理论参考.文中综述了利用枯草芽孢杆菌为...     

生物素对地衣芽孢杆菌生长和乳酸形成的影响

生物素对地衣芽孢杆菌生长和乳酸形成的影响胡尚勤（重庆师范学院生物系重庆６３００４７）地衣芽孢杆菌（Ｂａｃｉｌｕｓｌｉｃｇｅｎｉｆｏｒｍｉｓ）是我国９０年代首次从正常待产妇阴道拭子分离出来的一个无毒新菌株。现在国内已经用它来生产“整肠生”活菌剂。它是一...     

Isolation and characteristics of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase secreted by a recombinant strain of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> at various growth phases

The recombinant strain of Bacillus subtilis bearing B. intermedius glutamyl endopeptidase gene in multicopy plasmid Δ58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca²⁺ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied.     

高效回收苏云金杆菌质粒DNA的方法研究

介绍三种简易、快速和高效回收苏云金杆菌（Bacillus thuringiensis,简称Bt)质粒DNA的方法。这些方法省时、经济、适用范围广,回收的Bt质粒DNA质量高,可直接用于各种分子克隆操作。     

Metal Leaching and Reductive Dissolution of Iron from Contaminated Soil and Sediment Samples by Indigenous Bacteria and Bacillus Isolates

The purpose of this study was to leach Cu, Zn, As, and Fe from contaminated soil and sediment samples with indigenous heterotrophic bacteria isolated from the study sites. The sediment contained Fe in the form of goethite and low concentrations of other metals. The soil contained hematite and high concentrations of other metals. The environmental conditions affected the bacterial activity in the metals dissolution. As and Fe were the major metals leached from the sediment sample while a minor fraction of Cu was solubilized. Cu and Zn were the major metals leached from the soil sample while only a minor fraction of Fe was dissolved. As a control, a disinfectant was used for partial inactivation of indigenous bacteria. This treatment had a negative effect on the leaching of Fe, Zn and As from soil and sediment samples, but it increased Cu dissolution from the sediment. Bacterial different dissolution of Fe during soil and sediment bioleaching was also investigated with ferrihydrite. The iron concentration was much higher during ferrihydrite dissolution when indigenous bacteria from sediment were used compared to indigenous bacteria isolated from soil. The indigenous bacterial inoculum provided more biological and metabolic diversity which may account for the difference in reductive iron reduction from ferrihydrite. The Bacillus cultures isolated from soil and sediment samples showed similar efficiencies in reductive dissolution of ferrihydrite. The synergetic bacterial inhibition effect created by the environmental conditions can influence bioremediation effect.     

枯草芽孢杆菌B47菌株高产抗菌物质的培养基及发酵条件优化

枯草芽孢杆菌Bacillus subtilis B47菌株为番茄内生细菌, 也是玉米小斑病拮抗菌, 能产生对玉米小斑病菌有强烈抑制作用的抗菌物质。以B47菌株发酵液的无菌滤液对玉米小斑病菌的抗菌活性为检测指标, 测定B47菌株产抗菌物质培养所需的最佳碳、氮源和无机盐, 并通过正交试验法对该菌株产抗菌物质的培养基配方和摇瓶发酵条件进行优化。研究结果表明, B47菌株产抗菌物质最佳碳、氮源和无机盐分别为蔗糖、酵母浸膏和MgSO4·7H2O, 最优培养基是YSB (Yeast extract-sucrose-beef extract)培养基, 其配方为: 蔗糖2%, 酵母浸膏2%, 牛肉浸膏1.5%, MgSO4?7H2O 0.06%, FeSO4·7H2O 0.000 9%, 最优发酵条件组合为: 30 °C, pH 7.0, 170 r/min摇床培养6 d, 接种量为1%, 装液量为40 mL/200 mL。