味精废水综合处理的研究

提出了利用味精废水培养苏云金芽孢杆菌进而生产Bt生物农药的新的味精废水处理方法.对苏云金芽孢杆菌在味精废水中培养的培养基优化和深层培养条件及深层培养过程各参数的变化规律等进行了较为系统的研究,提出了进行工业化试验的培养工艺.     

微生物杀虫剂Bt53菌株的发酵培养基优化

运用正交试验L2 7(313 )设计法对鳞翅目昆虫高毒效的苏云金杆菌Bt5 3菌株进行培养基优化试验 ,在培养温度 30± 1℃ ,5 0ml/5 0 0ml三角瓶 ,摇床转速 180r/min条件下 ,苏云金杆菌 5 3菌株的发酵最佳培养基是 (% ) :碳源 0 5、氮源A 2 0、氮源B 0 10、氮源C 0 2 5、磷酸氢二钾 0 75、碳酸钙 0 15、硫酸镁 0 0 18及pH8 0。     

苏云金杆菌33菌株最佳培养基和发酵条件研究

应用正交试验L27(313)对鳞翅目昆虫高毒效的苏云金杆菌33菌株的发酵培养基进行研究,得到了适合其发酵培养的优化复合培养基配方—0.5％玉米粉 2．00％黄豆粉 0．15％酵母粉 0．25％鱼粉 0．075％蛋白胨 0．25％磷酸二氢钾 0．05％碳酸钙 0．035％硫酸镁。另外实验还证实适当增加通气量、发酵初始酸碱度控制在pH7．5、发酵温度30℃、转速180r／min、培养40h更适合该菌株液体深层发酵。     

苏云金杆菌SD—5菌株发酵生理学研究及生产

通过苏云金杆菌SD－5菌株对混合氨基酸的抗性及在3吨发酵罐中发酵生理学的研究,显示SD－5菌株具有优良的发酵性能。用正交试验设计方法筛选出SD－5菌株发酵培养基配方,菌数可达110－130亿／ml,芽孢率达95％以上。     

苏云金芽胞杆菌clpP基因缺失对碱刺激敏感性的影响

[目的]比较苏云金芽胞杆菌与枯草芽胞杆菌在碱性培养条件下生长情况,明确clpp基因在碱刺激条件下的作用.[方法]采用同源重组技术敲除苏云金芽胞杆菌HD73菌株clpP基因,通过在不同pH下生长曲线的测定明确了clpP基因缺失突变体对碱性环境的敏感性,测定clpp基因的缺失对芽胞形成率、芽胞萌发效率和盐胁迫的影响.[结果]苏云金芽胞杆菌在碱刺激后,当培养基pH值为8.9-9.1时可以恢复生长,而枯草芽胞杆菌在pH值为8.2-8.4时可以恢复生长,说明苏云金芽胞杆菌对碱性环境适应能力更强,这有助于作为病原菌的Bt适应昆虫中肠的碱性环境.clpp基因缺失对芽胞形成率和萌发效率没有明显的影响.在将培养基中NaOH终浓度调节至30 mmol/L NaOH时,clpp基因缺失突变体的生长较出发菌株慢.说明ClpP在苏云金芽胞杆菌对碱性环境的适应过程中具有重要作用.     

经高浓度味精废水驯化的B.t菌株伴胞晶体特性及其活性成分研究

对3株能在味精废水中生长的苏云金芽孢杆菌菌株T.4,G.1,S-2.5进行了废水茄子瓶培养基培养,应用液体双相法分离晶体,通过SDS－PAGE电泳确定了它们的分子量为120KD,104KD,67－68KD,43－45KD,选取菌珠G．1经分离后的晶体通过DEAE－纤维素离子交换层析和Sephadex G-100凝胶排阻层析分离到两上峰,分子量分别为120KD,68KD,致死中浓度LC50分别为0．609,＞25。     

产几丁质酶的苏云金杆菌菌株筛选及酶合成条件研究

从本室保存的 64株苏云金芽孢杆菌中 ,筛选出一株几丁质酶活力较高的菌株WB 50。产酶条件研究表明 :在pH 7.0的基础培养基中添加 2 .0 %的细粉几丁质 ,1.0 %的酵母膏 ,2 2 0rpm 30℃下培养 72小时 ,几丁质酶的产出最大。     

正交试验设计法考察苏云金杆菌“140”－51培养基

我们运用了正变试验设计法对经过复壮、紫外线照射处理的苏云金杆菌“１４０”－５１菌株进行培养基的考察,采用Ｌ２７（３１３）的正交表来安排培养基中影响发酵单位的几个主要因子［１．２．３］,其它试验条件固定为;培养温度３０±１℃,１５ｍｌ装量／５００ｍｌ三角瓶,摇床转速２１０ｒ／ｍｉｎ,ｐＨ值控制在每一配比培养基灭菌后为ｐＨ６．８～７．２范围内。     

甘蓝型油菜抗虫转基因植株及其抗性分析

通过油菜子叶外植体－农杆菌共培养法将苏云金杆菌杀虫蛋白基因导入甘蓝型油菜,获得抗虫的转基因植株。带有１￣２ｍｍ子叶柄的油菜子叶经农杆菌感染后,共培养２￣３天,然后转移到附加１５ｍｇ／Ｌ卡那霉素的ＭＳ选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含２０￣５０ｍｇ／Ｌ卡那霉素的选择培养基上继代培养,再转移到含２５ｍｇ／Ｌ卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫蛋白基因为探针,进行１     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌被鉴定为苏云金芽胞杆菌,鞭毛血清型H2,幕虫亚种;产生卵圆形伴胞晶体,伴胞晶体蛋白为100kD;测定了该蛋白 N－末端序列,该序列与炭疽芽胞杆菌的细胞表面S－层蛋白具92－93％相似性,根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱,分别克隆了该基因5’和3’端所在2．9kb XbaI片段和3．1kb Cla I DNA片段,彼此间具0．6kb重叠,通过拼接获得含完整ctc基因的克隆,含该基因的大肠杆菌与表达S－层蛋白的大肠杆菌具相似生长特征,初步表明CTC菌株的伴胞晶体由细胞表面S－层蛋白组成,苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体,由于S－层是细胞表面的结构成分,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑。     

Differences in adhesion, phagocytosis and virulence of clones from Entamoeba histolytica, strain HM1: IMSS

Orozco E., Suárez M. E. and Sánchez T. Differences in adhesion, phagocytosis and virulence of clones from Entamoeba histolytica, strain HM1: IMSS. International Journal for Parasitology15: 655–660. Clones isolated from Entamoeba histolytica, strain HM1: IMSS were tested for adhesion, phagocytosis and virulence after subculturing in liquid medium. Other clones were isolated from a subpopulation of strain HM1: IMSS, and highly phagocytic trophozoites were eliminated by irradiation, after incorporating bromodeoxiuridine into their DNA by phagocytosis of labelled bacteria. We thus obtained several clones from strain HM1: IMSS showing a different degree of phagocytosis. Some phagocytosis-deficient clones showed impairment in red blood cell adherence, while others showed a reduced intake of particles into their cytoplasm. The degree of phagocytosis always was associated with the virulence of the clone.     

Biosynthesis of gold nanoparticles by actinomycete Streptomyces viridogens strain HM10

Biosynthesis of gold nanoparticles by Streptomycetes from Himalayan Mountain was undertaken for the first time. Out of 10 actinomycete strains tested, four strains (D10, HM10, ANS2 and MSU) showed evidence for the intracellular biosynthesis of gold nanoparticles, among which the strain HM10 showed high potency. Presence of spherical and rod shaped gold nanoparticles in mycelium of the strain HM10 was determined by transmission electron microscopy (TEM) and X-ray diffraction analysis. The average particle size ranged from 18-20 nm. UV spectral analysis indicated that the reduction of chloroauric acid (HAuCl4) occurred within 24 h of reaction period. Further, the strain HM10 showed enhanced growth at 1 and 10 mM concentration of HAuCl4. The gold nanoparticles synthesized by the strain HM10 showed good antibacterial activity against S. aureus and E. coli in well-diffusion method. The potential actinomycete HM10 strain was phenotypically characterized and identified as Streptomyces viridogens (HM10). Thus, actinomycete strain HM10 reported in this study is a newly added source for the biosynthesis of gold nanoparticles.     

Azo Dye Decolorization under Microaerophilic Conditions by a Bacterial Mixture Isolated from Anthropogenic Dye‐Contaminated Soil

Soil samples isolated from dye-contaminated sites were exploited for isolation of dye decolorizing microorganisms. A novel bacterial mixture, RkNb1, was selected based on its efficiency, showing maximum and faster decolorization of textile dyes. Seven bacterial strains were isolated and identified from the bacterial mixture as Ochrobactrum intermedium (HM480365), Ochrobactrum intermedium strain M16-10-4 (HM030758), Enterococcus faecalis (HM480367), Arthrobacter crystallopoietes (HM480368), Kocuria flavus (HM480369), Bacillus beijingensis (HM480370), and Citrobacter freundii (HM480371) by 16S rRNA gene sequence analysis. This bacterial mixture showed 98.17% decolorization of Reactive Violet 5 (400 mg L^?1) within 8 h. The culture exhibited good decolorization ability at pH 8 and at a temperature of 37°C. Malt extract and peptone was found to enhance the decolorization rate of Reactive Violet 5. Plackett-Burman experimental design was used for elucidation of medium components affecting Reactive Violet 5 decolorization. Dye degradation products obtained during the course of decolorization were analyzed by high-performance thin-layer chromatography (HPTLC), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR). The potential of this bacterial mixture to decolorize Reactive Violet 5 dye from manufacturing industry effluent is to be carried out using appropriate bioreactors.     

Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.     

Properties of colony-stimulating factors produced by macrophage cell lines and hybrid cells

Colony-stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines (BAM1 and BAM3), and three hybrids (HM3-11, HM3-12, and HM3-14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222-16) were examined. HM3-11 and HM3-14 produce two molecular species of CSF, which are not found in the conditioned media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate-acetate (PMA), and zymosan-stimulated BAM3. HM3-12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species (peak 1-CSF) was characterized as a macrophage-colony-stimulating factor (M-CSF). The other CSF (peak 2-CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS-polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI-3 cells, the growth factor activity of peak 2-CSF binds to DEAE-Sephacel. Thus, peak 2-CSF is similar to a granulocyte-macrophage colony-stimulating factor (GM-CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1- and peak 2-CSF are of mouse origin.     

Chemoattractant activity of Entamoeba histolytica for human polymorphonuclear neutrophils

The ability of axenic Entamoeba histolytica trophozoites and amoebic protein preparations to stimulate chemotaxis of human polymorphonuclear neutrophils (PMN) was evaluated. Virulent E. histolytica (strain HM1:IMSS) stimulated chemotaxis (delta distance = 0.55 +/- 0.02 mm, P less than 0.01 vs. control medium). Sonicated (100 micrograms protein/ml) or homogenized (500 micrograms protein/ml) virulent amoebae also significantly stimulated PMN chemotaxis, whereas preparations of the nonpathogenic "Entamoeba-like" Laredo strain did not stimulate chemotaxis. Preparations of subcellular fractions of E. histolytica demonstrated maximal stimulation of PMN chemotaxis existed in nonvesiculated membranes and the supernatant from plasma membranes.     

Dimorphic cycle in Candida citri sp. nov., a novel yeast species isolated from rotting fruit in Borneo

Five dimorphic yeast strains were isolated from rotting lime fruits in Borneo. The sequences of the D1/D2 domains of the 26S rRNA genes, the internal transcribed spacer (ITS) chromosomal regions and the 18S rRNA genes were identical in the isolates and differed from the corresponding sequences of all known yeast species. Based on the sequence differences (12-15% in the D1/D2 domain) from the closest relatives and the different pattern of taxonomic traits, the new isolates are assigned the status of a new species, for which the name Candida citri sp. nov. is proposed. Its type strain is 11-469(T) , which has been deposited in Centralbureau voor Schimmelcultures (Utrecht, the Netherlands) as CBS 11858(T) , Culture Collection of Yeasts (Bratislava, Slovakia) as CCY 29-181-1(T) and the National Collection of Agricultural and Industrial Microorganisms (Budapest, Hungary) as NCAIM Y.01978(T) . MycoBank number: MB 519100. The GenBank accession numbers for nucleotide sequences of its D1/D2 domain, ITS and 18S regions are HM803241, HM803242 and HM803243, respectively. Candida citri produces invasive mycelium composed of true septate hyphae that grow towards nutrient-rich parts of the medium and develop large vacuoles at the nongrowing ends of their cells. The hyphae produce blastoconidia, which can establish satellite yeast colonies in the invaded solid substrate.     

Increased expression of integrins and decreased apoptosis correlate with increased melanocyte retention in cultured skin substitutes

Losses of human melanocytes (HM) in transplantation of cultured skin substitutes (CSS) may result from poor cellular attachments. To test this hypothesis, HM integrin expression was measured in four culture media: (a) melanocyte growth medium (MGM), an HM proliferation medium; (b) UCMC 160, a CSS maturation medium; (c) mMGM, modified MGM with 1.8 mM calcium; and (d) modified UCMC 160 with HM supplements (mUCMC 160). HM grew well in all media except UCMC 160. Increased expression of beta1, beta4, alpha3beta1 and alpha5 integrins on HM cultured in MGM and mMGM versus UCMC 160 was found by flow cytometry. Annexin V-allophycocyanin (APC) labeled HM in apoptosis and increased significantly in UCMC 160 (31.1%) compared with MGM (11.9%) or mMGM (13.9%). CSS were incubated in UCMC 160, mMGM or mUCMC 160 media, and grafted to athymic mice. In the mMGM group, grafts were darker as measured with a chromameter through 6 weeks and the average number of basal HM per field was greater at 12 weeks post-grafting. Increased graft loss was observed in the mMGM group which corresponded with the poor epidermal morphology in vitro. Although HM retention improved in vivo using mMGM to culture the CSS, the stability of the epidermis decreased. These results indicate that expression of integrins on HM in vitro correlates with HM retention in CSS and short-term survival after transplantation, but that long-term survival depends also on stable epithelium.     

Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain

Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase. HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation. The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272. Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method. The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272.