产氢红杆菌类胡萝卜素突变株的诱变和筛选

用紫外线照射和氯化锂夹层平板培养法对产氢红杆菌(Rhodobacter sp.R7)进行复合诱变,分离获得了一株产氢效率提高的类胡萝卜素突变株R726.该突变株在表观特征、光谱学特征、色谱特征、生长和产氢性能等方面与出发菌株有明显不同,但16S rDNA序列一致.R726菌株有550 nm类胡萝卜素特征性吸收峰,类胡萝卜素组成上比出发菌株少一黄色类胡萝卜素组分,生长和产氢性能均高于出发菌株,产氢效率比出发菌株提高了33.3%,类胡萝卜素含量比出发株提高了53.8%.     

常压室温等离子体诱变选育类胡萝卜素高产菌株及其性质研究北大核心CSCD

采用新型常压室温等离子体(Atmospheric and room temperature plasma,ARTP)诱变产类胡萝卜素菌株Deinococcus wulumuqiensis R12,通过深孔培养板进行高通量筛选,并结合菌株类胡萝卜素含量复筛获得突变菌株M1。与出发菌株相比,突变菌株M1的类胡萝卜素含量检测为612μg/g细胞干重(Dry cell weight,DCW),是出发菌株类胡萝卜素含量(212μg/g DCW)的2.8倍,且遗传性稳定;突变菌株M1类胡萝卜素的高铁离子还原能力(OD700=0.52)高于出发菌株类胡萝卜素的高铁离子还原能力(OD700=0.34),且M1的类胡萝卜素对DPPH自由基清除率为33.33%高于出发菌株类胡萝卜素的清除率(23.09%),结果显示突变菌株M1具有更强的抗氧化能力;在相同γ辐射与UV辐射剂量下突变菌株M1具有比出发菌株更高的存活率。研究表明突变菌株M1在类胡萝卜素产量、菌株抗氧化性能及抗辐射性能方面均优于出发菌株D.wulumuqiensis R12。     

常压室温等离子体诱变选育类胡萝卜素高产菌株及其性质研究

采用新型常压室温等离子体(Atmospheric and room temperature plasma,ARTP)诱变产类胡萝卜素菌株Deinococcus wulumuqiensis R12,通过深孔培养板进行高通量筛选,并结合菌株类胡萝卜素含量复筛获得突变菌株M1。与出发菌株相比,突变菌株M1的类胡萝卜素含量检测为612μg/g细胞干重(Dry cell weight,DCW),是出发菌株类胡萝卜素含量(212μg/g DCW)的2.8倍,且遗传性稳定;突变菌株M1类胡萝卜素的高铁离子还原能力(OD700=0.52)高于出发菌株类胡萝卜素的高铁离子还原能力(OD700=0.34),且M1的类胡萝卜素对DPPH自由基清除率为33.33%高于出发菌株类胡萝卜素的清除率(23.09%),结果显示突变菌株M1具有更强的抗氧化能力;在相同γ辐射与UV辐射剂量下突变菌株M1具有比出发菌株更高的存活率。研究表明突变菌株M1在类胡萝卜素产量、菌株抗氧化性能及抗辐射性能方面均优于出发菌株D.wulumuqiensis R12。     

产氢菌的复合诱变选育及突变株HCM-23的产氢特性

以厌氧产氢细菌Clostridium sp. H-61为原始菌株, 先后经亚硝基胍(NTG)、紫外(UV)诱变, 选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下, 其产氢量为3024 mL/L, 比原始菌株提高了69.89%; 其最大产氢速率为33.19 mmol H2/g DW·h, 比原始菌株(19.74 mmol H2/g DW·h)提高了68.14%。经过多次传代试验, 稳定性良好。其发酵末端产物以乙醇和乙酸为主, 属于典型乙醇型发酵代谢类型。其最适产氢初始pH为6.5, 最适生长温度为36℃, 以蔗糖为最佳碳源。与原始菌株相比, 突变株HCM-23的产氢特性发生了改变, 如生长延滞期延长, 可利用无机氮源等。     

产氢菌的复合诱变选育及突变株HCM-23的产氢特性

以厌氧产氢细菌Clostridium sp.H-61为原始菌株,先后经亚硝基胍(NTG)、紫外(UV)诱变,选育得到1株高产突变株HCM-23.在葡萄糖浓度为10 g/L的条件下,其产氢量为3024 mL/L,比原始菌株提高了69.89%;其最大产氢速率为33.19 mmol H2/g DW·h,比原始菌株(19.74 mmolH2/g DW·h)提高了68.14%.经过多次传代试验,稳定性良好.其发酵末端产物以乙醇和乙酸为主,属于典型乙醇型发酵代谢类型.其最适产氢初始pH为6.5,最适生长温度为36℃,以蔗糖为最佳碳源.与原始菌株相比,突变株HCM-23的产氢特性发生了改变,如生长延滞期延长,可利用无机氮源等.     

超高静压在琥珀酸生产菌株选育中的应用

采用超高静压对一株产琥珀酸放线杆菌A3进行诱变育种, 进一步提高其生产性能。考察了压力、变压速度、生长期对菌株致死率的影响。在压力为200 MPa, 50 MPa/min变压速度的诱变条件下对稳定期菌株进行诱变, 筛选到一株突变菌株产琥珀酸放线杆菌B19, 琥珀酸产量达到 32.2 g/L, 比出发菌株A3提高了17.9%, 代谢副产物乙酸的产量降低了11.5%, 经过6次传代表明突变菌株具有稳定的遗传特性。     

复合诱变选育高产GSH的菌株

目的:选育出GSH的高产菌株.方法:以产GSH的产朊假丝酵母(Candida utilis)为出发菌株,利用紫外-超声波复合诱变.结果:筛选得到ZnCl2抗性突变株UU3,GSH菌体含量为53.50mg·g-1,比出发菌株提高137.03%.结论:突变株UU3遗传性能稳定,可做进一步研究.     

超高静压在琥珀酸生产菌株选育中的应用

采用超高静压对一株产琥珀酸放线杆菌A3进行诱变育种,进一步提高其生产性能.考察了压力、变压速度、生长期对菌株致死率的影响.在压力为200 MPa,50 MPa/min变压速度的诱变条件下对稳定期菌株进行诱变,筛选到一株突变菌株产琥珀酸放线杆菌B19,琥珀酸产量达到32.2 g/L,比出发菌株A3提高了17.9%,代谢副产物乙酸的产量降低了11.5%,经过6次传代表明突变菌株具有稳定的遗传特性.     

莱氏野村菌紫外线诱变抗敌敌畏菌株的生理特性

【背景】夜蛾科害虫易对化学杀虫剂产生高抗性,但一些化学农药可以对部分虫生真菌的毒力作用效果起增幅作用,目前缺乏对莱氏野村菌(Nomuraea rileyi)的该方面研究。【目的】探究对常用有机磷杀虫剂敌敌畏具有较强耐药性的紫外线诱变莱氏野村菌突变菌株的生理特性,包括菌丝生长、产孢情况和产几丁质酶活性。【方法】在紫外线诱变莱氏野村菌结合药剂驯化筛选出的突变株Nr-UVY1和Nr-UVY6基础上,以添加不同含量敌敌畏的平板培养基对突变株进行菌丝生长抑制率、继代产孢量、几丁质酶活性测定。【结果】在含1291mg/L敌敌畏的培养基上,敌敌畏对出发菌株菌丝生长抑制率达到100%,突变株Nr-UVY1(42.38%)和Nr-UVY6(37.01%)均远低于出发菌株。处理浓度大于1291 mg/L时,2株突变株的菌丝生长抑制率曲线平稳上升且远低于出发菌株,说明突变株对敌敌畏的抗药性显著且稳定。在各浓度敌敌畏含量的培养基上,突变株Nr-UVY6得到的菌丝抑制率均小于Nr-UVY1,说明突变株Nr-UVY6对敌敌畏的抗药性更强。在同样的培养条件下,2株突变株的开始产孢时间略迟于出发菌株。突变株各代的产孢量均明显高于出发菌株,尤其是突变株Nr-UVY6各代的产孢量始终高于出发菌株2倍以上且更稳定。透明圈法测定几丁质酶活性,突变株Nr-UVY6高于Nr-UVY1和出发菌株。【结论】筛选出的经过紫外线诱变的突变菌株对敌敌畏的抗性远高于出发菌株,在生长和繁殖方面表现出更优秀的性状,并且由于其几丁质酶的活性高,可进一步探究其致病性。     

激光诱变青霉PT95原生质体选育类胡萝卜素高产菌株

采用激光对青霉PT95菌株的原生质体进行了诱变处理,选育到一株菌核生物量和类胡萝卜素含量均有显提高的突变菌株L05。与出发菌株相比,L05菌株的菌核生物量提高了98.6%,菌核中的类胡萝卜素含量提高了28.3%,在查氏平板上的类胡萝卜素产率提高了154.0%。所选育的L05菌株经3次传代培养,菌落没有发生扇形变异,菌核生物量和类胡萝卜素含量没有明显改变,说明该菌株具有良好的遗传稳定性。     

Isolation and Characterization of the Thylakoid Membranes from the NaCl-Resistant (NaClr) Mutant Strain of the Cyanobacterium Anabaena variabilis

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaCl^r mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaCl^r mutant strain and its wild-type counterpart respectively at wavelengths of 400–850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaCl^r mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaCl^r mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and β-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaCl^r mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaCl^r mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaCl^r mutant strain also showed morphological variations under NaCl stress. Received: 14 April 2000 / Accepted: 23 May 2000     

Isolation and characterization of pigment mutants from a filamentous cyanobacterium

Five strains of a pigment mutant were isolated following UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis from a non-nitrogen fixing mutant of the cyanobacteriumGloeotrichia ghosei. Two of them (B-1 and V-1) were isolated by UV mutagenesis and other three (B-3, B-7 and Br-6) by MNNG mutagenesis. Among the five strains cultures of three strains (B-1, B-3 and B-7) were typically blue-green in colour. Culture of strain V-1 was found to be violet-pink and of Br-6 was brownish in colour. The parent strain of these mutants was dark-blue in colour. Blue-green mutants showed the predominance of phycocyanin (610 nm) whereas violet-pink and brown strains showed the predominance of phycoerythrin (550 nm) in the absorption spectra of water-soluble pigments. In contrast to these strains their parent strain showed both the absorption peaks (at 550 and 610 nm). Occurrence of stable pigment mutants of a filamentous cyanobacterium indicates that the synthesis of water-soluble pigments is genetically controlled in these mutant strains.     

固氮红细菌(Rhodobacter azotoformans) 423 nm特征吸收峰成因与定位

【背景】在不产氧光合细菌中,因420-425nm特征峰位于类胡萝卜素(Carotenoid,Car)吸收区域,通常被认为是由Car积累引起,但固氮红细菌R7菌株呈现的423 nm特征峰不具备Car三指峰特征。【目的】阐明R7菌株423 nm特征吸收峰形成的物质基础及胞内定位。【方法】采用吸收光谱、薄层层析、高效液相色谱、质谱、超速离心和离子交换层析等方法阐明423 nm吸收峰形成原因。【结果】谷氨酸钠明显促进R7菌株活细胞呈现423 nm特征峰,色素提取液中该峰蓝移至415 nm,但其生长、细菌叶绿素(Bacteriochlorophyll,BChl)和Car含量大幅度降低,而添加酵母提取物则反之。色素组成分析表明,在检测到的色素成分中,只有镁卟啉单甲基酯Ⅸ (Magnesium Protoporphyrin Ⅸ Monomethylester,MPE)呈现415 nm特征吸收峰。MPE可定位于光合膜上并呈现出423 nm特征峰。对色素蛋白复合体(Pigment Protein Complex,PPC)的研究显示,添加谷氨酸和酵母提取物的菌体细胞虽然都检测到3种PPC组分[2个外周捕光复合体(Peripheral Light Harvesting Complex 2,LH2)和1个光反应中心(Reaction Center,RC)],但源自谷氨酸菌体细胞的RC和1个LH2则呈现423 nm特征吸收峰,表明R7菌株可产生2种不同类型的LH2,且MPE可定位于一种LH2和RC。【结论】R7菌株所呈现的423 nm特征峰不是由Car积累所致,而是由MPE积累所形成,且能与LH2和RC结合定位于光合膜上。MPE是BChl合成的中间产物,其合成受严格调控,不容易获得。MPE代谢调控的深入研究可为光合作用光氧化损伤与保护机理增添新内容。     

Laser mutagenesis of protoplasts of Penicillium sp. PT95 for the enhancement of carotenoid yield

Aims: To isolate the protoplasts from Penicillium sp. PT95 and carry out laser mutagenesis to attain high-yield mutant strain for carotenoid production. Methods and Results: The mycelial pellets of PT95 strain were digested with the lytic enzyme for 3 h in order to attain protoplasts. The prepared protoplasts were irradiated using helium neon (He–Ne) laser. Among all regenerated colonies isolated from irradiated protoplasts, five colonies proved to be able to form sclerotia. The five colonies were named as strains L01, L02, L03, L04 and L05, respectively. Whereas, among all regenerated colonies isolated from no-irradiated protoplasts, no colonies were found to form sclerotia. Strains L01, L02, L03, L04 and L05 showed higher carotenoid yield than the original strain in Czapek’s agar medium. Strain L05 gave the highest pigment yield of 381 μg per plate, which was 2·54 times higher than that of original strain. Conclusions: These results suggest that PT95 strain may be mutagenized using laser-irradiation to obtain higher-yield mutant strains for carotenoid production. Significance and Impact of the Study: These data prompted us to consider that several attempts should be made to improve carotenoid production in PT95 by strain selection using classical screening and mutagenesis techniques.     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

Molecular surface characterization of oral streptococci by Fourier transform infrared spectroscopy

In order to characterize the molecular composition of oral streptococci, infrared transmission spectroscopy on freeze-dried cells dissolved in KBr was used. All infrared spectra show similar absorption bands for the strains studied with the most important absorption bands located at 2930 cm-1 (CH), 1653 cm-1 (AmI), 1541 cm-1 (AmII) and two bands at 1236 cm-1 and 1082 cm-1, which were assigned to phosphate and sugar groups. However, calculation of absorption band ratios normalized with respect to the integrated intensity of the CH stretching region around 2930 cm-1, show significant differences between the strains. Both Streptococcus mitis strains possess high AmI/CH and AmII/CH absorption band ratios compared to the other strains. Streptococcus salivarius HBC12, a mutant strain devoid of all proteinaceous surface appendages, shows significantly lower AmI/CH and AmII/CH band ratios with respect to its parent strain S. salivarius HB. Two positive relationships could be established both between the AmII/CH absorption band ratio and the N/C elemental surface concentration ratio of the strains previously, determined from X-ray photoelectron spectroscopy (XPS) and also between AmI/CH and the fraction of carbon atoms at the surface involved in amide bonds, determined by XPS as well. From this comparison, it is concluded that transmission infrared spectroscopy can be employed as a technique to study the molecular surface composition of freeze-dried microorganisms.     

Lipid composition, lipid fluidity and radioresistance of Deinococcus radiodurans and two mutant strains

The lipid composition of D. radiodurans strain R1 and of two mutant strains has been studied in relation to membrane fluidity and sensitivity to X-ray radiation. No significant difference in the unsaturation degree of fatty acids was found between parental and mutant strains. An important decrease of carbohydrate-containing lipids was observed in the radiosensitive mutant strain. We also observed a higher fluidity in both mutant strains than in the parental one. Modification of membrane lipid fluidity by growing the parental strain at 39 degrees C did not lead to modified radioresistance. These results suggest that a particular chemical composition of the membrane leading to a special lipid phase may be an important parameter in controlling radiosensitivity.     

Studies on the role of carotenoid pigments in a chemoheterotrophic bacterium,corynebacterium poinsettiae

Summary The hypothesis that colored carotenoids can protect chemoheterotrophic microorganisms from damage by visible light has been investigated. Corynebacterium poinsettiae, a bacterium that forms the three carotenoid pigments lycoxanthin, cryptoxanthin and spirilloxanthin, was used as test organism. Non-pigmented cells, in which the normal carotenoids were largely replaced by the colorless C₄₀ polyene, phytoene, were obtained by two methods: isolation of a mutant with a block in carotenoid synthesis; and cultivation of the parent strain in the presence of diphenylamine, a specific chemical inhibitor of carotenoid synthesis.Comparative studies of the effects of visible light on dye-sensitized pigmented and non-pigmented cells showed that non-pigmented cells can be rapidly killed by exposures which are without effect on pigmented cells. Both physiological and genetic suppression of pigment synthesis produce photosensitivity. The non-pigmented mutant is killed by ultraviolet light at the same rate as the pigmented parent strain, indicating that the acquired photosensitivity of the former is specific for visible light.Herrn Prof. Dr. A. Rippel zum 70. Geburtstag gewidmet.