脱落酸高产菌的激光诱变筛选

对脱落酸产生菌分别进行了紫外诱变和紫外 -激光复合诱变筛选 ,结果表明紫外 -激光复合诱变正变率较高 ,正变幅度较大。从紫外 -激光复合诱变得到的突变株中筛选到一株遗传性状稳定的高产菌株 ,将其传代 5代 ,各子代发酵脱落酸的平均产量均在 2 .1mg·g-1 以上 ,比原始出发菌株产量提高了 32 %。     

解脂耶氏酵母诱变育种及发酵产α-酮戊二酸条件优化

对具有发酵产α-酮戊二酸能力的解脂耶氏酵母(Yarrowia Lipolytica)ZY-4进行了紫外诱变和NTG诱变育种,筛选得到产量提高的突变株,并对突变株的发酵培养基进行了优化,结果表明,紫外诱变和NTG诱变后筛选到的突变株分别比原始出发菌株产量提高了67.8%和110%。优化后发酵培养基成分为甘油8%,氯化铵5.0 g/L,硫胺素1.0μg/L,磷酸二氢钾1.0 g/L,七水硫酸镁0.5 g/L,培养基优化后α-酮戊二酸产量比原始出发菌株提高了232.4%。     

胞苷生产菌的选育

目的：筛选得到胞苷发酵单位较高的菌株,并对发酵过程作初步研究。方法：以胞苷脱氨酶缺失枯草芽孢杆菌DOS7为出发菌株,对其进行紫外诱变、5-氟胞苷（5FCR＋）和2-杂氮尿嘧啶抗性（2AU＋）抗性筛选。结果：通过紫外诱变和抗性筛选得到突变株DOS7-2-1000-15,抗5-氟胞苷和2-杂氮尿嘧啶的临界浓度分别为800mg/L和1 000mg/L。同时检测了抗5-氟胞苷突变株中CTP合成酶的活性,比原始菌株提高了12.4%,突变株DOS7-2-1000-15发酵过程结果为：36℃发酵72h能积累胞苷最高为3.5g/L。结论：筛选得到的突变株DOS7-2-1000-15的遗传稳定性较好,可稳定发酵。     

纳豆芽孢杆菌产VK2菌株的NTG与N＋注入复合诱变选育

以纳豆芽孢杆菌BN-2-6为出发菌株,利用亚硝基胍（NTG）和N＋注入复合诱变选育产维生素K2的突变株。经过NTG诱变后得到突变株BN-N30—1,其维生素K2的产量提高了53％,继而采用低能N＋注入技术进行处理得到突变株BN-P15—11-1,维生素K2的产量比BN—N30—1提高了96％,比原始菌株提高了166％。结果表明,对纳豆芽孢杆菌BN-2-6进行NTG和低能N＋注入复合诱变的效果明显,突变菌株维生素K2的产量显著提高。     

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.     

纤维素酶高产菌株的复合交替诱变选育

以里氏木霉ZM-4为出发菌株,研究了紫外诱变、硫酸二乙酯诱变以及紫外与硫酸二乙酯复合交替诱变等不同诱变方法对其产纤维素酶能力的影响,力求得到高产纤维素酶突变株.结果表明,复合交替诱变的正突变率最高,达45.98%.其中,突变株ZM4-F3具有最高的产酶能力,其滤纸酶活值达11.71U,比出发菌株ZM-4提高了19.75%.对ZM4-F3产纤维素酶酶系进行了详细分析,发现其葡聚糖内切酶、葡聚糖外切酶及β-葡萄糖苷酶酶活均较出发菌株ZM-4有显著提高,其中以β-葡萄糖苷酶酶活增幅最大,达58.3%.利用ZM4-F3降解稻草96h,还原糖产量达2.231g/L,比出发菌株提高了21.7%;利用ZM4-F3降解稻草144h,纤维素分解率和稻草分解率分别达53.01%和68.32%,比出发菌株分别提高了20.2%和14.0%.在对ZM4-F3进行6代连续培养后,仍能保持较高及较稳定的产酶能力,可以应用于工业生产.     

抗凝活性蛋白产生菌Termitomyces albuminosus的诱变育种

真菌Termitomyces albuminosusTA-SD能产生具有抗凝活性的蛋白质TA-P。本实验采用紫外诱变法对TA-SD菌株进行诱变育种,筛选高活性的突变株。出发菌株TA-SD紫外照射40s时,正突变率和抗凝活性提高的幅度最大,分别为33.3%和104.7%。N型突变株和放线菌酮抗性突变株的正突变率和活性提高的幅度均大于S型突变株和非抗性突变株。经筛选突变株SD-A-8的抗凝活性比出发菌株提高了2.3倍。该突变株传代稳定。     

一株感染家蚕的高致病性白僵菌的筛选及复合诱变

【背景】白僵蚕中的生物活性物质在医疗、保健品及化妆品行业有着广泛的应用。目前,许多人工养殖僵蚕基地在实际生产中使用的菌种多为未进行纯化优选的自然感病死亡僵蚕孢子粉且无固定的施用浓度,使得蚕的僵化死亡率难以保证。提高白僵菌菌株的致病力并筛选性状优良的高毒力菌株是工厂化生产白僵蚕研发的重要方向。【目的】利用紫外-微波复合诱变技术筛选高毒力菌株,为僵蚕工厂化生产提供优良菌株。【方法】利用孢子稀释法从山西省养殖农户中自然感染白僵菌的家蚕中分离获得一株原始白僵菌,运用紫外-微波复合的方式对该菌株进行诱变,并比较诱变前后菌株的产孢量及对家蚕的致病力。【结果】分离得到的原始菌株经鉴定为球孢白僵菌(Beauveria bassiana),命名为Beauveria bassiana Bb1003。通过对致死率和正突变率的考察,确定紫外-微波复合诱变的最佳诱变条件为紫外(功率为15 W)照射30 min,微波(功率为800 W、额定微波频率2 450 MHz)辐照60 s。筛选后得到6株复合诱变菌株(UMCM1、UMCM2、UMCM3、UMCM4、UMCM5和UMCM6)。菌株UMCM2对家蚕的僵化率高达...     

抗凝活性蛋白产生菌Termitomyces albuminosus的诱变育种

真菌Termitomyces albuminosus TA-SD能产生具有抗凝活性的蛋白质TA-P.本实验采用紫外诱变法对TA-SD菌株进行诱变育种,筛选高活性的突变株.出发菌株TA-SD紫外照射40s时,正突变率和抗凝活性提高的幅度最大,分别为33.3%和104.7%.N型突变株和放线菌酮抗性突变株的正突变率和活性提高的幅度均大于S型突变株和非抗性突变株.经筛选突变株SD-A-8的抗凝活性比出发菌株提高了2.3倍.该突变株传代稳定.     

不同来源溶藻菌的分离、鉴定及溶藻效果比较

为了探究从何种类型的自然生境中更易分离得到溶藻微生物,采用高氏1号培养基分别从水库底泥、湖泊底泥、农田土壤、林地土壤等四种来源共36份样品中分离了7 600株菌,并最终从中筛选得到了5株溶铜绿微囊藻(Microcystis aeruginosa)的溶藻菌,其中4株为假单胞菌(Pseudomonas sp.),1株为黄杆菌(Flavobacterium sp.),5株菌溶藻效率的变化范围为62%~95%。结果表明,当采用高氏1号培养基作为分离培养基时,湖泊底泥和水库底泥中的成功筛选概率最高,农田土壤次之,而林地土壤中则难以筛选得到,假单胞菌是较容易筛选得到的溶藻菌。     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

The isolation and genetic analysis of sporulation-deficient mutants in Saccharomyces cerevisiae

Sporulation-deficient mutants were isolated from a homothallic strain of Saccharomyces cerevisiae. Sporulation was induced in these mutants by procedures to sporulate the products of protoplast fusion between mutants and wild-type strains. Spores formed in this way were crossed to wild-type strains in order to analyze them genetically. Twenty-three genes essential to sporulation were identified by tetrad analysis and complementation tests. Gene symbols spoT1 to spoT23 were tentatively assigned to them. These mutants fell into four classes by examination of premeiotic DNA synthesis and meiotic nuclear division: (i) Premeiotic DNA synthesis did not occur (spoT1 - spoT11); (ii) premeiotic DNA synthesis occurred but meiosis I did not occur (spoT12 - spoT15); (iii) meiosis II did not occur (spoT16 - spoT18); (iv) meiosis II occurred but mature spores were not formed (spoT19 - spoT23). Genes spoT4, spoT8, spoT20, and spoT23 were mapped on chromosomes IV, II, XVI and XI, respectively. SpoT18-1 was a UAG nonsense mutation.     

一株溶藻细菌NP23的初步分离鉴别及其溶藻作用研究

从水体中分离得到一株具有溶藻能力的细菌,命名为NP23。经形态特征、生理生化鉴定和16S rDNA序列分析表明,该菌株属于肠杆菌属(Enterobacter)。研究了该菌株对湖泊中优势藻的溶藻效果,初步探讨了其溶藻方式及溶藻物质。结果表明,该菌株对小球藻、惠氏微囊藻、栅藻和蛋白核小球藻具有一定的去除效果,叶绿素a的去除率分别为64.1%、53.1%、87.2%和84.4%,而且在10-108CFU/mL菌浓度范围内,藻的去除率与菌液的浓度成正相关;该菌株对小球藻、栅藻和蛋白核小球藻是间接溶藻,对惠氏微囊藻是直接溶藻;该菌株对栅藻的溶藻物质是蛋白类物质,对蛋白核小球藻的溶藻因子是菌体胞外分泌的具有热稳定性的非蛋白类物质。     

Removal and biodegradation of nonylphenol by immobilized Chlorella vulgaris

The removal and biodegradation of nonylphenol (NP) by alginate-immobilized cells of Chlorella vulgaris were compared with their respective free cultures. The effects of four cell densities of 10(4) per algal bead were investigated, as were the four algal bead concentrations, with regard to the removal and biodegradation of NP. Although immobilization significantly decreased the growth rate and NP's biodegradation efficiency of C. vulgaris, NP removal over a short period was enhanced. The NP removal mechanism by immobilized cells was similar to that by free cells, including adsorption onto alginate matrix and algal cells, absorption within cells and cellular biodegradation. The optimal cell density and bead concentration for the removal and biodegradation of NP was 50-100×10(4) cells algal bead(-1) and 2-4 beads ml(-1) of wastewater, respectively. These results demonstrated that immobilized C. vulgaris cells under optimal biomass and photoautotrophic conditions are effective in removing NP from contaminated water.     

溶藻细菌筛选及溶藻活性物质对铜绿微囊藻生理活性的影响

从太湖水华水体中分离纯化细菌, 再将细菌的LB液体和固体斜面纯培养物分别收集后感染铜绿微囊藻(Microcystis aeruginosa)细胞, 从中筛选出7株具有溶藻活性的细菌, 并对其中一株溶藻细菌THW7的溶藻方式及溶藻活性物质对铜绿微囊藻生理活性的影响进行了初步研究。结果表明: 仅采用细菌的LB液体纯培养物进行溶藻细菌筛选会存在误筛或高估溶藻效率的风险, 而采用细菌的固体斜面纯培养物进行筛选则可避免以上风险; 溶藻细菌THW7通过分泌胞外活性物质的方式间接溶藻; 在THW7无菌滤液胁迫下, 铜绿微囊藻的生长受到显著抑制(P<0.01), 10d溶藻效率可达94.38%, 光合系统活性也显著降低(P<0.01), MDA含量积累, SOD、POD、CAT活性整体呈现先升高后降低的趋势且显著高于对照组(P<0.01)。推测菌株THW7分泌的溶藻活性物质可能作用于铜绿微囊藻细胞的光合系统Ⅱ, 阻碍电子传递, 抑制其光合作用过程, 并对藻细胞产生氧化损伤, 破坏藻细胞细胞膜的完整性, 从而实现溶藻作用。     

Genetic control of excision of Saccharomyces cerevisiae interstrand DNA cross-links induced by psoralen plus near-UV light.

Excision of interstrand DNA cross-links induced by 4,5',8-trimethyl psoralen plus 360-nm light was examined in wild type (RAD+) and various radiation-sensitive (rad) mutants of Saccharomyces cerevisiae known to be defective in the excision of UV light-induced pyrimidine dimers. Alkaline sucrose sedimentation of DNA after incubation of psoralen-plus-light-treated cells indicated little or no nicking of cross-linked DNA in rad1-2, rad2-5, rad3-2, rad4-4, rad10-2, and mms19-1 mutants. In the rad14-2 mutant, substantial nicking was observed but to a much lesser extent than in the RAD+ strains, whereas the rad16-1 mutant was as proficient in nicking as the RAD+ strain. Removal of cross-links was also examined in RAD+, rad3-2, and rad14-2 strains by determining the sensitivity of alkali-denatured and -neutralized DNA to hydrolysis by S1 nuclease. No cross-link removal was observed in the rad3-2 mutants, and the rad14-2 mutant was much less efficient than the RAD+ strain in removing cross-links.     

Reparation after the action of 8-methoxypsoralen and light (lambda=365 nm) on radiosensitive mutants of Saccharomyces cerevisiae yeasts]

The method of repeated irradiation allowed to study kinetics of excision of mono-adducts induced by 8-methoxypsoralen (8-MOP) plus light (lambda=365 nm) in DNA of UV-sensitive mutants rad4 and rad15 and X-ray sensitive mutants rad54, xrs2, xrs4. The survival of the mutant rad4 was not practically increased after incubation in complete liquid medium for 3 hours at 28 degrees C before the repeated irradiation. These data suggest that the mutant rad4 is characterized by nearly complete absence of the mono-adduct excision. The survival of mutants rad15 and rad54 in the same environment was increased less effectively than the survival of the control radioresistant strain, but the mutants xrs2 and xrs4 did not differ from the control strain. Possible causes of differences in survival between radiosensitive strains are discussed. The increased sensitivity of the excision defective strain (rad4) and of the postreplicative recombination defective strains (xrs2, xrs4, rad54) to the lethal effect of 8-MOP plus light (lambda=365 nm) suggests that two systems of reparation take part in the removal of photoproducts induced by 8-MOP in DNA of yeast cells.     

Bacteriophage T4 bypass31 mutations that make gene 31 nonessential for bacteriophage T4 replication: mapping bypass31 mutations by UV rescue experiments.

The product of gene 31 is normally required for assembly of the T4 capsid. Two mutations that each bypass that requirement are shown to be located at separate sites in gene 23, which encodes the major structural protein of the capsid. A second phenotypic effect that characterizes both bypass31 mutant strains is the ability to multiply in host-defective strains, such as hdB3-1 and groEL mutants, on which wild-type T4 is unable to assemble capsids. The genetic data indicate that both phenotypic effects are due to the bypass31 mutation. Elimination of the requirement for both the phage protein, gp31, and the host protein, GroEL, by either of two single mutations in gene 23 indicates that GroEL and gp31 are normally needed to interact with gp23 in capsid assembly of wild-type T4.     

Protein engineering of the 4-methyl-5-nitrocatechol monooxygenase from Burkholderia sp. strain DNT for enhanced degradation of nitroaromatics

4-Methyl-5-nitrocatechol (4M5NC) monooxygenase (DntB) from Burkholderia sp. strain DNT catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group. DntB is a flavoprotein that has a very narrow substrate range. Here, error-prone PCR was used to create variant DntB M22L/L380I, which accepts the two new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP). At 300 microM of 4NP, the initial rate of the variant expressing M22L/L380I enzyme (39 +/- 6 nmol/min/mg protein) was 10-fold higher than that of the wild-type enzyme (4 +/- 2 nmol/min/mg protein). The values of kcat/Km of the purified wild-type DntB enzyme and purified variant M22L/L380I were 40 and 450 (s(-1) M(-1)), respectively, which corroborates that the variant M22L/L380I enzyme has 11-fold-higher efficiency than the wild-type enzyme for 4NP degradation. In addition, the variant M22L/L380I enzyme has fourfold-higher activity toward 3M4NP; at 300 microM, the initial nitrite release rate of M22L/L380I enzyme was 17 +/- 4 nmol/min/mg protein, while that of the wild-type enzyme was 4.4 +/- 0.7 nmol/min/mg protein. Saturation mutagenesis was also used to further investigate the role of the individual amino acid residues at positions M22, L380, and M22/L380 simultaneously. Mutagenesis at the individual positions M22L and L380I did not show appreciable enhancement in 4NP activity, which suggested that these two sites should be mutated together; simultaneous saturation mutagenesis led to the identification of the variant M22S/L380V, with 20% enhanced degradation of 4NP compared to the variant M22L/L380I. This is the first report of protein engineering for nitrite removal by a flavoprotein.