用原生质体法诱变选育林可霉素高产菌株

去壁后的微生物原生质体外层仅存原生质体膜,在诱变过程中,直接与诱变剂接触,诱变剂迅速内溶并与核作用.弥补了常规诱变育种表型延迟和严重的遗传分离现象的缺点,我们以林可霉素生产菌G4为出发菌株,首先制备原生质体,再通过对原生质体进行紫外诱变,经过平皿再生,再经过摇瓶初筛、复筛,得到一株生产性能比出发菌株提高3.2%的突变株Y19.     

美伐他汀产生菌的原生质体诱变育种

以美伐他汀产生菌SIPI-8915为出发菌,研究了影响原生质体形成和再生的各种因素并选出最佳条件,其再生频率达到23.5%;并对原生质体进行紫外诱变育种,筛选到变异株.该菌株摇瓶发酵效价比对照提高30%,传代稳定好.     

赤霉素产生菌原生质体理化诱变-定向筛选模型的建立与应用

针对赤霉素工业生产菌株皆为不产生孢子的多核多细胞菌丝型,通过酶解手段溶去赤霉素菌丝体的细胞壁,使其成为单一游离的原生质体,在此基础上建立赤霉素产生菌原生质体抗药性致死突变标志诱变筛选模型,以解除高浓度反应底物及内源赤霉素对其产生菌生物合成的反馈抑制和阻遏,提高赤霉素生物合成酶的活力,从而获得赤霉素高产菌株。应用所建立的赤霉素诱变筛选模型,进行产生菌原生质体理化诱变-定向筛选,成功得到N7298等多株原生质体再生的抗药性突变高产菌株,其摇瓶效价比出发菌株N提高23.4%,在20m3罐生产水平提高24.1%。     

原生质体复合诱变选育刺芹侧耳木质素降解酶高产菌株

通过对刺芹侧耳(Pleurotus eryngiiGIM5.280)原生质体开展复合诱变及传代培养,以期得到遗传稳定的木质素降解酶高产菌株。结果表明,通过25s紫外诱变刺芹侧耳原生质体,正突变率达16%。对紫外诱变正突变株进行60Coγ二次复合诱变再生,获得五株正突变突出菌株。经过摇瓶发酵实验,发现这五株菌株产木质素降解酶能力较出发菌株明显提高。同时开展高产菌株的传代培养,检验其传代稳定性。连续传代四代测试结果表明,007号和167号菌株遗传的稳定性表现突出,发酵液中木质素降解酶产量稳定。尤其是007号菌株产酶可达到110U·mL-1,比出发菌株的酶活表达量提高54.3%。复合诱变能明显提高刺芹侧耳产木质素降解酶能力。     

抗药性基因突变筛选高产菌株

本文通过洁霉素对盐酸林可霉素产生菌 98-1菌株孢子的致死浓度测定 ,采用诱变剂EMS的四种不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含洁霉素 ( 2 0 0 0 0ug/ml)的高氏平板上 ,获得了大量的洁霉素抗性基因突变株 ,然后从 90 0株洁霉素抗性基因突变株中通过初筛获得高于诱变出发菌株产素能力的菌株 5 6株 ,再进一步通过摇瓶复筛 ,并结合菌丝生长及摇瓶代谢情况 ,获得优于出发菌株的诱变菌株 4株。将这 4个菌株连同出发菌株连续三批次进行摇瓶发酵 ,结果 4个突变株的产素能力 (产量 )及摇瓶代谢和菌丝生长情况均优于出发菌株 ,试验筛选出最优菌株 0 2 -0 3 -40 2。将 0 2 -0 3 -40 2进行罐上发酵生产试验 ,结果试验罐比对照罐平均效价提高 1 7.5 6% ,平均产量提高 1 9.3 4%。本文建立了林可霉素高产菌株的洁霉素抗性基因突变诱变快速高效的筛选方法     

微波诱变选育纤维素酶高产木霉

利用微波诱变的方法,对一株产纤维素酶的木霉进行了改造,提高了其对纤维素的降解能力。通过微波诱变筛选出5株纤维素酶高产菌株,其在摇瓶试验中的平均纤维素酶活力为野生型菌株的1.35倍,其中酶活力最高的菌株MW6,其纤维素酶产量为野生型菌株的1.4倍。固体发酵试验结果显示野生型菌株及诱变后的突变菌株在固体发酵中的酶活力均略低于摇瓶培养的酶活力,酶活力提高最快的菌株MW6,其固体培养的酶活力与液体摇瓶培养的差距最小。     

亚硝基胍-紫外复合诱变选育高产衣康酸菌株

为提高土曲霉KYK-031发酵生产衣康酸能力,对出发菌株开展诱变育种和高产能力选育研究。采用亚硝基胍（NTG）和紫外（UV）复合诱变方法,通过平板初筛和摇瓶复筛,筛选衣康酸高产突变株。结果表明,亚硝基胍在500μg/mL浓度下的最佳诱变时间为40 min,15 W紫外诱变的最佳诱变时间为2 min,亚硝基胍-紫外复合诱变后筛选得到一株衣康酸高产菌株KYK-1035,摇瓶发酵产量为73.7 g/L,比出发菌株（41.8 g/L）提高了76.3%,糖酸转化率为63.4%。采用亚硝基胍-紫外复合诱变,提高了出发菌株诱变的正突变率,为衣康酸发酵继续放大和工业化生产奠定了基础。     

应用原生质体诱变技术筛选井冈霉素高产菌种的研究

诱变育种是抗生素菌种选育中最常规而又最有效的方法，对微生物原生质体进行诱变育种是目前人们为获取高产菌种和保持高产菌种特性的一种行之有效的方法。本文研究井冈霉素产生菌原生质体制备及再生条件，并通过对原生质体进行紫外线诱变处理，筛选得到井冈霉素高产菌种PU-922，摇瓶发酵水平可达到每毫升26 586单位（以A组份计，补足蒸发量后数据，下同），比出发菌株提高34.5%。 1 材料与方法 1.1 菌株 井冈霉素产生菌UV-199，浙江钱江生物化学股份有限公司菌种室提供。 1.2 培养基及试剂 1.2.1 斜面及培养基 葡萄糖1.0%，L-天冬…     

N^＋离子注入选育纳他霉素高产菌株

以褐黄孢链霉菌GX-33为出发菌株,利用N^＋注入诱变,琼脂块初筛,结合了摇瓶复筛,获得了一株摇瓶效价能够稳定在3597-3625μg／mL水平的纳他霉素生产菌株,为后续生产提供了较优良的生产菌种。N^＋离子注入诱变纳他霉素产生菌在5×1015-1×1016N^＋／cm^2剂量这一区域内,存活率比较高,为理想的诱变剂量区域。     

He-Ne激光与紫外线复合诱变选育西罗莫司高产菌株

以吸水链霉菌为出发菌株,采用He-Ne激光-紫外复合诱变对其单孢子悬液进行诱变筛选高单位突变株,经复筛选育出具有较好遗传稳定性的高产突变株11-1-6#,摇瓶效价达到401.9u/mL,比出发菌株提高了79%。     

金属闪光涂料用水性丙烯酸树脂的合成及性能

采用分光光度计及其他涂膜性能测定方法研究了甲基丙烯酸乙酰乙酰氧基乙酯(AAEM)的用量对所合成的水性丙烯酸树脂的水溶性、粘度及其金属闪光涂料漆膜闪光指数等的影响。结果表明,随着AAEM单体用量提高,丙烯酸树脂的水溶性提高,粘度降低。当AAEM的质量分数为4%,其金属闪光涂料的固化漆膜具有高金属闪光指数、高抗冲、高硬度、良好的耐水、耐盐雾及耐老化等性能。     

高耐性水性环氧富锌底漆的制备与研究

为了制备高耐性的水性环氧富锌底漆,本文研究了环氧乳液粒径及其分布,活泼氢和环氧基的物质的量比对水性环氧富锌底漆的耐性尤其是耐盐雾、初期耐水性和耐闪锈性能的影响。实验结果表明,当环氧乳液粒径 ≤500 nm,且粒径分布均一,同时活泼氢和环氧基的物质的量比在 0. 8左右时,所得水性环氧富锌底漆的耐盐雾、初期耐水和耐闪锈性能达到最佳,其中耐中性盐雾可达 3 000 h,初期耐水性大于 480 h。此外,本文还研究了分散体系和防闪锈剂对耐盐雾、耐水和耐闪锈性能的影响,结果表明,选择高分子分散剂和与体系相容性好的防闪锈剂时,上述性能达到最佳。     

恩拉霉素高产菌株的筛选

从原始生产菌BKSFJ06出发,对其进行了紫外诱变、氯化锂与紫外复合诱变、亚硝基胍诱变和亚硝基胍与紫外的复合诱变及恩拉霉素抗性筛选工作,用HPLC检测方法,以恩拉霉素的产量为指标,筛选高产菌株。最终筛选出的最优突变菌株ERS42-101108产量提高了50%,且该菌株具有良好的遗传稳定性。通过实验内容可以看出,通过亚硝基胍和紫外复合诱变的方法,抗自身产物定向筛选出的突变株具有较优良的性质。     

L-phenylalanine production by auxotrophic regulatory mutants ofEscherichia coli—L-phenylalanine production by mutants ofE. coli

Various regulatory mutants ofEscherichia coli have been isolated using phenylalanine and tyrosine analogues. It has been found that the growth of wild type strain ofE. coli W3110 was strongly inhibited by phenylalanine analogues. Regulatory mutants resistant to phenylalanine analogue could accumulate b-phenylalanine at concentrations of 5–6 g/l. However, L-phenylalanine accumulation was increased significantly up to 11.4 g/1 using a tyrosine auxotrophic mutant resistant to phenylalanine analogue such as β-2-thienyl-DL-alanine.     

Allosteric p97 Inhibitors Can Overcome Resistance to ATP-Competitive p97 Inhibitors for Potential Anticancer Therapy

Abstract : A major challenge of targeted cancer therapy is the selection for drug-resistant mutations in tumor cells leading to loss of treatment effectiveness. p97/VCP is central regulator of protein homeostasis and a promising anticancer target because of its vital role in cell growth and survival. One ATP-competitive p97 inhibitor, CB-5083, has entered clinical trials. Selective pressure on HCT116 cells dosed with CB-5083 identified five different resistant mutants. Identification of p97 inhibitors with different mechanisms of action would offer the potential to overcome this class of resistance mutations. Our results demonstrate that two CB-5083 resistant p97 mutants, N660 K and T688 A, were also resistant to several other ATP-competitive p97 inhibitors, whereas inhibition by two allosteric p97 inhibitors NMS-873 and UPCDC-30245 were unaffected by these mutations. We also established a CB-5083 resistant cell line that harbors a new p97 double mutation (D649 A/T688 A). While CB-5083, NMS-873, and UPCDC-30245 all effectively inhibited proliferation of the parental HCT116 cell line, NMS-873 and UPCDC-30245 were 30-fold more potent in inhibiting the CB-5083 resistant D649 A/T688 A double mutant than CB-5083. Our results suggest that allosteric p97 inhibitors are promising alternatives when resistance to ATP-competitive p97 inhibitors arises during anticancer treatment.     

Isolation of directed evolution mutants of chloroperoxidase resistant to suicide inactivation by primary olefins

Wild-type chloroperoxidase catalyzes the efficient chiral epoxidation of secondary olefins but is rapidly inactivated in a mechanism-based suicide reaction when incubated with hydrogen peroxide and primary olefins. Directed evolution mutants of chloroperoxidase have now been isolated that are resistant to suicidal inactivation. Plasmid vectors containing error-prone copies of the chloroperoxidase gene and a hygromycin B resistance marker gene have been used to transform Caldariomyces fumago spheroplasts and produce mutant libraries. The mutant library clones were screened for their ability to resist mechanism-based inactivation by allylbenzene. Four generations of PCR-based random mutagenesis and screening yielded mutants that were completely resistant to the suicide-inactivation reaction. Rather surprisingly, the fourth generation mutant developed enhanced epoxidation activity in addition to resistance to allylbenzene inactivation. These initial results suggest that the directed evolution technique can be used to produce chloroperoxidase variants that can further exploit the potential of chloroperoxidase for the synthesis of chiral intermediates.     

Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the K(m) values for NAD (nicotinamide adenine dinucleotide) and/or the k(cat) values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.     

Jakob数在高温高压闪蒸过程中的作用及影响

基于新型高温高压喷雾闪蒸实验台进行变工况实验,选取量纲1的Jakob数（Ja）,即相变过程可用显热与蒸发潜热之比作为闪蒸过程的特征数,研究不同实验条件下闪蒸效率随Ja的变化规律。发现Ja越大,闪蒸效率越高,闪蒸越剧烈。闪蒸效率随液体初始温度的提高而增大,随闪蒸压力的提高而减小,这两种现象均与Ja的物理意义密切相关。闪蒸效率和Ja均是过热度的增函数。在过热度相同的前提下,较高的初始温度和闪蒸压力的组合更有利于闪蒸。综合实验数据提出闪蒸效率与Ja之间的经验公式。实验结果在高温高压闪蒸领域有较强通用性,可为其在工业上的应用提供参考。     

Effect of processing conditions on flash generation

Reduction of flash during injection molding is of great concern for manufacturers of electrical parts. In a previous report, we proposed a theoretical model for flash generation. The present study investigates the effect of processing conditions on flash generation and then attempts to analyze the flash data in terms of the flash model. Specifically, we carry out flow analyses to predict the temperature and pressure of materials when the flash generates. With the thus‐computed parameters, and material properties such as melt viscosity and crystallization temperature, a flash parameter defined in the model is calculated. In the end, it is shown that the flash parameter is correlated with the length of flash generated under various processing conditions. POLYM. ENG. SCI. 45:238–247, 2005. © 2005 Society of Plastics Engineers     

低闪点原油作发电机燃料油方案研究

低闪点的原油作主机燃料油存在重大的安全隐患,会影响主机的正常运行。为了避免低闪点原油带来的风险,需提高原油的闪点,目前提高闪点的方法主要有闪蒸法和蒸馏法,其中闪蒸法有能耗小、安全性高、处理效率高等优点。本文以南海某油田油品进行分析,经厂家调研及模拟分析得出运用闪蒸法能显著提高原油闪点,达到了主机燃料油对闪点的要求,解决了燃油外购的弊端,对类似油田的开发具有重大的借鉴意义。