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61.
62.
用改良的MTT法测定rhG-CSF活性 总被引:1,自引:1,他引:1
MTT测定法是根据线粒体脱氢酶催化MTT形成蓝色甲■的多少来检测活细胞数和功能状态的,但原始方法中存在着一些问题,如敏感性偏低、有机溶剂产生蛋白质沉淀以及产物的溶解度偏低等。为了摸索测定 rhG-CSF活性的最适条件,我们以 NFS-60细胞为对象,比较了多种溶解缓冲液,并且对细胞数、MTT浓度及保留时间、溶解液用量等条件进行了选择。结果表明,DMF-20%SDS和 20%SDS的效果最好,测定时细胞数为每孔 1000个细胞,所加 MTT浓度为 1mg/ml,保留时间为 4 h,溶解液的用量为每孔 100μl。 相似文献
63.
64.
The effect of Ce3+ on the chlorophyll (chl) of spinach was studied in pot culture experiments. The results showed that Ce3+ could obviously stimulate the growth of spinach and increase its chlorophyll contents and photosynthetic rate. It could also
improve the PSII formation and enhance its electron transport rate of PSII as well. By inductively coupled plasma-mass spectroscopy
and atom absorption spectroscopy methods, it was revealed that the rare-earth-element (REE) distribution pattern in the Ce3+-treated spinach was leaf>root>shoot in Ce3+ contents. The spinach leaves easily absorbed REEs. The Ce3+ contents of chloroplast and chlorophyll of the Ce3+-treated spinach were higher than that of any other rare earth and were much higher than that of the control; it was also
suggested that Ce3+ could enter the chloroplast and bind easily to chlorophyll and might replace magnesium to form Ce-chlorophyll. By ultraviolet-visible,
Fourier transform infrared, and extended X-ray absorption fine structure (EXAFS) methods, Ce3+-coordinated nitrogen of porphyrin rings with eight coordination numbers and average length of the Ce-N bond of 0.251 nm. 相似文献
65.
Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis
Wenqing Chen Dongjing Qu Lipeng Zhai Meifeng Tao Yemin Wang Shuangjun Lin Neil P. J. Price Zixin Deng 《蛋白质与细胞》2010,1(12):1093
Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β -1″,11′-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunA– tunL ). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies. 相似文献
66.
【目的】筛选能抗营养阻遏产漆酶的黄孢原毛平革菌,论证其产漆酶的确定性及抗营养阻遏产木质素酶的可行性,为白腐菌产酶代谢调控、木质素降解机理的研究奠定基础。【方法】利用重复紫外诱变法,以愈创木酚富氮鉴别培养基筛选目标菌株;比较不同营养条件下菌体生长与产酶动力学差异研究产酶营养调控机理;通过热处理、排除锰离子和加入过氧化氢酶等不同措施论证黄孢原平毛平革菌能否产生漆酶。【结果】3种不同方法均证实选育到的pcR5305和pcR5324菌株在限氮与富氮条件下均能产生漆酶,pcR5305和pcR5324在限氮条件下产漆酶分别达到203.5、187.6 U/L;在富氮条件下为220.6、183.9 U/L,而原菌株pc530在两种条件下都基本不产生漆酶。二菌株产漆酶调控方式不同,pcR5305漆酶产生与菌体生长同步,而pcR5324漆酶产生却受营养氮阻遏。二菌株同时具有抗营养阻遏高产木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)(分别为LiP 1343.2、MnP 252.2 U/L;LiP 1169.5、MnP 172.4 U/L)的能力。【结论】筛选到的黄孢原毛平革菌变异菌株能产漆酶,同时表现了抗营养阻遏产漆酶、木质素过氧化物酶和锰过氧化物酶的能力,具有重要的生产应用与理论研究价值,为白腐菌产酶代谢调控机理研究提供了原始菌株并奠定了良好的基础。 相似文献
67.
枯草芽孢杆菌渗透压调节基因proB的克隆和表达 总被引:8,自引:0,他引:8
用PCR扩增的方法从耐盐的枯草杆菌中克隆出一个13kb长的DNA片段,经功能检测,证明正向插入片段与大肠杆菌的脯氨酸营养缺陷特性(proB-)能够营养互补。含有该重组质粒的大肠杆菌DH5α在基本培养基上的耐盐能力从2%提高至4%。通过引物步行法测定了该插入片段的核苷酸序列。利用DNAsis软件进行序列分析发现,该片段第122~1235bp核苷酸编码一个由370个氨基酸组成的蛋白质分子,其上游存在非典型的-10区,典型的-35区和核糖体结合位点,起始密码子处有最佳翻译起始效率的侧翼核苷酸序列。将其与Genebank中的已知基因的序列和编码的氨基酸序列进行同源性比较,结果表明该片段与枯草杆菌168的核苷酸序列、氨基酸序列的同源性分别为81%和90%。证明该基因确实是一个proB基因。通过与三十个不同种属微芽生物proB基因的氨基酸序列比较,发现该蛋白存在有可能与形成酶的活性中心和三维结构有密切关系的几个绝对保守的区域。 相似文献
68.
Meng F Han Y Teng W Li Y Li W 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2011,123(8):1459-1465
Soybean aphid (Aphis glycines Matsumura) results in severe yield loss of soybean in many soybean-growing countries of the world. A few loci have been previously
identified to be associated with the aphid resistance in soybean. However, none of them was via isoflavone-mediated antibiosis
process. The aim of the present study was to conduct genetic analysis of aphid resistance and to identify quantitative trait
loci (QTL) underlying aphid resistance in a Chinese soybean cultivar with high isoflavone content. One hundred and thirty
F5:6 derived recombinant inbred lines from the ‘Zhongdou 27’ × ‘Jiunong 20’ cross were used. Two QTL were directly associated
with resistance to aphid as measured by aphid damage index. qRa_1, close to Satt470 on soybean linkage group (LG) A2 (chromosome
8), was consistently detected for 3- and 4-week ratings and explained a large portion of phenotypic variations ranging from
25 to 35%. qRa_2, close to Satt144 of LG F (chromosome 13), was detected for 3- and 4-week ratings and could explain 7 and
11% of the phenotypic variation, respectively. These two QTL were highly associated with high isoflavone content and both
positive alleles were derived from ‘Zhongdou 27’, a cultivar with higher isoflavone content. The results revealed that higher
individual or total isoflavones contents in soybean lines could protect soybean against aphid attack. These two QTL detected
jointly provide potential for marker-assisted selection to improve the resistance of soybean cultivars to aphid along with
the increase of isoflavone content. 相似文献
69.
Xia M Hou C Demong D Pollack S Pan M Brackley J Singer M Matheis M Cavender D Wachter M 《Bioorganic & medicinal chemistry letters》2008,18(12):3562-3564
The synthesis and biological evaluation of a series of substituted dipiperidine alcohols are described. Structure-activity relationship studies led to the discovery of potent CCR2 antagonists displaying IC(50) values in the nanomolar or subnanomolar range. The cinnamoyl compounds had higher binding affinities than the corresponding urea analogs. 相似文献
70.
ABSTRACT: BACKGROUND: Lipase from Rhizopus chinensis is a versatile biocatalyst for various bioconversions and has been expressed at high-level in Pichia pastoris. However, the use of R. chinensis lipase in industrial applications is restricted by its low thermostability. Directed evolution has been proven to be a powerful and efficient protein engineering tool for improvement of biocatalysts. The present work describes improvement of the thermostability of R. chinensis lipase by directed evolution using P. pastoris as the host. RESULTS: An efficient, fast and highly simplified method was developed to create a mutant gene library in P. pastoris based on in vivo recombination, whose recombination efficiency could reach 2.3 x 105 /mug DNA. The thermostability of r27RCL was improved significantly by two rounds of error-prone PCR and two rounds of DNA shuffling in P. pastoris. The S4-3 variant was found to be the most thermostable lipase, under the conditions tested. Compared with the parent, the optimum temperature of S4-3 was two degrees higher, Tm was 22 degrees higher and half-lives at 60degreesC and 65degreesC were 46- and 23- times longer. Moreover, the catalytic efficiency kcat/Km of S4-3 was comparable to the parent. Stabilizing mutations probably increased thermostability by increasing the hydrophilicity and polarity of the protein surface and creating hydrophobic contacts inside the protein. CONCLUSIONS: P. pastoris was shown to be a valuable cell factory to improve thermostability of enzymes by directed evolution and it also could be used for improving other properties of enzymes. In this study, by using P. pastoris as a host to build mutant pool, we succeeded in obtaining a thermostable variant S4-3 without compromising enzyme activity and making it a highly promising candidate for future applications at high temperatures. 相似文献