Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

重组大肠杆菌发酵生产核酸疫苗的初步研究

初步摸索大肠杆菌DH 5α生产核酸疫苗的发酵条件。通过均匀试验设计确定,当胰蛋白胨与酵母粉的质量比为1∶1.2,并且磷酸盐的加量较大时菌体的质粒产量较高。在菌体生长刚进入对数生长期时采用指数式流加葡萄糖与采用恒速流加葡萄糖,菌体得率相同,而产物收率前者高于后者。在菌体生长进入平稳期时将温度由37°C升高到42°C,在菌体密度没有改变的情况下,质粒产量得到提高,达到75 m g/L。     

运用交流阻抗技术直接监测DNA杂交

介绍了基于交流阻抗技术构建非标记型脱氧核糖核酸（DNA）杂交传感器的方法.以24个碱基长度的寡聚DNA作为实验对象,将5′端巯基化的单链寡聚DNA（SH-ssDNA）探针与巯基乙酸（RSH）同时自组装到金电极表面,形成杂交识别层,利用交流阻抗技术测量出杂交前后金电极表面电子传递电阻R_et的增量作为杂交信号.实验中对DNA探针的自组装时间、杂交温度、杂交时间和阻抗测量液等实验条件进行了观察和优化;通过选择自组装液中SH-ssDNA探针和RSH的浓度,减少DNA在金电极表面的非特异性吸附,同时保证金电极表面自组装的SH-ssDNA探针有合适的疏密度,提高了杂交效率.在各优化条件下,无需扩增杂交信号,此非标记型DNA杂交传感器的检测下限为3.0×10^-14mol/L;和完全互补序列相比,一个和三个碱基错配序列分别产生55.6%和1.3%的杂交信号.     

硅胶破碎法抽提真菌染色体DNA

在进行基因组步行PCR克隆真菌木聚糖酶基因的研究中,探索并建立了一套可在普通分子生物学实验室采用的高得率、高质量真菌染色体DNA的抽提方法.采用液氮研磨和硅胶破碎相结合提高染色体DNA得率,采用亚精胺法纯化提高DNA的纯度.抽提的染色体DNA用琼脂糖凝胶电泳、限制酶切、PCR反应及紫外吸收光谱等方法进行了鉴定,结果显示此方法可以获得分子量大、样品纯的染色体DNA,可有效地用于PCR扩增,并能被限制酶有效地消化.     

抗癌药物8-氮鸟嘌呤与DNA相互作用的电化学研究

研究了抗癌药物8-氮鸟嘌呤(8-AG)与DNA在pH=3.6条件下相互作用的电化学行为.DNA的存在能导致8-AG氧化峰电流降低,通过测定DNA加入前后的一些电化学参数,推测8-AG与DNA在该条件下通过静电作用结合生成了一种1∶2非电活性的超分子化合物,其结合常数是1.38×1010.同时,根据加入DNA前后8-AG峰电流的降低,测定了模拟样品中的DNA,效果令人满意.     

重症急性胰腺炎病人外周血单个核细胞线粒体DNA拷贝数的意义

目的探讨重症急性胰腺炎患者外周血单个核细胞线粒体DNA拷贝数与患者病情严重程度、预后的相关性。方法分离研究对象（重症急性胰腺炎患者34名,健康对照组17名）外周血单个核细胞,采用荧光实时定量PCR检测各组PBMC线粒体DNA相对舍量,其与严重程度、预后的相关性。结果重症急性胰腺炎患者线粒体DNA含量较健康对照组明显降低（P〈0．05）。患者单个核细胞线粒体DNA拷贝教与APACHEⅡ评分呈负相关（r=-0．425）其中并发症组mtDNA拷贝数显著低于无并发症组（P〈0．05）。结论重症急性胰腺炎患者外周血单个核细胞中线粒体DNA含量显著下降,与患者APACHEⅡ评分呈负相关,且低水平的线粒体DNA拷贝数提示并发症发生率的升高,因此,线粒体DNA拷贝数可作为潜在评估患者病情危重程度及预后的指标,这一发现值得进一步大型,前瞻性的研究。     

Genus Baseodiscus (Nemertea: Heteronemertea): Molecular identification of a new species in a phylogenetic context

A new heteronemertean, Baseodiscus jonasii sp. nov., is described from Guadalcanal, Solomon Islands. It resembles B. delineatus in inner morphology but can be distinguished from this species by its different colour pattern and differences in the nucleotide sequence of the mitochondrial 16S rRNA gene. The monophyletic status of the genus is investigated by reconstructing the phylogeny of six specimens from four species assigned to this genus, together with 22 specimens from nine other heteronemertean genera, using parsimony and Bayesian analysis. The results imply that Baseodiscus is a monophyletic group while several other heteronemertean genera are non‐monophyletic.     

The morphological and molecular analyses of a new South American urban tardigrade offer new insights on the biological meaning of the Macrobiotus hufelandi group of species (Tardigrada: Macrobiotidae)

Worldwide knowledge of tardigrade fauna is still limited, and many areas such as South America are not well studied. The collection of new substrates in Argentinean urban areas provided an opportunity to describe the new tardigrade species Macrobiotus kristenseni sp. nov. This species has been studied with an integrative taxonomic approach, analysing its morphology by light and scanning electron microscopy, and considering two genes (cox1 and 18S rRNA) for DNA barcoding and phylogenetic purposes. The species belongs to the Macrobiotus hufelandi group of species, and it is characterized by egg processes in the shape of elongated cones with truncated and enlarged apexes, and by a high genetic distance with respect to closely related species (cox1: 18.2–33.4%). Morphological and molecular data show that the Macrobiotus hufelandi group of species has to be considered a true biological entity; one of the more widespread tardigrade lineages in continental environments. http://www.zoobank.org/urn:lsid:zoobank.org:pub:2CA51ABA-5570-4AFB-8182-CE499D97B0F1     

Nine new species of the subgenus Stegana (Oxyphortica) from the Oriental region,with morphological and molecular evidence based on Chinese species (Diptera: Drosophilidae)

Nine new species of the subgenus Stegana (Oxyphortica) have been discovered from the Oriental Region: S. (O.) chuanjiangi Zhang and Chen, sp. nov., S (O.) dainuo Wang and Chen, sp. nov., S. (O.) dawa Zhang and Chen, sp. nov., S. (O.) laohlie Zhang and Chen, sp. nov., S. (O.) luchun Wang and Chen, sp. nov., S. (O.) mengwan Wang and Chen, sp. nov., S. (O.) triodonta Cheng and Chen, sp. nov. from southern China; S. (O.) dehiscens Cheng and Chen, sp. nov. and S. (O.) spinosa Cheng and Chen, sp. nov. from eastern Malaysia. One newly recorded species from China: S. (O.) maichouensis Sidorenko is re-described. The DNA sequences of the mitochondrial COI gene with BOLD Process ID and GenBank accession numbers are provided for the above seven new species and five known species from China: S. (O.) acutipenis Xu, Gao and Chen, S. (O.) adentata Toda and Peng, S. (O.) hirtipenis Xu, Gao and Chen, S. (O.) latipenis Xu, Gao and Chen, and S. (O.) maichouensis Sidorenko. Intra- and interspecific pairwise K-2P distances among these species are summarized and the phylogenetic relationships are reconstructed based on the neighbour-joining (NJ) analyses.

www.zoobank.org/urn:lsid:zoobank.org:pub:2FA403F6-5535-40A4-9FD0-ADB9913EDE83.