Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display

To develop a novel yeast whole-cell biocatalyst by yeast surface display technology that can hydrolyze chitin, the chitinaseC gene from Serratia marcescens AS1.1652 strain was cloned and subcloned into the yeast surface display plasmid pYD1, and the recombinant plasmid pYD1/SmchiC was electroporated into Saccharomyces cerevisiae EBY100 cell. Aga2p-SmChiC fusion protein was expressed and anchored on the yeast cell surface by induction with galactose, which was verified by indirect immunofluorescence and Western blotting. The chitinolytic activity of the yeast whole-cell biocatalyst or partially purified enzyme was detected by agar plate clear zone test, SDS-PAGE zymography and dinitrosalicylic acid method. The results showed that the chitinaseC gene from S. marcescens AS1.1652 strain was successfully cloned and expressed on the yeast cell surface, Aga2p-SmChiC fusion protein with molecular weight (67 kDa) was determined. Tests on the effect of temperature and pH on enzyme activity and stability revealed that the yeast whole-cell biocatalyst and partially purified enzyme possessed both thermal stability and activity, and even maintained some activity under acidic and weakly alkaline conditions. The optimum reaction temperature and pH value were set at 52 °C and 5.0, respectively. Yeast surface display technology succeeded in preparing a yeast whole-cell biocatalyst with chitinolytic activity, and the utilization of chitin could benefit from this process of enzyme preparation.     

Structural and functional analysis of a new subfamily of glycosyltransferases required for glycosylation of serine-rich streptococcal adhesins

Serine-rich repeat glycoproteins (SRRPs) are a growing family of bacterial adhesins found in many streptococci and staphylococci; they play important roles in bacterial biofilm formation and pathogenesis. Glycosylation of this family of adhesins is essential for their biogenesis. A glucosyltransferase (Gtf3) catalyzes the second step of glycosylation of a SRRP (Fap1) from an oral streptococcus, Streptococcus parasanguinis. Although Gtf3 homologs are highly conserved in SRRP-containing streptococci, they share minimal homology with functionally known glycosyltransferases. We report here the 2.3 ? crystal structure of Gtf3. The structural analysis indicates that Gtf3 forms a tetramer and shares significant structural homology with glycosyltransferases from GT4, GT5, and GT20 subfamilies. Combining crystal structural analysis with site-directed mutagenesis and in vitro glycosyltransferase assays, we identified residues that are required for UDP- or UDP-glucose binding and for oligomerization of Gtf3 and determined their contribution to the enzymatic activity of Gtf3. Further in vivo studies revealed that the critical amino acid residues identified by the structural analysis are crucial for Fap1 glycosylation in S. parasanguinis in vivo. Moreover, Gtf3 homologs from other streptococci were able to rescue the gtf3 knock-out mutant of S. parasanguinis in vivo and catalyze the sugar transfer to the modified SRRP substrate in vitro, demonstrating the importance and conservation of the Gtf3 homologs in glycosylation of SRRPs. As the Gtf3 homologs only exist in SRRP-containing streptococci, we conclude that the Gtf3 homologs represent a unique subfamily of glycosyltransferases.     

桉树人工林林下植被、地面覆盖物与土壤物理性质的关系

对儋州市东成镇的7个不同类型的桉树(Eucalyptussp.)人工林和一个马占相思林(Acaciamangium)林下的土壤含水量、土壤温度、土壤容重、土壤坚实度(硬度)以及土壤的机械组成等5个土壤物理指标进行研究。结果表明,有林下植被的桉树人工林地的土壤含水量比无林下植被的桉树人工林地的高出11.2~62.9 g.kg-1;同一样地内,有枯枝落叶覆盖物的土壤含水量平均比没有枯枝落叶覆盖物的土壤含水量高27.6~38.2 g.kg-1;有林下植被的桉树人工林地土壤温度比无林下植被的桉树人工林地和空旷弃荒地低,但却比无林下植被、树冠大、荫蔽度大的马占相思林地的高。有林下植被的桉树人工林地的土壤硬度要比无林下植被的桉树人工林地、马占相思林地和空旷弃荒地的小5.49~18.76 kg.cm-3。林下植被覆盖率高的桉树人工林地,其容重比有林下植被的桉树人工林地、无林下植被的桉树人工林地、马占相思林地、牧草实验地样地小0.33~0.48 g.cm-3。这说明桉树人工林下植被和枯枝落叶是影响土壤含水量、土壤硬度、土壤温度、土壤容重等土壤物理性质的重要因子。因此,为了有效改善桉树林的立地条件以及能更好地经营桉树人工林,在...     

不同动物源性细胞系对猪丁型冠状病毒的易感性

[目的]探究猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)能否在不同动物源性细胞系中感染和增殖.[方法]本研究将PDCoV四川分离株CHN-SC2015接种来自仓鼠、家禽、猴、人和猪的12种细胞系,将病毒在每种细胞系中盲传至少5代并通过逆转录-聚合酶链式反应(RT-PCR)、实时荧光定量...     

转基因定量检测用质粒分子标准物质研究进展

近10年来,应用于转基因植物产品定量检测的质粒分子标准物质凭借其易于富集,快捷高效等优点,成为各国检测机构的研究热点。对国内外转基因植物核酸定量检测用的质粒分子标准物质研究进展进行总结和评述,并且对该类标准物质在研制过程中的问题进行分析,同时展望了该类标准物质的发展与应用。     

用做标记的两种酵母菌完整染色体DNA的简化制备法

已知分子量大小的染色体ＤＮＡ分子标记是脉冲电泳研究中用以估算样本染色体ＤＮＡ分子大小必不可少的参照物。对用做标记的啤酒酵母（Ｓａｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）和粟酒裂殖酵母（Ｓｃｈｉｚｏｓａｃｈａｒｏｍｙｃｅｓｐｏｍｂｅ）完整染色体ＤＮＡ几种制备方法的比较研究以及改进研究表明,液氮冷冻研磨法,凝胶包埋法以及凝胶包埋破壁法效果均很好,都得到大量染色体ＤＮＡ,且彼此间无明显差异。表明液氮冷冻研磨法和凝胶包埋法制备上述两种酵母菌染色体ＤＮＡ分子标记是两种理想的方法,可完全取代凝胶包埋破壁法,即缩短处理时间又降低成本。此外,相同的电泳样本块在不同的电泳条件下,所得结果有一定的差异,表明电泳条件是影响电泳结果的一个重要因素     

以D-果糖为原料利用新型异构酶转化生产D-阿洛糖

稀少糖是自然界中含量稀少、化学合成困难的一类低热量单糖。D-阿洛糖是一种重要的稀少己醛糖,其具有减少活性自由基、抑制癌细胞增殖等独特的生理学功能。因此,以微生物发酵生产D-阿洛酮糖-3-差向异构酶(DPE)和L-鼠李糖异构酶(L-RhI)转化生产D-阿洛糖,成为近几年来国际研究的热点之一。文中分别克隆了来源于解纤维梭菌Clostridium cellulolyticum H10的DPE基因以及来源于枯草芽胞杆菌Bacillussubtilis 168的L-RhI基因,并分别使其在宿主菌B.subtilis及大肠杆菌Escherichia coli BL21(DE3)中得到了表达。进一步利用镍亲和层析和阴离子交换色谱等手段对这两种酶进行了纯化,并对这两种纯化后酶的转化能力进行了分析测定。结果表明,以D-果糖为原料利用两种异构酶依次转化获得D-阿洛酮糖及D-阿洛糖,其两步转化效率分别为27.34%和34.64%。     

基于能值分析和生态用地分类的中国生态系统生产总值核算研究

生态系统生产总值是生态系统为人类提供的产品与服务价值的总和,研究与建立一个独立的核算一个国家或地区的生态系统生产总值的方法与体系,对科学评价与合理利用自然资源、助推生态文明建设等具有重要意义。将生态系统生产总值分为供给价值、文化价值、承载价值与调节价值等四大类,基于能值分析方法和生态用地分类体系,计算得出全国及各地区生态系统生产总值,并将其与国内生产总值进行比较,为自然资源资产负债核算和综合生态系统管理等研究提供理论和方法借鉴。研究结果表明:(1)生态用地分类体系包括湿地、森林、草地和其他生态土地4个一级类型、19个二级类型。其中,湿地、森林、草地可统称为基础性生态用地。2008年,中国生态用地总量为763.95万km2,约占陆域国土面积的80%,其中基础性生态用地约为530.8万km2。(2)2008年,中国国土生态系统生产总值约为19万亿美元,是当年国内生产总值4.71万亿美元的4倍,人均生态系统总值约为1.45万美元/人。从生态系统生产总值来看,广东、山东、河南位居前三,西藏最低;从人均生态系统生产总值来看,西藏和内蒙古居前,甘肃最低。