瑞氏木霉β-内切葡聚糖酶的纯化及其部分酶学性质研究

 利用盐析、SephadexG-75和DEAE-SephadexA50层析,从瑞氏木霉TrichodermareeseiQM9414发酵液中纯化了2个具有β-内切葡聚糖酶活性的组分Eg1和Eg2,分子质量分别为65.47ku和57.04ku.其最适温度分别为50℃和55℃,最适pH分别为4.8和5.0,米氏常数Km分别为3.76×10^-2g/L和4.20×10^-2g/L.根据其酶学性质,这是一类不同于已有的β-内切葡聚糖酶,为瑞氏木霉T.reeseiQM9414所产β-内切葡聚糖酶的进一步研究奠定了基础.     

匍枝根霉内切葡聚糖酶基因在毕赤酵母中的克隆及表达

从匍枝根霉TP-02的cDNA文库中克隆得到其内切葡聚糖酶基因,并在毕赤酵母中进行表达.该内切葡聚糖酶基因大小为954bp,将其与pPIC9K质粒经EcoRⅠ和NotⅠ双酶切后,利用T4连接酶进行连接.将线性化后的重组质粒转化毕赤酵母GS115,并利用MD平板和刚果红平板进行筛选.阳性克隆经甲醇诱导培养84h后,产生的内切葡聚糖酶酶活力达到峰值为1.754IU/mL.     

毛霉Amylomyces.rouxii HN0512中内切葡聚糖酶的诱导、纯化及性质分析

Amylomyces.rouxii HN0512经羧甲基纤维素钠(CMC-Na)诱导,可产内切葡聚糖酶,通过正交试验优化诱导培养基,结果在培养基为1％羧甲基纤维素钠、2％麸皮汁、0.2％蛋白胨(以上均为质量分数)时可以诱导出较高活性的酶,用HiTrap QFF阴离子交换层析从培养液上清中纯化得到了具有内切葡聚糖酶活性的P3组分,活性组份的SDS-PAGE分析电泳呈现一条带,经AlphaEaseFCTM软件预测P3的分子质量为18.7 ku;酶反应的最适温度和pH分别为60℃和5.0,在pH 4.0 -7.0范围内稳定性最好,在70℃还能保持50％以上的酶活力;金属离子Fe3+、Ba2+、Fe2和表面活性剂尿素对酶起到了激活作用;反应动力学参数Km和Vmax分别为11.68 mg· mL-1、0.56 μmol·L-1·min-1.本研究获得了一株产内切葡聚糖酶新菌种,并为进一步用基因工程方法克隆并构建重组内切葡聚糖酶基因工程菌奠定了重要的基础.     

白蚁头部纤维素酶基因的克隆与表达

对中国广泛分布的黑翅土白蚁(Odontotermes formosanus)头部内切β-1,4葡聚糖酶(endo-beta-1,4-glucanase)基因进行克隆,并在原核生物大肠杆菌体内进行了表达.根据Genbank上已报道的endo-beta-1,4-glu-canase基因设计引物,通过RT-PCR获得1个完整的内切β-1,4葡聚糖酶基因.通过基因测序以及基因比对发现与Genbank上发表的内切β-1,4葡聚糖酶同源性高达96.40%.将获得的基因导入大肠杆菌体内进行诱导表达,表达出的蛋白经Western-blot检测,为该基因所表达的目的蛋白.实验为进一步对内切β-1,4葡聚糖酶的实际应用奠定一定的实验基础.     

产朊假丝酵母细胞壁33kD蛋白的酶学特性研究

培养产朊假丝酵母菌收获菌体,经裂解酶处理细胞壁,离心后得到上清粒酶液,粗酶波经DEAE-纤维素层析、Sepharose4B凝胶过滤纯化。对33kD蛋白的酶学性质研究表明,反应最适pH为5．5,反应的最适温度为45℃。热稳定性较差,50℃温度下放置1h活力下降50％。主要水解β-1,3-倍苦键,具有较强的专一性,是一种典型的葡聚糖内切酶（对pNPG无作用）。     

巨大芽孢杆菌AP25内切葡聚糖酶基因的克隆及序列测定

从土壤中分离到一株产纤维素酶的巨大芽孢杆菌AP25,经羧甲基纤维素平板检测,该菌可产生葡聚糖内切酶。根据GenBank登录的β-1,4内切葡聚糖酶基因（DQ782954．1,M28332．1,AY859492．1）的同源性序列,利用PCR方法克隆到该酶的基因,并对其进行测序。测序结果显示其全长为1500bp,推测其含499个氨基酸。与GenBank中的已知葡聚糖内切酶相比较,发现该酶的氨基酸序列与Bacillus subtilis的β-1,4内切葡聚糖酶基因同源性达到达94％。     

β-1,4-内切葡聚糖酶基因在毕赤酵母中的表达研究

根据芽孢杆菌β-1,4-内切葡聚糖酶基因序列设计引物,以pHBM102为模板,扩增得到β-1,4-内切葡聚糖酶GluD基因片段,克隆至毕赤酵母(Pichia pastoris)表达载体pHBM905上,获得重组毕赤酵母表达载体pHBM905D,将此质粒分别转化毕赤酵母GS115,KM71和SMD1168菌株,筛选获得GS115(pHBM905D),KM71(pHBM905D),和SMD1168(pHBM905D),平板诱导培养表明,GS115(pHBM905D)所产生的水解圈最大,酶活力最高.摇瓶诱导培养,表达β-1,4-内切葡聚糖酶,此酶的最适反应温度为65℃,最适反应pH值6.4,在诱导8 d后酶活力达到最高为0.185 8 U/mL.     

内切葡聚糖酶的纯化及其基本性质

纤维素酶是自然界碳素循环中的一种关键酶,它可以将纤维素性材料,转化为可直接利用的葡萄糖,并可通过其它酶和酶系统的作用转化为乙醇及蛋白质。纤维素酶是多酶体系,包含外切葡聚糖酶、内切葡聚糖酶(Cx)及β-葡萄糖苷酶。其中内切葡聚糖酶含有若干种同工酶,能将可溶性纤维素转化为还原寡糖,是纤维素酶系中的重要组分,为了深入研究Cx酶的性质,本文对其中一种Cx酶进行了分离纯化,并对其基本性质进行了研究。 1实验部分     

短小芽孢杆菌C-9葡聚糖内切酶基因的克隆及其在酿酒酵母中的表达

从短小芽孢杆菌(Bacillus pumilus)C-9中克隆得到葡聚糖内切酶基因,该基因包含1980 bp核苷酸,编码559个氨基酸,其N端有一个预测的29个氨基酸的信号肽序列;以YIp5质粒为骨架,构建rDNA介导的多拷贝整合载体,实现葡聚糖内切酶在酿酒酵母中的高效、稳定表达。与对照菌株相比,含有葡聚糖内切酶基因的重组酿酒酵母可以在以羧甲基纤维素为唯一碳源的培养基上生长。     

从黑曲霉发酵粉中分离纯化一种内切β-葡聚糖苷酶

通过硫酸铵分级沉淀 ,CM -SephadexC - 2 5 ,DEAE -SephadexA - 5 0 ,POROS 2 0HQ离子交换色谱分离方法 ,从黑曲霉发酵粉中分离纯化了一种新的内切 β -葡聚糖苷酶 .该酶在还原条件下分子量为 39.2kD ,最适pH为 4.0 ,最适温度为 5 5℃ ,Km 为 7.41mg mL .     

Markedness and its Interpretation in Language Use

Language markedness is a common phenomenon in languages, and is reflected from hearing, vision and sense, i.e. the variation in the three aspects such as phonology, morphology and semantics. This paper focuses on the interpretation of markedness in language use following the three perspectives, i.e. pragmatic interpretation, psychological interpretation and cognitive interpretation, with an aim to define the function of markedness.     

Lower Paleozoic oil relationships within Williston Basin, Canada

The Williston Basin is a significant petroleum province, containing oil production zones that include the Middle Cambrian to Lower Ordovician, Upper Ordovician, Middle Devonian, Upper Devonian and Mississippian and within the Jurassic and Cretaceous. The oils of the Williston Basin exhibit a wide range of geochemical characteristics defined as "oil families", although the geochemical signature of the Cambrian Deadwood Formation and Lower Ordovician Winnipeg reservoired oils does not match any "oil family". Despite their close stratigraphic proximity, it is evident that the oils of the Lower Palaeozoic within the Williston Basin are distinct. This suggests the presence of a new "oil family" within the Williston Basin. Diagnostic geochemical signatures occur in the gasoline range chromatograms, within saturate fraction gas chromatograms and biomarker fingerprints. However, some of the established criteria and cross-plots that are currently used to segregate oils into distinct genetic families within the basin do not always meet with success, particularly when applied to the Lower Palaeozoic oils of the Deadwood and Winnipeg Formation.     

An Analysis of Catherine＇s Character——Wuthering Heights

Wuthering Heights, Emily Bronte＇s only novel, was published in December of 1847 under the pseudonym Ellis Bell. The book did not gain immediate success, but it is now thought one of the finest novels in the English language. Catherine is the key character of this masterpiece, because everybody and everything center on her though she had a short life. We can understand this masterpiece better if we know Catherine well.     

Characterization of Ordovician carbonate reservoirs, southeastern Saskatchewan, Canada

The discovery of the prolific Ordovician Red River reservoirs in 1995 in southeastern Saskatchewan was the catalyst for extensive exploration activity which resulted in the discovery of more than 15 new Red River pools. The best yields of Red River production to date have been from dolomite reservoirs. Understanding the processes of dolomitization is, therefore, crucial for the prediction of the connectivity, spatial distribution and heterogeneity of dolomite reservoirs.The Red River reservoirs in the Midale area consist of 3～4 thin dolomitized zones, with a total thickness of about 20 m, which occur at the top of the Yeoman Formation. Two types of replacement dolomite were recognized in the Red River reservoir: dolomitized burrow infills and dolomitized host matrix. The spatial distribution of dolomite suggests that burrowing organisms played an important role in facilitating the fluid flow in the backfilled sediments. This resulted in penecontemporaneous dolomitization of burrow infills by normal seawater. The dolomite in the host matrix is interpreted as having occurred at shallow burial by evaporitic seawater during precipitation of Lake Almar anhydrite that immediately overlies the Yeoman Formation. However, the low δ18O values of dolomited burrow infills (-5.9‰～ -7.8‰, PDB) and matrix dolomites (-6.6‰～ -8.1‰, avg. -7.4‰ PDB) compared to the estimated values for the late Ordovician marine dolomite could be attributed to modification and alteration of dolomite at higher temperatures during deeper burial, which could also be responsible for its 87Sr/86Sr ratios (0.7084～0.7088) that are higher than suggested for the late Ordovician seawaters (0.7078～0.7080). The trace amounts of saddle dolomite cement in the Red River carbonates are probably related to "cannibalization" of earlier replacement dolomite during the chemical compaction.     

Design and analysis of generic LBS application developing platform

Location based services is promising due to its novel working style and contents.A software platform is proposed to provide application programs of typical location based services and support new applications developing efficiently. The analysis shows that this scheme is easy implemented, low cost and adapt to all kinds of mobile nework system.     

橡胶颗粒沥青混合料低温抗裂性试验研究

以AC-13级配为基础,将橡胶颗粒代替部分集料掺入混合料中,以低温弯曲试验为评价方法对不同橡胶颗粒掺量下沥青混合料的低温抗裂性进行研究,并引入应变能密度值对混合料的低温抗裂性进行综合评价.试验结果表明:橡胶颗粒沥青混合料试件的破坏微应变均超过2 300,满足冬寒区的技术指标;无论是否掺加橡胶颗粒,随着温度的下降,沥青混合料破坏时的最大弯拉强度增大,弯拉应变降低,劲度模量增大;弯曲应变能密度在胶粒掺量为1%左右时具有较大的弯曲应变能密度值,此时橡胶颗粒沥青混合料具有较好的低温抗裂性.     

低渗透非均质砂岩油藏启动压力梯度研究

理论推导与室内实验相结合,建立了低渗透非均质砂岩油藏启动压力梯度确定方法。首先借助油藏流场与电场相似的原理,推导了非均质砂岩油藏启动压力梯度计算公式。其次基于稳定流实验方法,建立了非均质砂岩油藏启动压力梯度测试方法。结果表明:低渗透非均质砂岩油藏的启动压力梯度确定遵循两个等效原则。平面非均质油藏的启动压力梯度等于各级渗透率段的启动压力梯度关于长度的加权平均;纵向非均质油藏的启动压力梯度等于各渗透率层的启动压力梯度关于渗透率与渗流面积乘积的加权平均。研究成果可用于有效指导低渗透非均质砂岩油藏的合理井距确定,促进该类油藏的高效开发。     

Quantitative trait loci （QTLs） for quality traits related to protein and starch in wheat

Quality traits in wheat （Triticum aestirum L.） were studied by quantitative trait locus （QTL） analysis in a recombinant inbred line （RIL） population, a set of 131 lines derived from Chuan 35050 × Shannong 483 cross （ChSh）. Grains from RILs were assayed for 21 quality traits related to protein and starch. A total of 35 putative QTLs for 19 traits with a single QTL explaining 7.99-40.52% of phenotypic variations were detected on 10 chromosomes, 1D, 2A, 2D, 3B, 3D, 5A, 6A, 6B, 6D, and 7B. The additive effects of 30 QTLs were positive, contributed by Chuan 35050, the remaining 5 QTLs were negative with the additive effect contributed by Shannong 483. For protein traits, 15 QTLs were obtained and most of them were located on chromosomes 1 D, 3B and 6D, while 20 QTLs for starch traits were detected and most of them were located on chromosomes 3D, 6B and 7B. Only 7 QTLs for protein and starch traits were co-located in three regions on chromosomes 1D, 2A and 2D. These protein and starch trait QTLs showed a distinct distribution pattern in certain regions and chromosomes. Twenty-two QTLs were clustered in 6 regions of 5 chromosomes. Two QTL clusters for protein traits were located on chromosomes 1D and 3B, respectively, three clusters for starch traits on chromosomes 3D, 6B and 7B, and one cluster including protein and starch traits on chromosome 1D.     

On Tragic Consciousness In The Works By Ernest Hemingway

As an American modern novelist who were famous in the literary world, Hemingway was not a person who always followed the trend but a sharp observer. At the same time, he was a tragedy maestro, he paid great attention on existence, fate and end-result. The dramatis personae's tragedy of his works was an extreme limit by all means tragedy on the meaning of fearless challenge that failed. The beauty of tragedy was not produced on the destruction of life, but now this kind of value was in the impact activity. They performed for the reader about the tragedy on challenging for the limit and the death.     

The Analects and the Moment in Peking——The influence of a Confucian education system on the personality formation,the way of thinking and the fate of the main characters

0.Preface China has long been boasting of its colorful and immense culture which comprises of Taoism,Confucianism and Buddhism.The interwoven influence among the religions on Chinese way of thinking,on the nation＇s behavior in their social life throughout the long history is beyond list.