人源EC-SOD在毕赤酵母中的构建与表达

利用基因工程技术构建表达载体并电转化至毕赤酵母中,成功实现人源EC-SOD在毕赤酵母X33中的表达.重组蛋白经盐酸羟胺法测得25 mL摇瓶发酵液上清酶活为508 U·mg~(-1),5 L发酵液上清酶活为909U·mg~(-1).发酵液上清用硫酸铵沉降后,使用DEAE弱阴离子交换树脂初步分离纯化出较纯的EC-SOD,测得比活为1 700 U·mg~(-1),并进行氯化硝基四氮唑蓝(NBT)活性定性染色.结果表明,毕赤酵母成功胞外表达具有一定活性的SOD蛋白.     

猪胃蛋白酶原A在毕赤酵母中的表达

利用已有的猪胃蛋白酶原AcDNA,构建毕赤酵母pPIC3.5K-pA胞内表达载体.经纤维蛋白消化试验和干酪素琼脂平板法筛选,获得两株高表达的重组子,研究它们在不同pH值、不同诱导时间、不同起始浓度和不同甲醇诱导浓度等条件下表达水平的高低.结果表明:在pH值为7、诱导72h、起始诱导浓度的OD600为2、甲醇诱导浓度为1%时,重组胃蛋白酶活力最高,达到6.46U/mL.     

大黄鱼生长激素在毕赤酵母中的优化表达

尝试利用毕赤酵母的表达系统制备大黄鱼生长激素(Pseudoscraena crocea,growth hormone,pcGH).根据酵母偏好密码子设计优化的基因序列,通过化学方法合成pcGH基因,构建表达载体pPICZ A - GH,利用化学转化技术,将该基因整合到巴斯德毕赤酵母X -33菌株染色体上并得以成功表达,...     

毕赤酵母表达猪胃蛋白酶的研究

利用已有的猪胃蛋白酶原A cDNA,构建毕赤酵母pPIC3．5K—pA胞内表达载体．经纤维蛋白消化试验和干酪素琼脂平板法筛选,获得2株高表达的重组子,并研究了在不同pH、不同诱导时间、不同起始浓度和甲醇诱导浓度等条件下表达水平的高低．结果表明：在pH7、诱导72h、起始诱导浓度为OD600为2、甲醇诱导浓度为1％时,重组胃蛋白酶活力最高,达到6．46U／mL．     

克鲁维酵母中外切菊粉酶基因在毕赤酵母中的表达研究

用PCR的方法从克鲁维酵母(Kluyveromyces marxianus)ATCC12424中克隆出外切菊粉酶基因.将该基因克隆到毕赤酵母表达载体pHBM905C、pHBM906上,构建了外切菊粉酶毕赤酵母表达载体pHBM1200、pHBM1201.将两者转化毕赤酵母GS115,得到重组菌株GS115(pHBM1200)、GS115(pHBM1201),将这两种重组菌株进行摇瓶发酵,测得带α-信号肽的菌株GS115(pHBM1200)表达的外切菊粉酶最高酶活为89.43 U/mL,带自身信号肽的菌株GS115(pHBM1201)表达的外切菊粉酶最高酶活为14.828 U/mL.SDS-PAGE电泳表明,外切菊粉酶的表观分子量为90 kD.将外切菊粉酶用去糖基化酶endoH处理后,SDS-PAGE电泳表明,分子量为60 kD,和预计的分子量一致.     

密码子优化的植酸酶基因appA-P在毕赤酵母中的高效表达

根据毕赤酵母(Pichia pastoris)密码子使用偏爱性,对Escherichia coli来源的高比活植酸酶基因(appA)全面地进行密码子优化,人工合成了植酸酶基因(appA-P),并将该基因克隆到毕赤酵母诱导型分泌表达载体pHBM905A上,获得重组质粒pHBM905A-appA-P,通过PEG1000转化法将线性化的重组质粒转化毕赤酵母GS115菌株,筛选得到一个高效表达植酸酶的重组菌株GS115-appA-P,在25℃摇瓶培养168 h后酶活力达到422 U/mL,该植酸酶最适反应温度为60℃,最适反应pH为4.5,在20~50℃、pH1.0~6.0范围内较稳定.     

朱黄青霉α-葡聚糖酶在毕赤酵母中的高效表达

【目的】提高朱黄青霉（Penicillium minioluteum ）α-葡聚糖酶（Dextranase）基因在毕赤酵母（Pichia pastoris）中的表达水平。【方法】根据毕赤酵母的密码子偏爱对酶基因进行优化与合成。优化后的基因片段与载体 pGAPZαA 连接,转化毕赤酵母 KM71 H。【结果】获得组成型分泌表达α-葡聚糖酶的工程菌 KM71 H／pGAPZαA-dex。发酵工艺试验中,摇瓶培养144 h,酶活为153 U／mL。6．8 L发酵罐补料分批培养92 h,酶活达到1218 U／mL。【结论】该工程菌以甘油作为碳源,发酵调控简单,产酶水平较高,具有适用于大规模生产的潜力。     

α-葡聚糖酶在毕赤酵母中的组成型表达

为避免α-葡聚糖造成的制糖过程中的糖分损失、制炼难度增加以及糖产品质量下降,利用α-葡聚糖酶分解而直接去除α-葡聚糖是目前最佳的选择.文中扩增了α-葡聚糖酶基因dex,构建组成型表达载体pGAPZαA-dex;以毕赤酵母KM71H为受体菌,将表达载体整合进入受体菌基因组,构建了组成型表达α-葡聚糖酶的毕赤酵母工程菌.摇瓶发酵120h,α-葡聚糖酶酶活达到60.4U/mL,从而实现了α-葡聚糖酶在毕赤酵母中的组成型表达,使发酵过程无需使用甲醇进行诱导,提高了生产和使用安全性.     

南极假丝酵母脂肪酶B在毕赤酵母中的分泌型表达及酶学性质初探

将南极假丝酵母脂肪酶B(CALB)基因克隆到毕赤酵母表达载体pPICZαA(含pAOX1启动子)和pGAPZαA(含pGAP启动子)中,转入毕赤酵母表达宿主KM71H,经三丁酸甘油酯平板活性筛选获得高效表达的酵母工程菌株.分泌型表达CLAB酵母工程菌的酶活较高,经摇瓶发酵96h后,CLAB活力达到11.1U/mL.经发酵罐高密度发酵84h后,CLAB活力达到179.2U/mL,蛋白质量浓度为1.36mg/mL.体积分数12%SDS-PAGE凝胶电泳显示重组CALB相对分子量为36kDa.该酶最适温度为40℃,最适pH值为8.0.在pH7.5~9.5较稳定,经55℃保温1h,残余酶活还有48.5%.5mmol/LMg2+、Ni+、Co2+和体积分数0.10%SDS对CALB酶活影响很小,而Zn2+强烈抑制酶催化反应,体积分数0.05%TritonX-100对CALB稍有激活作用.     

扩展青霉FS1884脂肪酶基因的定点诱变及其活性分析

为提高扩展青霉FS1884脂肪酶活性,采用一步突变法对脂肪酶(PEL)的基因进行体外M220V定点诱变,然后将其连接到表达载体pPIC3.5K中, 构建重组表达载体pPIC3.5K-M220V-PEL,电转化毕赤酵母GS115中,经甲醇诱导后脂肪酶成功分泌表达.重组脂肪酶的活性分析表明:突变体在毕赤酵母中得到活性表达,获得脂肪酶表达产物PEL-M220V-GS,在35 ℃下具有最高酶活为3 099 U/mg, 比野生型提高了5%.     

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.     

A computer generator for randomly layered structures

AcomputergeneratorforrandomlylayeredstructuresYUJia shun1,2,HEZhen hua2(1.TheInstituteofGeologicalandNuclearSciences,NewZealand;2.StateKeyLaboratoryofOilandGasReservoirGeologyandExploitation,ChengduUniversityofTechnology,China)Abstract:Analgorithmisintrod…     

海南岛地体及其毗邻陆缘晚中生代—新生代古地磁研究和构造演化

本文叙述了对海南岛及其毗邻大陆边缘白垩纪到第四纪地层岩石进行古地磁研究的全部工作过程。通过分析岩石中剩余磁矢量的磁偏角及磁倾角的变化,提出海南岛白垩纪以来经历的构造演化模式如下:早期伴随顺时针旋转而向南迁移,后期伴随逆时针转动并向北运移。联系该地区及邻区的地质、地球物理资料,对海南岛上述的构造地体运动提出以下认识:北部湾内早期有一拉张作用,主要是该作用使湾内地壳显著伸长减薄,形成北部湾盆地。从而导致了海南岛的早期构造运动,而海南岛后期的构造运动则主要是受南海海底扩张的影响。海南地体运动规律的阐明对于了解北部湾油气盆地的形成演化有重要的理论和实际意义。     

Exciton Seebeck in Molecular Systems

Various applications relevant to the exciton dynamics,such as the organic solar cell,the large-area organic light-emitting diodes and the thermoelectricity,are operating under temperature gradient.The potential abnormal behavior of the exicton dynamics driven by the temperature difference may affect the efficiency and performance of the corresponding devices.In the above situations,the exciton dynamics under temperature difference is mixed with     

Achieving Unusual States of Matter Under High Pressure

The elongation method,originally proposed by Imamura was further developed for many years in our group.As a method towards O(N)with high efficiency and high accuracy for any dimensional systems.This treatment designed for one-dimensional(ID)polymers is now available for three-dimensional(3D)systems,but geometry optimization is now possible only for 1D-systems.As an approach toward post-Hartree-Fock,it was also extended to     

《Chinese Science Bulletin》SUBJECT INDEX TO VOLUME 59(2014)

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An on-line scaling method for improving scalability of a database cluster

The explosive growth of the Internet and database applications has driven database to be more scalable and available, and able to support on-line scaling without interrupting service. To support more client's queries without downtime and degrading the response time, more nodes have to be scaled up while the database is running. This paper presents the overview of scalable and available database that satisfies the above characteristics. And we propose a novel on-line scaling method. Our method improves the existing on-line scaling method for fast response time and higher throughputs. Our proposed method reduces unnecessary network use, i.e. , we decrease the number of data copy by reusing the backup data. Also, our on-line scaling operation can be processed parallel by selecting adequate nodes as new node. Our performance study shows that our method results in significant reduction in data copy time.     

An improved R-tree based on childnode''s probability

R-Tree is a good structure for spatial searching. But in this indexing structure,either the sequence of nodes in the same level or sequence of traveling these nodes when queries are made is random. Since the possibility that the object appears in different MBR which have the same parents node is different, if we make the subnode who has the most possibility be traveled first, the time cost will be decreased in most of the cases. In some case, the possibility of a point belong to a rectangle will shows direct proportion with the size of the rectangle. But this conclusion is based on an assumption that the objects are symmetrically distributing in the area and this assumption is not always coming into existence. Now we found a more direct parameter to scale the possibility and made a little change on the structure of R-tree, to increase the possibility of founding the satisfying answer in the front sub trees. We names this structure probability based arranged R-tree (PBAR-tree).     

An Index mechanism for multi-scale view in spatial databases

There are numerous geometric objects stored in the spatial databases. An importance function in a spatial database is that users can browse the geometric objects as a map efficiently. Thus the spatial database should display the geometric objects users concern about swiftly onto the display window. This process includes two operations:retrieve data from database and then draw them onto screen. Accordingly, to improve the efficiency, we should try to reduce time of both retrieving object and displaying them. The former can be achieved with the aid of spatial index such as R-tree, the latter require to simplify the objects. Simplification means that objects are shown with sufficient but not with unnecessary detail which depend on the scale of browse. So the major problem is how to retrieve data at different detail level efficiently. This paper introduces the implementation of a multi-scale index in the spatial database SISP (Spatial Information Shared Platform) which is generalized from R-tree. The difference between the generalization and the R-tree lies on two facets: One is that every node and geometric object in the generalization is assigned with a importance value which denote the importance of them, and every vertex in the objects are assigned with a importance value,too. The importance value can be use to decide which data should be retrieve from disk in a query. The other difference is that geometric objects in the generalization are divided into one or more sub-blocks, and vertexes are total ordered by their importance value. With the help of the generalized R-tree, one can easily retrieve data at different detail levels.Some experiments are performed on real-life data to evaluate the performance of solutions that separately use normal spatial index and multi-scale spatial index. The results show that the solution using multi-scale index in SISP is satisfying.     

Integrating GIS Web services based on mediating architecture

The geographic information service is enabled by the advancements in general Web service technology and the focused efforts of the OGC in defining XML-based Web GIS service. Based on these models, this paper addresses the issue of services chaining,the process of combining or pipelining results from several interoperable GIS Web Services to create a customized solution. This paper presents a mediated chaining architecture in which a specific service takes responsibility for performing the process that describes a service chain. We designed the Spatial Information Process Language (SIPL) for dynamic modeling and describing the service chain, also a prototype of the Spatial Information Process Execution Engine (SIPEE) is implemented for executing processes written in SIPL. Discussion of measures to improve the functionality and performance of such system will be included.