嗜热脂肪地芽孢杆菌NAD激酶克隆表达及酶学性质研究

NAD激酶能催化NAD生成NADP。本研究采用PCR技术从嗜热脂肪地芽孢杆菌基因组中获得NAD激酶基因,以pET30a（＋）为表达载体、E．coliBL21（DE3）为宿主菌,实现其在大肠杆菌中异源表达,并进行酶学性质研究。结果显示,嗜热脂肪地芽孢杆菌中NAD激酶编码基因大小为816bp,酶分子量大约为35kD。酶学性质分析表明,来源于嗜热脂肪地芽孢杆菌的NAD激酶最适反应温度和pH分别为35℃、pH7．5,在35qC中保温2h后仍能保持80％左右的活性。Mn2＋、Ca2＋对该酶有较强的激活作用,在最适反应条件下该酶的比活力为4．43U／mg。动力学性质分析结果显示NAD激酶对底物NAD催化的k和圪。,分别为1．46mmol／L和0．25tzmol／（L·min）。NAD激酶在大肠杆菌的异源表达为以NAD为底物生物合成NADP提供了更多生物资源。     

响应面-满意度函数优化重组大肠杆菌产NMN转移酶发酵条件

重组大肠杆菌Escherichaia coli能高效表达NMN转移酶,以此为出发菌株,以菌体生长量OD600和NMN转移酶的活力为响应值,对重组大肠杆菌产NMN转移酶的发酵条件进行优化.首先以Plackett-Burman实验设计优化筛选出3个主要影响因子:胰蛋白胨、甘油、MgSO4;随后以Box-Behnken中心组合设计建立上述3个因子对OD600和NMN转移酶活力水平的数学模型;最后通过满意度函数获得最佳发酵条件为:酵母粉30 g/L,胰蛋白胨10.5 g/L,甘油3.49 mL/L,MgSO40.45 g/L,K2 HPO440.5 g/L,KH2 PO46.0 g/L,NH4 Cl 1.5 g/L,NaCl 0.6 g/L,接种量1.5%,诱导时间12 h.在该优化条件下,菌体生长和产酶水平均获得了显著的提升.重组NMN转移酶的活力水平从8.85 U/mg提高到15.48 U/mg,菌体生长量OD600从4.85提高到6.01,提高幅度分别为74.92%和23.92%.     

深海放线菌生淀粉酶基因的克隆表达及酶学特性研究

从深海放线菌Streptomyces sp.SCSIO03032基因组中扩增到1条含淀粉结合域的水解糖苷13家族基因amy032,该基因编码氨基酸与已知蛋白一致性最高为67%。将amy032插入表达载体pET32a启动子下游,构建重组载体pET-amy。重组质粒导入大肠杆菌Rosseta(DE3)菌株中,SDS-PAGE分析结果显示目的基因成功实现异源表达。Ni-NTA对重组酶进行纯化,并对其酶学性质进行表征。结果表明:重组淀粉酶AMY032的最适作用温度为50℃,最适pH为8.0,以可溶性淀粉为底物时的比酶活为(276±57)U/mg,Km为0.02g/L,Vmax为70mg/(L·min)。Ca2+能提高该酶的催化活性,Ni2+、Cu2+、Zn2+和Mn2+对该酶有抑制作用。AMY032对生玉米淀粉和生大米淀粉具有水解活性,其比酶活分别为(49±12)U/mg和(39±11)U/mg;扫描电镜结果显示AMY032使生玉米淀粉的表面产生明显凹陷。     

蜡样芽孢杆菌亮氨酸脱氢酶基因在大肠杆菌中的表达及重组酶性质

本研究采用PCR技术从蜡样芽孢杆菌Bacillus cereus基因组DNA中克隆出亮氨酸脱氢酶基因,构建重组表达质粒p ET28α(+)-ldh,实现在大肠杆菌中的高效表达,并分析重组亮氨酸脱氢酶的酶学性质。结果表明,从Bacillus cereus成功克隆的亮氨酸脱氢酶编码基因约为1 000 bp,表达的重组亮氨酸脱氢酶相对分子质量约为40 k Da。酶学研究结果表明:该酶的最适反应温度为37℃,其热稳定性好,30℃的半衰期长达330 h;最适反应p H为9.5;在p H 7.0~8.0的缓冲液中保存24 h后仍保持原有酶活力的80%以上;金属离子Fe2+对该酶具有明显的促进作用,而EDTA强烈抑制亮氨酸脱氢酶的活性。动力学分析结果表明该酶对底物NADH催化的Km和Vmax分别为0.635 mmol/L和1.54μmol/(L·min)。亮氨酸脱氢酶基因在大肠杆菌中的成功表达为手性氨基酸的生物合成提供了可能。     

一种不对称水解（R,S）-2,6-二甲基苯基氨基丙酸甲酯的新酯酶基因的克隆表达和酶学性质研究

本文将来自反硝化无色杆菌Achromobacterdenitrificans1104的酯酶基因EHest,转化大肠杆菌中,成功表达了具有不对称水解农药甲霜灵的中间体(R,S)-2,6-二甲基苯基氨基丙酸甲酯( MAP )活性的酯酶EHesterase。用重组酯酶EHesterase催化MAP 的水解,底物浓度50 g/L,反应1h的转化率29.5%,产物( R-酸)的eep 是85.1%。该酶的最适反应pH和温度分别为9.0和50℃,在50℃以下和pH5~9之间具有较好的稳定性。该酶水解MAP 的米氏动力学参数Vm、Km 分别是0.733 g/(L·min)和7.49 g/L。加入10%DMSO对酶EHesterase的立体选择性和催化速度有一定的促进作用。 Cu2+、Fe3+对酶活有明显抑制作用。该酶水解MAP 的活性与水解p-对硝基苯乙酸酯的活性数量级相当,是水解橄榄油活性的333倍。     

芽胞杆菌(Bacillus sp.)YX-1耐有机溶剂葡萄糖脱氢酶的基因克隆与酶学性质

[目的] 从芽胞杆菌(Bacillus sp.)YX-1基因组中克隆出一种有机溶剂耐受型的葡萄糖脱氢酶基因,实现了该基因在大肠杆菌中的高效表达,研究了重组蛋白的酶学性质.[方法] 依据芽胞杆菌属中葡萄糖脱氢酶氨基酸序列的保守性,设计合理引物,钓取来源于Bacillus sp.YX-1的葡萄糖脱氢酶基因,构建诱导型表达载体pET28a-gdh,于大肠杆菌中进行表达.镍柱亲和层析法纯化重组蛋白,考察了重组蛋白的酶学性质.[结果] 葡萄糖脱氢酶基因全长为786 bp,编码261个氨基酸.酶学研究结果表明:该酶最适反应温度为45℃,最适pH值为8.0;具有良好的有机溶剂耐受性,于50％的辛烷、环己烷、癸烷中室温放置1h后,酶活仍能保持90％以上;具有较宽的底物谱,对多种糖均具有一定的催化活性,其中催化D-葡萄糖的活力最高,产生还原型辅酶因子;对辅酶NADH和NADPH具有相似的依赖性,对NAD+和NADP+的催化比活分别为8.37 U/mg和8.62 U/mg.[结论]利用生物信息学成功地挖掘出Bacillus sp.YX-1一种耐有机溶剂的葡萄糖脱氢酶,为氧化还原酶在有机相反应中的的辅酶再生循环提供了新型的生物催化剂.     

嗜酸喜温硫杆菌硫加氧还原酶基因的克隆、表达及酶活性研究

旨为研究嗜酸喜温硫杆菌硫加氧还原酶的生化特性,以嗜酸喜温硫杆菌TST3的基因组DNA为模板,PCR扩增出硫加氧还原酶基因(sor),构建了表达载体p ET-sor,转化到Escherichia coli BL21(DE3)后,获得了重组菌株E.coli BL21(p ET-sor2)。SDS-PAGE实验证明,经IPTG诱导,该重组菌可以表达目的蛋白SOR。对诱导条件进行优化,并在最优的条件下诱导SOR酶表达,再经超声波破碎重组菌,上清液通过Ni+-NTA亲和层析柱纯化获得重组酶,其催化氧化反应的比酶活、Km值和Vmax分别为0.70U/mg,15.672×10-2 g/m L和12.755×10-5 mol/(L·min),而催化的还原反应的比酶活、Km值和Vmax分别为2.21 U/mg,0.507×10-2g/m L和4.876×10-5 mol/(L·min)。     

乙酸钙不动杆菌磷脂酶C在大肠杆菌中重组表达、纯化及酶学性质分析

【目的】构建乙酸钙不动杆菌(Acinetobacter calcoaceticus)ATCC17902磷脂酶C(Phospholipase C,PLC)的重组大肠杆菌菌株、纯化重组酶并进行酶学性质分析比较。【方法】以A.calcoaceticus ATCC17902基因组DNA为模板,PCR扩增得到两段磷脂酶C基因(PLC1、PLC2),构建重组大肠杆菌表达质粒并转化大肠杆菌BL21(DE3)中,实现PLC1、PLC2基因的表达。IPTG诱导表达后,经镍柱亲和层析纯化重组蛋白。【结果】成功构建两株产磷脂酶C的重组大肠杆菌并纯化,样品经SDS-PAGE分析在80 kDa附近均出现显著的特异性条带。NPPC法测得PLC1、PLC2酶活分别为31160±418 U/mg、13640±354 U/mg,最适反应温度分别为65、50℃,最适pH值分别为8、7.5。在低于30℃时,pH值7-8时,PLC1、PLC2重组酶较稳定,40℃处理30min,PLC1酶活稳定而PLC2残余酶活低于25%。Mg2+、Ca2+增强PLC1、PLC2的活性,Zn2+增强PLC1酶活性却抑制PLC2酶活。底物特异性分析表明PLC1、PLC2均水解磷脂酰肌醇(Phosphatidylinositol,PI),对其他种类磷脂不能水解或水解程度很低。【结论】本文首次实现了A.calcoaceticus ATCC17902来源的磷脂酶C的重组表达与功能验证,为其它食品安全性微生物来源的磷脂酶C的研究提供了一定的借鉴意义。     

产酸克雷伯氏菌M5al普鲁兰酶的异源表达及酶学性质研究

普鲁兰酶(EC 3.2.1.41)是一类淀粉脱支酶,能够特异性水解淀粉中的α-1,6-糖苷键,从而提高淀粉的利用率,在以淀粉为原料的食品、纺织、生物燃料和洗涤剂等行业中具有重要的应用价值。本研究以产酸克雷伯氏菌Klebsiella oxytoca M5al基因组DNA为模板,将PCR扩增得到的普鲁兰酶基因pul A克隆至表达载体p ET28a(+),构建好的重组质粒转化大肠杆菌Escherichia coli BL21(DE3),在培养基中添加0.5 mmol/L异丙基硫代半乳糖苷(IPTG)的条件下对该酶基因进行诱导表达,经镍柱纯化获得重组普鲁兰酶用于酶学性质研究。SDS-PAGE及Western Blot检测显示普鲁兰酶基因pul A在上述大肠杆菌宿主中成功获得了表达。该重组酶最适反应p H5.5,最适温度60℃。金属离子对酶活性有一定影响。Mn2+对酶活促进作用显著;Fe3+、Mg2+、Fe2+对酶活只有微弱的促进作用,而Cu2+对酶活造成强烈抑制。来源于Klebsiella oxytoca M5al的普鲁兰酶最适催化条件符合工业生产中淀粉糖化工艺的要求,具有应用于淀粉工业的潜力。     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37℃下在pH8～10的缓冲液中保温1h酶活几乎不改变。重组酶反应的最适温度为75℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的KmKG为7.559mmol/L,VmaxKG为0.086mmol/(L·min),KmAsp为2.031mmol/L,VmaxAsp为0·024mmol/(L·min)。Ca2 、Fe3 、Mn2 等金属离子对酶活性有微弱抑制作用。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.