乙酰短杆菌中核苷磷酸化酶的性质研究

目的：以乙酰短杆菌完整细胞为酶源,研究不同条件下核苷磷酸化酶的性质。方法：将乙酰短杆菌湿茵体置于不同保藏温度及在不同种类缓冲溶液中考察其稳定性;在有或无核保护下核苷磷酸化酶对热的稳定性;并设计核苷的磷酸解反应或合成反应,测定核苷磷酸化酶的活力及酶促反应的袁观米氏常数。结果：乙酰短杆菌中的核苷磷酸化酶经低温保藏可以保持较长时间的稳定性;茵体在60℃处理1小时即失去核苷磷酸化酶的活力,但是添加胸腺嘧啶有明显的保护作用;茵体中核苷磷酸化酶的合成能力明显大于磷酸解能力;对尿苷和5-甲基尿苷的表观米氏常数和最大反应速率分别为16．7、11．4mm01／L,0．0063、0．0041mmol／L．min。结论：含核苷磷酸化酶的乙酰短杆菌完整细胞作为酶源,在低温下可以长时间保藏,反应中的碱基对核苷磷酸化酶的抗热性有益,该菌种可以作为工业上核苷磷酸化酶的来源。     

合成唾液酸乳糖重组大肠杆菌的构建

唾液酸寡糖具有抗感染、提高免疫力、促进双歧杆菌增殖等功能,对其微生物合成方法的研究具有重要的理论和应用价值。克隆和表达来源于Campylobacter jejuni的N-乙酰葡萄糖胺异构酶基因(neuC)、乙酰神经氨酸合成酶基因(neuB)、CMP-乙酰神经氨酸合成酶基因(neuA)和来源于Nesseria meningitidis的唾液酸转移酶基因(nst),利用表达载体pSTV29,在大肠杆菌Escherichia coli JM109内构建合成唾液酸乳糖的代谢途径。在接种量2%、装液量50 mL/250 mL三角瓶、摇床转速180 r/min、温度34℃的条件下发酵30 h,并以10 g/L乳糖为底物,利用HPLC检测到唾液酸乳糖的合成量为2.45 g/L,为人乳唾液酸寡糖及其类似物的异体合成提供了新的思路。     

RP-HPLC法测定长期盐胁迫下盐爪爪和盐穗木中的甜菜碱

通过对影响甜菜碱提取和测定的各种因素进行研究,建立了一种准确、快速的测定长期盐胁迫下盐爪爪和盐穗木中甜菜碱的HPLC方法。样品采用超声波甲醇提取,并依次通过强阴阳离子交换树脂(Dowex 50×8和Dowex 50×2)进行纯化,得到的样品采用反相C18柱进行分离,色谱条件:流动相为50 mmol/L、pH 4.5的KH2PO4溶液;波长200 nm下紫外测定,流速0.7 mL/min;此方法的检出限为0.02μg/mL,平均回收率为97.2%。通过对0~500 mmol/L NaCl胁迫下的盐爪爪和盐穗木中甜菜碱进行测定,甜菜碱的含量均呈现先增加后减少的趋势,盐爪爪在NaCl浓度为200 mmol/L时达到最大,而盐穗木在NaCl浓度为300 mmol/L时达到最大。     

圆红冬孢酵母菌发酵产油脂培养基及发酵条件的优化研究

采用均匀设计和单因子试验法,系统考察了圆红冬孢酵母菌(Rhodosporidiumtoruloides)在不同碳氮比条件下产油发酵情况以及添加无机盐对产油发酵的影响,通过均匀设计软件对二次多项回归方程求解及单因素分析得知在培养基组成分别为葡萄糖70g/L,硫酸铵0.1g/L,酵母粉0.75g/L,磷酸二氢钾0.4g/L,七水硫酸镁1.5g/L,初始pH6.0,在灭菌(121℃15min)后添加ZnSO41.91×10-6mmol/L、CaCl21.50mmol/L、MnCl21.22×10-4mmol/L、CuSO41.00×10-4mmol/L。发酵摇瓶装液量为250mL三角瓶装培养基50mL,接种量为10%(种龄28h)。在上述条件下,30℃振荡(200r/min)培养120h,所得菌体油脂含量高达76.1%,脂肪得率系数可达22.7。     

利用固定化原生质体生产L—谷氨酸

在含有生物素(50μg/L)的培养基中培养的黄色短杆菌(Brevibacterium flavum),经溶菌酶处理后制得其原生质体,再固定在琼脂—乙酰纤维素基质的滤器中。该固定化原生质体可用于葡萄糖、尿素为原料的间歇系统中生产L—谷氨酸。在最佳条件下,其L—谷氨酸的生产能力高于固定化细胞的2.5倍,最大生产能力开始可达到1.5mg/ml。该固定化原生质体能够重复使用六次,其生产能力还能保持初始生产能力的70%左右。     

ATP合成菌株的构建及用于联合生产S-腺苷甲硫氨酸

为降低S-腺苷甲硫氨酸的生产成本,构建了同时表达腺苷激酶、腺苷酸激酶和乙酸激酶3种酶的重组大肠杆菌菌株用于ATP的合成,并对ATP的转化条件进行了优化,优化后的反应体系为:腺苷30 mmol/L,乙酰磷酸二锂盐135 mmol/L,硫酸镁5 mmol/L,硼砂50 mmol/L,菌体2 g/L(湿重),反应液初始pH7.5,反应温度为35℃,反应时间为3 h,反应转化率可以达到99%以上。按照上述反应体系进行5 L放大,反应结束后再投入65 mmol/L D,L-甲硫氨酸和50 g/L(湿重)表达腺苷甲硫氨酸合成酶的重组大肠杆菌菌体,并补加15 mmol/L硫酸镁,转化18 h S-腺苷甲硫酸浓度能达到8.7 g/L,转化率达到72.5%。     

短小芽胞杆菌产碱性木聚糖酶发酵条件的研究

碱性木聚糖酶在碱性条件下催化水解木聚糖,广泛应用于造纸、纺织等领域.着重对短小芽胞杆菌M-11产碱性木聚糖酶的发酵条件进行初步的探索.研究了菌株的生长曲线、确定最佳接种龄为16 h、最佳接种量为1％;确定最适碳源浓度为7％、最适单一氮源为氯化铵、其浓度为1.0％、最适无机盐为氯化铁、其浓度为3 mmol/L;在此基础之上进行6因素3水平的正交试验,确定最适产酶培养基组成:麸皮5％,接种量3％,氯化铵1.2％,氯化铁3.5 mmol/L,硫酸镁0.03％,氯化钠5 mmol/L,磷酸氢二钾0.4％;最适培养条件:接种龄16 h,初始pH 8.0,温度37℃,300 mL摇瓶装液量50 mL,摇床转速220 r/min,发酵周期48 h.通过对发酵条件的优化使发酵液酶活达613 IU/mL.无机氮源为其最适氮源,因此短小芽胞杆菌M-11在碱性木聚糖酶的产品开发上优于短小芽胞杆菌M -26.     

钝齿棒杆菌中异源表达N-乙酰鸟氨酸脱乙酰基酶合成L-鸟氨酸的研究

目的:对一株产鸟氨酸的钝齿棒杆菌Corynebacterium crenatum SYPA5-5/△proB/△argF(SYPO-1)进行代谢工程改造,筛选不同细菌来源的N-乙酰鸟氨酸脱乙酰基酶在大肠杆菌中克隆与表达,纯化后对其进行酶学性质的比较;将黏质沙雷氏菌Serratia marcescens Y213来源的Smarg E基因编码的N-乙酰鸟氨酸脱乙酰基酶在L-鸟氨酸生产菌株C.crenatum SYPO-1中过量表达,进一步提高L-鸟氨酸的产量。方法:通过利用pDXW10穿梭质粒对不同来源的N-乙酰鸟氨酸脱乙酰化酶进行克隆表达和酶学性质比较,选择性质最优来源的N-乙酰鸟氨酸脱乙酰基酶编码基因Smarg E在产L-鸟氨酸重组钝齿棒杆菌中表达,考察重组菌株发酵过程中参数的变化。结果:来源于S.marcescens Y213的N-乙酰鸟氨酸脱乙酰基酶比酶活最高为798.98U/mg,最适pH为7,最适温度为37℃,0.1mmol/L的Mg~(2+)、Li~+、Mn~(2+)促进酶的比酶活提高了50%;在钝齿棒杆菌中表达N-乙酰鸟氨酸脱乙酰基酶酶活达到128.4U/ml,显著提高了钝齿棒杆菌中胞内乙酰基循环水平;5L发酵罐发酵重组菌株96h,L-鸟氨酸的产量达到38.5g/L,比出发菌株,L-鸟氨酸的产量提高了33.2%,产率达0.401g/(L·h)。结论:筛选出最佳来源的N-乙酰鸟氨酸脱乙酰基酶,在鸟氨酸生产菌株C.crenatum(SYPO-1)中过量表达,可以促进鸟氨酸的前体物质N-乙酰鸟氨酸的快速消耗,实现鸟氨酸的积累。     

小菜蛾幼虫肠道细菌肉杆菌的代谢表型分析

【目的】肠道细菌肉杆菌Carnobacterium maltaromaticum是小菜蛾Plutella xylostella幼虫肠道的可培养优势细菌,本研究旨在阐明肉杆菌的代谢表型特征。【方法】采用BIOLOG细胞表型芯片技术系统地研究了肉杆菌的细胞表型;采用PM1-PM10代谢板,对肉杆菌的950种代谢表型进行了测定。【结果】肉杆菌能代谢34.74%的碳源、99.47%的氮源、100%的硫源和79.66%的磷源;高效代谢的碳源为有机酸类和糖化合物类,高效代谢的氮源为氨基酸类和肽类。该肠道细菌未表现出生物合成途径。肉杆菌具有广泛的适应性,能在分别具有高达10%氯化钠、6%氯化钾、5%硫酸钠、20%乙二醇、6%甲酸钠、7%尿素、8%乳酸钠、200 mmol/L磷酸钠(p H 7.0)、200 mmol/L苯甲酸钠(p H 5.2)、100 mmol/L硫酸铵(p H 8.0)、100 mmol/L硝酸钠和100 mmol/L亚硝酸钠的渗透溶液中正常代谢,不能在9%-12%的乳酸钠渗透溶液中代谢;其适应p H值范围为5-10,最适约为10.0。在多种氨基酸的作用下,肉杆菌仅有脱氨酶活性,而无脱羧酶活性。【结论】肉杆菌的代谢特征增加了我们对该肠道细菌的认识,同时为其功能的研究和其与宿主小菜蛾的互作研究提供了信息基础。     

两株内生芽孢杆菌对盐胁迫下大豆幼苗超氧化物歧化酶和过氧化物酶活性影响

【背景】盐胁迫环境严重影响大豆幼苗生长,内生菌可提高作物的抗逆性。【目的】探究接种内生枯草芽孢杆菌127和解蛋白芽孢杆菌133对盐胁迫下大豆幼苗体内超氧化物歧化酶(superoxide dismutase,SOD)和过氧化物酶(peroxidase,POD)活性的影响。【方法】以“徐豆20”为实验材料,采用盆栽实验法,设置对照组、盐胁迫组和盐胁迫接菌组,在人工气候培养条件下,用不同NaCl浓度(50、100、150、200、250和300 mmol/L)处理大豆幼苗,并接种不同OD600值(OD0.33、OD0.50和OD0.75)的菌悬液。【结果】培养14 d,接种枯草芽孢杆菌127的菌悬液OD0.33和OD0.75分别在盐浓度300 mmol/L和100 mmol/L时,SOD活性均为1.04 U/g-FW;接种解蛋白芽孢杆菌133的菌悬液OD0.50在盐浓度300 mmol/L胁迫下POD活性最高为7 820 U/(g·min),对大豆幼苗修复效果较显著。培养28 d,接种枯草芽孢杆菌127的菌悬液OD0.50,在150 mmol/L时SOD活性最高(0.88 U/g-FW);接...     

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.