C-端引入二硫键对黑曲霉木聚糖酶XynZF-2热稳定性的影响

同源比对黑曲霉XZ-3S木聚糖酶基因xyn ZF-2氨基酸序列,模拟构建木聚糖酶三维结构,确定能够提高酶热稳定性的最佳突变位点。在C-端引入二硫键,突变xyn ZF-2 205位点的色氨酸和52位点的丙氨酸为半胱氨酸,获取突变基因T205C-A52C,表达于大肠杆菌BL21(DE3)。酶学性质比较发现,突变酶Xyn ZFT205C-A52C的最适温度为50℃,比原酶Xyn ZF-2提高了10℃;50℃保温5 min,突变酶相对酶活性为55.36%,原酶相对酶活性为32.62%;原酶与突变酶最适p H均为5.0,但相同p H下突变酶的相对酶活性较原酶高;突变酶的p H稳定区间由原酶的5.0~9.0扩大为3.0~9.0。因此,定点突变T205C和A52C在C-端引入二硫键能提高黑曲霉木聚糖酶Xyn ZF-2热稳定性及p H稳定性。     

N端二硫键对11家族木聚糖酶热稳定性的影响

以来源于米曲霉Aspergillus oryzae的11家族常温木聚糖酶AoXyn11A为母本,将其N端替换成同一家族耐热木聚糖酶EvXyn11TS的对应片段,构建出耐热杂合木聚糖酶AEx11A。将AoXyn11A和AEx11A基因分别在毕赤酵母GS115中进行表达并分析比较温度对表达产物酶活性的影响。结果表明,AEx11A的最适温度Topt为75℃,在70℃的半衰期t1/270为197 min,较AoXyn11A(Topt=50℃,t1/270=1.0 min)显著提高。通过对AEx11A结构的同源建模及其与AoXyn11A结构的比对,发现在AEx11A的N端引入了一个二硫键(Cys5–Cys32)。利用定点突变法将其5位的半胱氨酸突变为苏氨酸(C5T),去除该二硫键,以探讨其对AEx11A热稳定性的影响。分析表明,突变酶(AEx11AC5T)的Topt由突变前的75℃降为60℃,其t1/270和t1/280也分别由197 min和25 min缩短为3.0 min和1.0 min。     

K178M、S170F、G180V突变对木聚糖酶XynZF-2热稳定性的影响

利用生物信息学分析黑曲霉木聚糖酶Xyn ZF-2,选择α-螺旋178位点、170位点和180位点氨基酸进行定点突变(K178M,S170F,G180V)获得突变木聚糖酶基因xyn MFV,构建重组表达载体转化大肠杆菌E.coli BL21(DE3)诱导表达。酶学性质分析对比发现,突变酶最适温度(50℃)比原酶(40℃)提高了10℃;40℃条件下保温1 h突变酶Xyn MFV相对酶活力下降到热处理前的56.0%,原酶Xyn ZF-2下降到42.0%;45℃时突变酶Xyn MFV的半衰期t_(1/2)为21 min,与原酶Xyn ZF-2(t_(1/2)=7 min)相比较提高了14 min。结果表明,K178M、S170F、G180V突变木聚糖酶可以提高Xyn ZF-2最适温度和热稳定性。     

常压室温等离子体快速诱变绿色糖单孢菌筛选 木聚糖酶高产菌株及其酶学性质研究

[目的]利用常压室温等离子体快速诱变绿色糖单孢菌,筛选耐热耐碱木聚糖酶高产菌株,并对其进行酶学性质分析,确保其适用于生物制浆漂白工艺.[方法]采用刚果红平板水解圈法结合摇瓶发酵胞外酶测定法进行菌株筛选,并通过DNS木聚糖酶活性测定等方法对来源于不同突变株的木聚糖酶进行酶学性质分析对比.[结果]筛选出遗传稳定性良好的两株木聚糖酶高产菌株AT24和AT22-2,以麦草浆为诱导底物的粗酶液中,突变株AT24及AT22-2所产的木聚糖酶活性分别为512.74、552.70U/mL,分别为原始菌株S.v的16和17倍的.来源于突变株AT22-2的木聚糖酶的最适反应pH为9.5,最适反应温度为90℃,在50℃-90℃温度范围内具有良好的热稳定性,在100℃条件下处理30 min剩余酶活仍为68％;突变株AT24所产木聚糖酶的最适反应温度为60℃,最适pH为10.0,在60℃-80℃的高温环境下,突变株AT24所产的木聚糖酶具有良好的热稳定性.[结论]突变株AT22-2所产具有耐碱耐高温性质的木聚糖酶,在应用领域尤其在纸浆造纸行业具有较大的潜在应用价值.     

木霉菌株T6木聚糖酶固态发酵条件和酶学性质研究*

研究了碳源和氮源、起始pH、接种量及温度等条件对一野生型木霉Trichoderma sp.T6菌株固态发酵产木聚糖酶的影响。在28℃培养4d后,酶活力可达1918IU/g干培养物。酶的最适反应温度为50℃,最适反应pH4.5。不同温度保温1h后,测定酶的半失活温度为47.7℃,酶的pH稳定性也进行了研究。     

嗜酸青霉酸性木聚糖酶产生特性及部分酶学性质

从煤矿酸性废水中分离到一株产木聚糖酶青霉,通过酸性液体培养研究了菌体生长对pH的响应及木聚糖酶的产生特征,并测定了木聚糖酶的部分应用性质.结果表明:实验菌株嗜酸,菌丝生长最适pH为2.0,孢子萌发生长适宜pH为3.0～4.0;木聚糖诱导菌体在生长稳定期大量产生木聚糖酶,蛋白胨是菌体产酶的适宜氮源;菌株所产木聚糖酶属于酸性木聚糖酶,反应最适pH 3.5、最适温度50 ℃～55 ℃,pH 2.0时酶活达到最高活力的72%,在最适反应条件下保温60 min,残余酶活接近70%,适用于较强酸性的高温加工环境.     

木霉菌株T6木聚糖酶固态发酵条件和酶学性质研究

研究了碳源和氮源、起始pH、接种量及温度等条件对一野生型木霉Trichoderma sp.T6菌株固态发酵产木聚糖酶的影响。在28℃培养4d后,酶活力可达1918IU/g干培养物。酶的最适反应温度为50℃,最适反应pH4.5。不同温度保温1h后,测定酶的半失活温度为47.7℃,酶的pH稳定性也进行了研究。     

木霉菌株T6木聚糖酶固态发酵条件和酶学性质研究

研究了碳源和氮源、起始pH、接种量及温度等条件对一野生型木霉Trichodermasp.T6菌株固态发酵产木聚糖酶的影响。在28℃培养4d后,酶活力可达1918IU／g干培养物。酶的最适反应温度为50℃,最适反应pH4．5。不同温度保温1h后,测定酶的半失活温度为47．7℃,酶的pH稳定性也进行了研究。     

硫色曲霉木聚糖酶基因xynA的克隆、表达及酶学性质分析

目的:分析硫色曲霉产木聚糖酶的酶学性质。方法:利用RT-PCR克隆了硫色曲霉的木聚糖酶基因xynA,构建了该基因的原核表达载体,并获得了诱导表达。结果:纯化的木聚糖酶的最适催化温度为50℃,最适反应pH值为2.4,在30～50℃保温30min对酶活性没有大的影响。结论:木聚糖酶在低pH值下的催化活性使之在饲料工业中具有较好的应用前景。     

黑曲霉产木聚糖酶固态发酵条件及部分酶学性质的研究

实验以棉粕和玉米秆为主要原料,采用单因素和正交实验方法对黑曲霉固态发酵产木聚糖酶的培养条件进行了优化,为了获得高酶活产品的发酵条件。结果表明,最适培养基组分为棉粕和玉米秆的比例为3∶2,固水比为1∶1.2,尿素的最适添加量为2%(以干重计),KH2PO4的最适添加量为0.2%。在此条件下,菌株产酶活性可达6 529U/g干曲。该酶的最适反应温度为55℃,最适pH为5.0,pH稳定范围较宽,在30℃、pH 3.5~6.0范围内处理100min,酶活保持在85%以上,但耐热性不是很理想,在60℃保温30min残余酶活只有17%。     

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.