黑曲霉菊糖酶的纯化及性质

黑曲霉(Aspergillus niger)319发酵液经硫酸铵分级沉淀、DEAE-纤维素52离子交换层析和Sephadex G-100分子筛层析,得到了电泳纯的菊糖酶组分。提纯倍数为67,收率为25.5％。菊糖酶的最适pH为5.0,最适温度为60℃。此酶为单亚基蛋白,凝胶过滤法测得分子量为28000,含糖13.9％,用等电聚焦法测得等电点为5.4,该酶对温度有较高的稳定性,对pH的稳定范围较窄。Hg²⁺、Pb²⁺和Cu²⁺对该酶有强烈的抑制作用。此酶对菊糖有较强的底物专一性,产物为果糖,但它也可作用于蔗糖,I/S值为0.348。当以菊糖为底物时,K_m为6.25mmol/L,V_m为67.11 μmol·mg~(-1)·min~(-1)。     

黑曲霉纤维素酶的化学组成

本文测定的黑曲霉纤维素酶各组分均为糖蛋白,但含糖量和组成各不相同,含糖量分别为：β－葡萄糖苷酶１１．３％,内切β－葡聚糖酶１１．８％,β－葡聚糖纤维二糖水解酶ＣＢＨ—１７．２％、ＣＢＨ－Ⅱ５．８％。各酶组分的氨基酸组成有一定的差异,但也有一些共同之处,即都含有较多的酸性氨基酸（Ａｓｐ、Ｇｌｕ）,碱性氨基酸（Ｈｉｓ、Ａｒｇ）含量较低。分析酶组分间的相似性时发现,β－葡聚精纤维二糖水解酶的ＣＢＨ－Ⅰ组分与内切β－葡聚糖酶在各种氨基酸的含量上较为相似,而同属于β－葡聚糖纤维二糖水解酶的两个组分ＣＢＨⅠ和ＣＢＨ—Ⅱ在氨基酸的含量上有一定的差异。     

黑曲霉几丁质和壳聚糖的研究

通过正交试验确定黑曲霉的最佳培养条件。由培养的黑曲霉湿菌体制备几丁质,最经济的方法是电解法;制备壳聚糖则采用间歇提取法,所得干燥壳聚糖产品的壳聚糖含量为９２．２９％,游离氨基为９６．１１％,１％壳聚精的１％醋酸溶液运动粘度为５．３３ｘ１０^－６ｍ^２／ｓ,粘均分子是为８．０２４ｘ１０^４,灰分为９．２４％,产品得率为９．７２％,探讨了从柠檬酸厂废弃酸渣中分离黑曲霉菌丝体的方法,得率为３０％左右。     

黑曲霉蛋白酶缺陷突变体的分离和鉴定

对黑曲霉(Aspergillus niger)3.795进行紫外诱变和筛选,获得了胞外蛋白酶缺失的菌株。其中突变株37951、3795123和3795130的生长特性与原株基本相同,胞外蛋白酶的活性明显低于原株,葡萄糖淀粉酶的活性与原株基本相同。突变株3795123和3795130的胞外蛋白酶活性分别为原株的54%和84%,可作为外源基因高效分泌表达的宿主菌。     

黑曲霉纤维素酶系中β-葡聚糖纤维二糖水解酶的提纯和性质

通过硫酸铵分段,Sephadex G-100凝胶过滤及DEAE-SephadexA-25(A-50)离子交换柱层析等提纯步骤,从黑曲霉(Aspergillus niger)培养液中分离到β-葡聚糖纤维二糖水解酶的两个组分(Ⅰ-3a,Ⅰ-3b),经凝胶电泳鉴定均为单一带,Ⅰ-3a和Ⅰ-3b的最适pH分别为4.0及4.5,最适温度50℃及45℃,在pH2.0—8.0之间稳定,保温1h时的半失活温度t 1/2分别为52℃及48℃。SDS-凝胶电泳法测得Ⅰ-3a和Ⅰ-3b的分子量分别为57000及55000;聚丙稀酰胺凝胶等电聚焦法测得二者的等电点分别为3.2和3.0。在所测定的化学试剂中,Ag~+、Hg~(2+)和Cu~(2+)对该酶均有较强的抑制作用。     

黑曲霉突变株葡萄糖淀粉酶的底物特异性

黑曲霉(Aspergillus niger)突变株T-21葡萄糖淀粉酶(GAI)仅能水解多种淀粉及麦芽低聚糖生成唯一产物β-葡萄糖,其水解麦芽糖及麦芽三糖的速度分别为200和570mg葡萄糖·h~(-1)·mg~(-1).GAI水解α-1,4键的速度比水解α-1.6键快100多倍.除了马铃薯淀粉外,对其它淀粉及麦芽低聚糖几乎都能100%地水解,但不能水解环状糊精,其水解各麦芽低聚糖的最先产物都比原底物少一个葡萄糖单位,说明GAI为一外切型淀粉酶.GAI对麦芽糖、麦芽三糖、可溶性淀粉、糯米淀粉、糊精及糖原的Km值分别1.92mmol/L、0.38mmol/L、0.053%、0.045%、0.059%、及0.076%,V_(max)分别为590、1370、1270、1520、1120和1220mg葡萄糖·h~(-1)·mg~(-1).D-葡萄糖酸-δ-内酯及麦芽糖醇对此酶分别具有反竞争性抑制和混合性抑制.     

黑曲霉M89菊粉酶的提纯与性质

黑曲霉(Aspergillusniger）M89菊粉酶经硫酸铵分级盐析、SephadexG-200凝胶过滤、DEAE-纤维素离子交换层析、聚丙烯酞胺凝胶电泳（PAGE）制备分离,提纯到4个菊粉酶组分EⅠ、EⅡ、EⅢ和EⅣ。用SDS-PAGE测定分子量分别为102．6、97．9、61．2和36．5kD;用等电聚焦电泳测得其等电点分别为4．15、4．24、4．48和4．15。4个组分的最适反应温度均为55～60℃;EⅠ的最适pH为pH4．0,其余3个组分为pH4．5．各组分的热稳定性有一定差异,分子量越小的组分,热稳定性越好,55℃处理90min,EⅠ有一定的热失活,其余3个组分无活力丧失,4个组分都是外切酶。     

黑曲霉A3木聚糖酶酶学性质研究*

黑曲霉A3（AspergillusnigerA3）的固体培养物浸出液,经过多步分离纯化后,获得三个组份的木聚糖酶,称为xⅠ、xⅡ和xⅢ。经7％凝胶浓度的盘状电泳分析均为单一组份。经等电聚焦电泳测xⅠ、xⅡ和xⅢ的等电点分别为6.8、5．5和6.1。SDS－PAGE测得亚基分子量（Da）分别为xⅠ,42000;xⅡ,20000;xⅢ,3000。三个酶组份的最适反应温度分别为xⅠ,40℃i;xⅡ,50℃;xⅢ,50℃。最适反应pHxⅠ,3．5;xⅢ,4．5;xⅢ,5．0。保温一个小时后,酶的半失活温度分别为xⅠ,55.6℃;xⅡ,54.8℃;xⅢ,46.6℃。金属离子Ag+、Hg2+、Ni2+和脲对不同的酶组份具有一定的影响。     

黑曲霉A3木聚糖酶酶学性质研究

黑曲霉A3(AspergillusnigerA3)的固体培养物浸出液,经过多步分离纯化后,获得三个组份的木聚糖酶,称为xⅠ、xⅡ和xⅢ.经7%凝胶浓度的盘状电泳分析均为单一组份.经等电聚焦电泳测xⅠ、xⅡ和xⅢ的等电点分别为6.8、5.5和6.1.SDS-PAGE测得亚基分子量(Da)分别为xⅠ,42000;xⅡ,20000;xⅢ,31000.三个酶组份的最适反应温度分别为xⅠ,40℃;xⅡ,50℃;xⅢ,50℃.最适反应pHxⅠ,3.5;xⅡ,4.5;xⅢ,5.0.保温一个小时后,酶的半失活温度分别为xⅠ,55.6℃;xⅡ,54.8℃;xⅢ,46.6℃.金属离子Ag+、Hg2+、Ni2+和脲对不同的酶组份具有一定的影响.     

EFFECTS OF DIFFERENT LEVELS OF INBREEDING ON PROGENY FITNESS IN PLANTAGO CORONOPUS

Inbreeding depression (δ) is a major selective force favoring outcrossing in flowering plants. Many phenotypic and genetic models of the evolution of selfing conclude that complete outcrossing should evolve whenever inbreeding depression is greater than one-half, otherwise selfing should evolve. Recent theoretical work, however, has challenged this view and emphasized (1) the importance of variation in inbreeding depression among individuals within a population; and (2) the nature of gene action between deleterious mutations at different loci (epistasis) as important determinants for the evolution of plant mating systems. The focus of this study was to examine the maintenance of inbreeding depression and the relationship between inbreeding level and inbreeding depression at both the population and the individual level in one population of the partially self-fertilizing plant Plantago coronopus (L.). Maternal plants, randomly selected from an area of about 50 m² in a natural population, were used to establish lines with expected inbreeding coefficients (f) of 0, 0.25, 0.50, 0.75, and 0.875. Inbreeding depression was estimated both in the greenhouse and at the site of origin of the maternal plants by comparing growth, survival, flowering, and seed production of the progeny with different inbreeding coefficients. No significant inbreeding depression for these fitness traits was detected in the greenhouse after 16 weeks. This was in strong contrast to the field, where the traits all displayed significant inbreeding depression and declined with increased inbreeding. The results were consistent with the view that mutation to mildly deleterious alleles is the primary cause of inbreeding depression. At the family level, significantly different maternal line responses (maternal parent × inbreeding level interaction) provide a mechanism for the invasion of a selfing variant into the population through any maternal line exhibiting purging of its genetic load. At the population level, evidence for synergistic epistasis was detected for the probability of flowering, but not for total seed production. At the family level, however, a significant interaction between inbreeding level and maternal families for both traits was observed, indicating that epistasis could play a role in the expression of inbreeding depression among maternal lines.     

对硫磷真菌降解酶的分离纯化和性质

黑曲霉AspergillusnigerY－8在液体培养基中30℃培养5d,其菌体用超声波破碎后,经硫酸铵沉淀、DEAE-纤维素层析、Sephadex－100凝胶过滤,得到凝胶电泳均一的对硫磷降解酶,其比活为6.94,提纯倍数为13.6倍,收率为17．4％。酶作用的最适温度是50℃,最适pH为7.5,在40℃以下和pH6.0～9.0之间稳定,此酶为单亚基蛋白,凝胶过滤法测得分子量为42000,含糖14.6％,SDS、Hg2+、Ag+、Fe3+对酶有强烈的抑制作用,金属螫合剂EDTA对酶活无影响。此酶对甲基对硫磷、敌敌畏、亚胺硫磷也有较好的降解作用,当以对硫磷为底物时,Km为0.43mmol／L,Vmax为1.24μmol·mg-1·min-1。     

黑曲霉N25植酸酶phyA基因在巴斯德毕赤酵母中高效表达的研究

从黑曲霉N25(A.niger China strain)中提取出染色体DNA,根据已经测定出的植酸酶phyA基因全序列设计了一对引物,采用高保真度的聚合酶Advangage-HF扩增到了去除信号肽和内含子后约1.4kb片段,对该片段进行了克隆及序列测定。将该序列与植酸酶phyA基因全序列进行了比较。以此片段构建成功了pPIC9K-phyA载体(命名为pPNP-1),并转化毕赤巴斯德酵母。经G418抗性筛选、酶活性测定、Southern印迹和Western印迹,获得了高效表达的转化子PP-NP-1(23869.4u/ml)、PP-NP-2(20533.0u/ml)、PP-NP-3(35646.7u/ml),其酶活性分别是出发菌株的酶活(513.4u/ml)的46.46倍、39.99倍和69.46倍,且转化子具有很好的遗传稳定性。     

GENETIC DRIFT IN ANTAGONISTIC GENES LEADS TO DIVERGENCE IN SEX‐SPECIFIC FITNESS BETWEEN EXPERIMENTAL POPULATIONS OF DROSOPHILA MELANOGASTER

Males and females differ in their reproductive roles and as a consequence are often under diverging selection pressures on shared phenotypic traits. Theory predicts that divergent selection can favor the invasion of sexually antagonistic alleles, which increase the fitness of one sex at the detriment of the other. Sexual antagonism can be subsequently resolved through the evolution of sex‐specific gene expression, allowing the sexes to diverge phenotypically. Although sexual dimorphism is very common, recent evidence also shows that antagonistic genetic variation continues to segregate in populations of many organisms. Here we present empirical data on the interaction between sexual antagonism and genetic drift in populations that have independently evolved under standardized conditions. We demonstrate that small experimental populations of Drosophila melanogaster have diverged in male and female fitness, with some populations showing high male, but low female fitness while other populations show the reverse pattern. The between‐population patterns are consistent with the differentiation in reproductive fitness being driven by genetic drift in sexually antagonistic alleles. We discuss the implications of our results with respect to the maintenance of antagonistic variation in subdivided populations and consider the wider implications of drift in fitness‐related genes.     

黑曲霉菊糖酶的分离纯化及其活性测定

从一株黑曲霉p319的菊芋培养液中,采用硫酸铵盐析、SephadexG-25、DEAE-cellulose-52、SephadexG-100柱层析等方法,分离纯化得到三个菊糖酶组份EI、EII、EⅢ,三者经聚丙烯酰胺凝胶电泳验证均达到均一。并对其活性进行了初步测定,三者比活分别达到29．8u／mg,4．9u／mg,1．2u／mg。     

EVOLUTION OF THE GENETIC ARCHITECTURE UNDERLYING FITNESS IN THE PITCHER-PLANT MOSQUITO,WYEOMYIA SMITHII

We examined the genetic basis for evolutionary divergence among geographic populations of the pitcher-plant mosquito, Wyeomyia smithii, using protein electrophoresis and line-cross analysis. Line-cross experiments were performed under both low density, near-optimal conditions, and at high, limiting larval densities sufficient to reduce fitness (r_c) in parental populations by approximately 50%. We found high levels of electrophoretic divergence between ancestral and derived populations, but low levels of divergence between two ancestral populations and between two derived populations. Assessed under near-optimal conditions, the genetic divergence of fitness (r_c) between ancestral and derived populations, but not between two derived populations or between two ancestral populations, has involved both allelic (dominance) and genic (epistatic) interactions. The role of dominance and epistasis in the divergence of r_c among populations affects its component traits in a pattern that is unique to each cross. Patterns of genetic differentiation among populations of W. smithii provide evidence for a topographically complex “adaptive landscape” as envisioned by Wright in his “shifting balance” theory of evolution. Although we cannot definitively rule out the role of deterministic evolution in the divergence of populations on this landscape, ecological inference and genetic data are more consistent with a stochastic than a deterministic process. At high, limiting larval density, hybrid vigor is enhanced and the influence of epistasis disappears. Thus, under stressful conditions, the advantages to fitness due to hybrid heterozygosity can outweigh the deleterious effects of fragmented gene complexes. These results have important implications for the management of inbred populations. Outbreeding depression assessed in experimental crosses under benign lab, zoo, or farm conditions may not accurately reveal the increased advantages of heterozygosity in suboptimal or marginal conditions likely to be found in nature.     

柠檬酸产生菌黑曲霉诱变选育的研究

本文以分离到的一株黑曲霉Aspergillus nigerwl-1为出发苗株,采用硫酸二乙酯(DES)、亚硝基胍(NTG)等化学绣变剂处理。选育出一株产柠檬酸较高的变异株A.niger WZ—12,柠檬酸产率由2.9％提高到7.6％,对糖的转化率为63.3％,且不生成其它有机杂酸。     

黑曲霉突变株葡萄糖淀粉酶的化学组成及其光谱学性质

黑曲霉突变株葡萄糖淀粉酶中一型GAI舍糖量为17.6%。氨基酸分析表明,天门冬氨酸和谷氨酸(包括酰胺)占20.3%,苏氨酸和丝氨酸占25.1%,三种碱性氨基酸占6%。紫外光谱在278nm和250nm处分别有最大和最小吸收;其荧光光谱的最大激发波长和发射波长分别为284nm与342nm;远紫外CD谱表现为一双负峰;在溶液中的构象是α-螺旋10.6%,β折叠16.3%和无规卷曲73.1%。     

植酸钠对黑曲霉柠檬酸发酵产酸的促进效应

研究了植酸钠对黑曲霉柠檬酸发酵产酸的促进效应。在葡萄糖全合成培养基中添加１％的植酸钠,可使产酸比对照提高２．４倍;在薯粉、玉米粉等天然培养基中添加１％植酸钠,柠檬酸产量分别提高１．７倍和１．３倍。酶活性测定分析表明,植酸钠对柠檬酸代谢途径中的几种关键酶的活性有影响。     

黑曲霉及其多糖组分诱导红豆杉叶子产生抗病反应之比较

用一种从中国红豆杉树皮内分离得到的黑曲霉及其酸解物多糖组分处理离体红豆杉叶子,发现经过两种不同处理的叶子干重和叶绿素含量均比对照下降。而丙二醛含量上升,但黑曲霉处理的呈现更为明显的趋势,说明多糖和黑曲霉孢子对叶片均有损伤,但多糖造成的损伤较小,多糖组分处理提高酚类物质和可溶性蛋白的含量并激活过氧化物酶和苯丙氨酸解氨酶的活性。而黑曲霉却导致了相反的现象,因此认为多糖组分具有诱导红豆杉叶子产生抗病反应的能力。而黑曲霉却具有致病性。