东方肉座菌EU7-22木聚糖酶和木糖苷酶基因克隆及生物信息学研究

东方肉座菌EU7-22具有高产半纤维素酶的能力。根据已报道的同属里氏木霉及绿色木霉木聚糖酶,木糖苷酶相关基因序列,设计PCR引物扩增出东方肉座菌内切木聚糖酶(XYNⅠ,XYNⅡ)及β-木糖苷酶(β-BXL)基因。序列经NCBIBlast分析:东方肉座菌xynⅠ基因与里氏木霉xyn1基因(X69573.1)的同源性最高达到91%;xynⅡ基因与绿色木霉xyn2基因(EF079061)同源性最高达到93%;β-bxl基因与里氏木霉β-bxl1基因(Z69257.1)的同源性最高达到94%。生物信息学分析表明内切木聚糖酶Ⅰ和Ⅱ均属于糖基水解酶家族11,N末端前19个氨基酸均为信号肽。内切木聚糖酶Ⅰ分子量为24.13kD,等电点为7.87,含有3个糖基化位点;内切木聚糖酶Ⅱ分子量为24.44kD,等电点为4.86,含有1个糖基化位点;β-木糖苷酶属于糖基水解酶家族3,分子量为87.27kD,等电点为5.49,N末端前20个氨基酸为信号肽,含有8个糖基化位点。利用SWISS-Model对木聚糖酶,木糖苷酶蛋白质三级结构进行了预测和模拟。对木聚糖酶和木糖苷酶基因及其编码蛋白质的生物信息学分析,为进一步研究这些基因的表达与调控、构建高效利用纤维素组份的工程菌株奠定基础。     

绿色木霉木聚糖酶的纯化和性质

绿色木霉木聚糖酶经分离纯化后,获得三个组分木聚糖酶,称为XⅠ,XⅡ和XⅢ,它们最反应温度分别为60℃、60℃、50℃,pH分别为5．5、5．0、0、4．5,pⅠ分别为XⅠ3．8,XⅡ3．4,XⅢ3．6。半失活温度分别为XⅠ37℃,XⅡ44℃,XⅢ40℃。     

嗜冷嗜酸β-1,4-木聚糖酶及其钙离子依赖型碳水化合物结合模块

【目的】β-1,4-木聚糖酶是木聚糖降解的关键酶之一,嗜冷嗜酸木聚糖酶在功能性低聚木糖的制备中具有重要作用,但相关报道较少。【方法】从太平洋火色杆菌(Flammeovirga pacifica)菌株WPAGA1基因组发掘到一条新型的木聚糖酶序列,经基因合成、质粒构建和表达,并对其进行分离纯化及酶学性质研究。【结果】该木聚糖酶(Xyl4513)具有2个保守结构域,一个属于糖苷水解酶11家族(glycoside hydrolase family 11,GH11)催化模块(Xyl4513-T),另一个属于碳水化合物结合模块(carbohydrate-binding module,CBM) 60家族(CBM4513),这是一种非常罕见的GH11家族木聚糖酶含有CBM的现象。纯化后的Xyl4513最适反应温度和pH值分别为30℃、3.0,这一特性说明Xyl4513为嗜冷嗜酸β-1,4-木聚糖酶;而截短的木聚糖酶Xyl4513-T最适反应温度和pH值分别为20℃、4.0,且催化效率(k_cat/K_m)较前者下降了20%,说明CBM4513对酶稳定性和催化效...     

粗毛栓菌木聚糖酶的纯化及性质

以麦草粉为基质培养粗毛栓菌Trametes gallica,浸提固态培养物得浸提液,后经超滤浓缩、硫酸铵盐析、Phenyl-Sepharose CL-4B疏水层析、DEAE Sepharose fast flow阴离子交换层析和Sephadex G-150分子筛层析等分离与纯化步骤,获得部分纯化的木聚糖酶,其回收率和纯化倍数分别为1.45%和15.6。进一步经活性-PAGE回收,获得三种SDS-PAGE电泳纯级的木聚糖酶同工酶组分:XⅠ、XⅡ和XⅢ(按等电点从大到小排列)。三种组分分子量均约为19.0kDa;等电点分别为:5.6、4.7和4.0;含糖量分别为:0.25%、0.63%和3.4%;XⅠ既能降解木聚糖,又能降解纤维素;XⅡ的最适作用pH值为5.0,最适作用温度45℃;Mg2+、Fe2+对XⅡ有激活作用;Mn2+和Co2+有抑制作用;测得XⅡ的Km值为0.75mg/mL,Vmax为5,000mmoL/min·mg。     

嗜冷嗜酸β-1,4-木聚糖酶及其钙离子依赖型碳水化合物结合模块

【目的】β-1,4-木聚糖酶是木聚糖降解的关键酶之一,嗜冷嗜酸木聚糖酶在功能性低聚木糖的制备中具有重要作用,但相关报道较少。【方法】从太平洋火色杆菌(Flammeovirga pacifica)菌株WPAGA1基因组发掘到一条新型的木聚糖酶序列,经基因合成、质粒构建和表达,并对其进行分离纯化及酶学性质研究。【结果】该木聚糖酶(Xyl4513)具有2个保守结构域,一个属于糖苷水解酶11家族(glycoside hydrolase family 11,GH11)催化模块(Xyl4513-T),另一个属于碳水化合物结合模块(carbohydrate-binding module,CBM)60家族(CBM4513),这是一种非常罕见的GH11家族木聚糖酶含有CBM的现象。纯化后的Xyl4513最适反应温度和pH值分别为30℃、3.0,这一特性说明Xyl4513为嗜冷嗜酸β-1,4-木聚糖酶;而截短的木聚糖酶Xyl4513-T最适反应温度和pH值分别为20℃、4.0,且催化效率(kcat/Km)较前者下降了20%,说明CBM4513对酶稳定性和催化效率有较大影响。Ca^(2+)、Mg2+和Ni2+对酶催化活性均有明显促进作用,其中Ca^(2+)效果更为明显。仅当含有Ca^(2+)时,CBM4513才对β-1,4-木聚糖具有特异性结合能力,属于Ca^(2+)依赖型CBM,其最大结合量为9.13μmol/g。【结论】本文获得了一种新型的嗜冷嗜酸木聚糖酶和相应的Ca^(2+)依赖型CBM,进一步丰富了它们的基因和蛋白资源。     

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.     

链霉菌zxy19木聚糖酶酶学性质及酶基因克隆

采用平板筛选法,从红树林放线菌中筛选到一株有较强木聚糖酶活的菌株zxy19,其16SrDNA序列与Streptomyces sampsonii的同源性仅为96%.该菌株木聚糖酶活为852.41 IU/mL,酶反应最适pH值为7,最适反应温度为60℃.用针对木聚糖酶基因保守结构域的一对简并引物扩增到该酶基因部分序列,进而通过反向PCR扩增到了完整的酶基因,对该基因序列分析结果表明此木聚糖酶基因属于糖基水解酶家族11的成员,酶蛋白氨基酸序列与已报道序列同源性最高为79%(Streptomyces lividans xylanase B).构建了该酶重组表达质粒pET-28a-xyl 696,经过IPTG诱导实现了该酶蛋白在大肠杆菌BL21(DE3)中的异源表达,且通过镍柱纯化后的表达产物具有生物学活性.     

黑曲霉A3木聚糖酶酶学性质研究

黑曲霉Ａ３（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ Ａ３）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     

同步分泌高效纤维素酶和木聚糖酶菌株YB的鉴定及其酶学性质研究

筛选和鉴定可降解木质纤维素的真菌,并研究其产酶特征。采用刚果红平板涂布法,从荔枝腐叶中筛选具有木质纤维素降解能力的真菌,结合ITS-rDNA序列分析进行鉴定,初步测定其产酶条件,然后采用DEAE Sepharose Fast Flow阴离子交换层析与Sephadex G-100凝胶层析对硫酸铵沉淀的粗酶液进行分离纯化,对其开展酶学性质研究。结果显示,筛选出一株可降解木质纤维素降解的菌株YB,鉴定为绿木霉(Trichoderma virens)。在发酵过程中,纤维素酶和木聚糖酶的最大活力分别为313.53±26.78 U/mL和18 120.87±500.37 U/mL。分离纯化得到纤维素酶(CMC酶)Ⅰb、Ⅳ和木聚糖酶Ⅰa;通过SDS-PAGE检测,其分子量分别为58.5 kD、22.8 kD和44.5 kD。3种酶的最适酶促反应条件均为:50℃,pH 5.0。其中,木聚糖酶能有效降解玉米芯木聚糖为木糖和多种木寡糖。菌株Trichoderma virens YB可分泌高效木质纤维素降解酶,具有应用于木聚糖酶和木寡糖生产的潜力。     

A new fungal genus, Teracosphaeria, with a phialophora-like anamorph (Sordariomycetes, Ascomycota)

The new genus and species Teracosphaeria petroica is described for a perithecial ascomycete and its anamorph occurring on decayed wood collected in New Zealand. The fungus produces immersed, non-stromatic ceratosphaeria-like perithecia in nature, with hyaline, septate ascospores produced in unitunicate, non-amyloid asci. The anamorph produced in vitro is phialophora-like with lightly pigmented phialides terminating in flaring, deep collarettes that are often noticeably brown with conspicuous periclinal thickening. Phylogenetic analysis of LSU rDNA sequence data indicates that this fungus is distinct from morphologically similar fungi classified in the Chaetosphaeriales, the Trichosphaeriales or the Magnaporthaceae. It forms a monophyletic group with recently described, chaetosphaeria-like ascomycetes, such as the pyrenomycete genus Mirannulata, and shows affinity with the anamorphic species Dictyochaeta cylindrospora. The usefulness of describing anamorph genera for morphologically reduced anamorphs, when anamorph characteristics are actually part of the holomorph diagnosis, is discussed. An apparently contradictory example of the so-called Cordana and Pseudobotrytis anamorphs of Porosphaerella spp. is also discussed.     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Evolutionary relationships among aquatic anamorphs and teleomorphs: Lemonniera, Margaritispora, and Goniopila

The hypothesis that similar conidial morphologies in aquatic hyphomycetes are a result of convergent evolution was tested using molecular sequence data. Cladistic analyses were performed on partial sequences of 28S rDNA of seven species of Lemonniera, one species of Margaritispora and one species of Goniopila. Lemonniera has tetraradiate conidia with long arms, whereas Margaritispora and Goniopila have typically globose (isodiametric) conidia, with short conical protuberances in a stellate or quadrangular arrangement. Lemonniera and Margaritispora have phialidic conidiogenesis and both produce dark, minute sclerotia in culture whereas Goniopila has holoblastic conidiogenesis and does not produce sclerotia in culture. Goniopila produces a microconidial phialidic synanamorph in culture. All three genera have schizolytic conidial secession. Molecular analyses demonstrate that Lemonniera species are placed in two distinct clades: one within Leotiomycetes; the other within Pleosporales, Dothideomycetes. Margaritispora is placed with Lemonniera species within Leotiomycetes. Goniopila and Lemonniera pseudofloscula are placed within Dothideomycetes. No morphological character was entirely congruent with the molecular derived phylogeny. This suggests that for the group of species studied, conidial shape is not a reliable indicator of phylogeny but more likely the result of convergent evolution in response to the aquatic environment.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C₁₈ column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5–40.8 mg/m³), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

Taxonomic notes on Pogonieae ( Orchidaceae ): Cleistesiopsis, a new genus segregated from Cleistes , and description of two new South American species, Cleistes batistana and C. elongata

Summary  A new genus, Cleistesiopsis, was segregated from Cleistes based on morphological and molecular characteristics, and two new species of Cleistes: C. batistana and C. elongata, both occurring in the Brazilian Central Plateau (Central-Western Brazil), are described and illustrated. Furthermore, a key to genera currently recognised within Pogonieae is presented.

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Resumo  Um novo gênero, Cleistesiopsis, é segregado de Cleistes com base em caracteres morfológicos e moleculares, e duas novas espécies de Cleistes: C. batistana e C. elongata, que ocorrem no Planalto Central, no Centro-Oeste do Brasil, s?o descritas e ilustradas. Além disso, uma chave para os gêneros que atualmente comp?em a tribo Pogonieae é apresentada.

     

木薯UGT41基因对细菌性枯萎病的抗性研究北大核心CSCD

糖基转移酶广泛存在于植物中,其中UDP依赖型糖基转移酶(UDP-glycosyltransferases,UGTs)基因家族是糖基转移酶中的一大类。该研究以华南124木薯品种(Manihot esculenta cv.SC124)为材料,利用RT-PCR技术克隆木薯MeUGT41基因,以病毒诱导干扰木薯MeUGT41基因的表达量,并对基因干扰植株进行细菌性枯萎病抗性评价,为研究MeUGT41基因在木薯抵抗细菌性枯萎病的抗病机理奠定基础。结果表明:(1)地毯草黄单胞菌(Xamthomonas axonopodis pv.Manihotis,Xam)可显著诱导木薯MeUGT41基因表达。(2)成功构建MeUGT41的病毒诱导基因沉默(VIGS)载体,将干扰载体转化至木薯叶片进行MeUGT41基因沉默,荧光定量PCR检测结果显示,木薯叶片中MeUGT41基因的表达量显著下降。(3)Xam侵染实验表明,干扰抑制MeUGT41基因表达可显著降低木薯植株叶片对Xam病菌侵染的抵抗能力。研究认为,木薯叶片中MeUGT41基因具有抵抗Xam病菌侵染的能力。