产朊假丝酵母细胞壁对铜离子吸附机理研究

比较了产朊假丝酵母细胞与分离纯化的细胞壁对铜离子吸附能力。观察铜离子浓度、温度和pH值对产朊假丝酵母吸附铜离子的影响,探讨细胞壁在酵母吸附重金属离子过程中的作用机理。结果表明,细胞壁是酵母吸附重金属离子的主要部位。细胞壁的蛋白酶酶解实验证明,对胰蛋白酶不敏感的细胞壁嵌合蛋白是铜离子吸附的主要位点。     

冷激蛋白的研究进展

冷激蛋白（CSPs）广泛存在于革兰氏阳性菌和阴性菌中,它是细胞在应对冷刺激时所产生的一系列7 ku左右的蛋白质,结构上富含芳香族氨基酸,起着重要的分子伴侣作用,能够增强细胞抵御冷激环境胁迫的能力.以大肠杆菌、枯草杆菌、嗜热链球菌、沙门氏杆菌等为例,介绍各种冷激蛋白在产生、结构、调控等方面的异同,以及它在生产、生活中的应用价值.     

草菇乙酰木聚糖酯酶的生物信息学分析

以草菇（Volvariella volvacea）乙酰木聚糖酯酶AXE、AXEII为研究对象,采用生物信息学方法对其核苷酸及编码的蛋白氨基酸序列、组成成分、信号肽、跨膜结构域、疏水性/亲水性、蛋白质三级结构等进行预测和分析,并构建了乙酰木聚糖酯酶蛋白家族的系统进化树。结果表明,草菇的AXE蛋白由349个氨基酸残基组成,编码蛋白质的分子量为37.55 ku,等电点为8.58,属稳定性蛋白质。AXEII由253个氨基酸残基组成,编码蛋白质的分子量为27.70 ku,等电点为5.82,属稳定性蛋白质。两者均为亲水性蛋白,存在信号肽,AXE与AXEII的潜在磷酸化位点总数分别为15个和11个。草菇的AXE与灰盖鬼伞（Coprinopsis cinerea）亲缘关系较近,AXEII与裂褶菌（Schizophyllum commune）亲缘关系较近。     

伪狂犬病病毒闽A株gE基因去信号肽片段在Pichia pastoris中的表达

伪狂犬病病毒囊膜糖蛋白E是一种在伪狂犬病根除计划中具有重要作用的糖蛋白.将伪狂犬病病毒闽A株gE基因去信号肽片段克隆到巴斯德毕赤酵母（Pichia pastoris）表达载体pPICZαA中,获得的重组表达载体pPICZαA-FL电击转化野生型酵母菌SMD1168后,得到多株酵母工程菌SMD1168/pPICZαA-FL.经高浓度Zeocin^TM筛选、表型鉴定、工程菌的诱导表达及表达产物的鉴定,最后得到高效表达gE基因去信号肽片段的酵母工程菌SMD1168/pPICZαA-FL-7.工程菌72 h培养上清的SDS-聚丙烯酰胺凝胶电泳与蛋白质印迹结果显示,gE基因去信号肽片段表达产物大小约为80 ku,比预期的63.8 ku大.凝胶薄层扫描结合Bradford蛋白质总含量测定结果表明,表达产物占工程菌培养上清总蛋白的13.49%,表达量可达11.7 mg/L.间接ELISA结果表明重组表达产物具有良好的抗原性,能够有效地区分伪狂犬病病毒gE标准阳性与阴性血清.     

植物细胞壁研究进展

植物细胞壁是一种复杂的网状结构,其成分包含纤维素、半纤维素、果胶和少量的结构蛋白等。在植物细胞生长过程中,细胞能产生伸展素蛋白,打断纤维素和半纤维素之间的氢键,引起细胞膨压驱动的细胞壁扩张。成熟细胞壁扩张性的丧失是由于细胞壁硬化作用而对扩张性蛋白的作用不敏感造成的,细胞壁成熟过程中很多不同的连接会同时发生,当细胞壁基质多聚体分子之间的连接增加到一定的程度。细胞壁的伸长就会被完全抑制。     

产朊假丝酵母细胞和细胞壁对铜离子吸附条件研究

观察温度和pH 值对产朊假丝酵母细胞与分离纯化的细胞壁对铜离子吸附的影响,探讨细胞壁在酵母吸附重金属离子过程中的作用pH 升高,细胞和细胞壁对铜离子的吸附能力都提高,吸附最适pH 为6-0 。温度升高可提高细胞和细胞壁的吸附能力,最适温度为50 ℃。细胞壁是铜离子吸附的主要部位,细胞壁嵌合蛋白(33 ×103蛋白) 起重要作用。     

亲和层析法分离鉴定人类端粒酶复合体及其蛋白质组分分析

为分离纯化人类端粒酶复合体并对其蛋白质组分进行分析 ,自行设计一套特异的以端粒重复序列为配体 ,以亲和磁珠为介质 ,以竞争性碱基序列为洗脱原则的寡核苷酸亲和纯化法 ,对He La细胞端粒酶复合体进行分离纯化 .并采用近年发展起来的主要用于检测序列特异性 DNA结合蛋白的凝胶迁移阻滞分析法对纯化产物进行鉴定分析 .应用 SDS- PAGE对纯化蛋白质亚基组分进行分析与鉴定 ,并采用电泳迁移率和已知蛋白质分子质量标准的对数作图分析测得所得蛋白质亚基成分的相对分子质量 .结果表明 ,纯化产物以 TRAP法检测酶活性可见典型的梯形条带 ;比活性为每 mg蛋白质的 cpm值为 1 80× 1 0 9,纯化倍数 1 80 0 ,得率 90 % .凝胶迁移阻滞法分析显示特异的凝胶迁移阻滞性电泳条带 ;以 SDS- PAGE检测得到 4种蛋白质亚基成分 ,其蛋白质相对分子质量分别为 2 2 0 ku、2 1 2 ku、1 1 6ku和 43ku.由上述可见 ,采用自行设计的寡核苷酸亲和纯化法获得了人端粒酶复合体及 4种蛋白质亚基组分 .     

快速检测植物类囊体膜蛋白体内磷酸化的方法

借助特异的磷酸蛋白探针,建立了快速检测植物类囊体膜蛋白体内磷酸化的方法,可以检测到光照处理的豌豆叶圆片类囊体膜中8条磷酸化蛋白带存在,它们的分子质量分别为65、45、36、33、30、29、20和10 ku.进一步使用光系统Ⅱ反应中心蛋白和捕光色素复合物Ⅱ(LHCⅡ)的特异抗体,确定了上述磷酸化蛋白的归属,分别是磷酸化D1和（或）D2的聚合体（65 ku）、CP43（45 ku）、D2（36 ku）、D1（33 ku）,LHCB1（30 ku）,LHCB2（29 ku）和psbH gene产物（10 ku）,20 ku小肽尚不清楚其来源.并与其他几种检测磷酸化蛋白的方法进行了比较.     

猕猴脑胱天蛋白酶-3活化及其靶蛋白的体外研究

凋亡的主要生化过程包括胱天蛋白酶的活化及其对细胞内蛋白质的选择性切割.在已知的胱天蛋白酶中,可被多种凋亡刺激信号激活的胱天蛋白酶-3备受注目.为进一步揭示灵长类动物神经组织中未知的胱天蛋白酶-3靶蛋白,采用成年猕猴脑组织粗提物作为无细胞体系,通过加入granzyme B引发凋亡途径的部分反应,如胱天蛋白酶-3的活化及随后发生的蛋白质水解.经蛋白质印迹分析发现,与granzyme B共孵育后,猕猴脑胱天蛋白酶-3以两步方式从酶原转化为活性酶.对猕猴脑组织自身蛋白质的进一步分析显示,多聚ADP-核糖聚合酶(PARP)被水解为长85 ku的片段,此片段提示胱天蛋白酶-3的特异切割活性.此外,神经元凋亡抑制蛋白(NAIP)也被切割,产生长约40 ku的小片段,但是它的出现不被胱天蛋白酶-3特异性抑制剂Ac-DEVD-CHO阻断,因此可能是granzyme B直接作用于NAIP所致.以上结果提示,凋亡相关酶切反应可在成年猕猴脑组织提取物中得到重现;NAIP可能是granzyme B而非胱天蛋白酶-3的作用靶点.     

猕猴脑胱天蛋白酶-3活化及其靶蛋白的体外研究(英)

凋亡的主要生化过程包括胱天蛋白酶的活化及其对细胞内蛋白质的选择性切割.在已知的胱天蛋白酶中,可被多种凋亡刺激信号激活的胱天蛋白酶-3备受注目.为进一步揭示灵长类动物神经组织中未知的胱天蛋白酶-3靶蛋白,采用成年猕猴脑组织粗提物作为无细胞体系,通过加入granzyme B引发凋亡途径的部分反应,如胱天蛋白酶-3的活化及随后发生的蛋白质水解.经蛋白质印迹分析发现,与granzyme B共孵育后,猕猴脑胱天蛋白酶-3以两步方式从酶原转化为活性酶.对猕猴脑组织自身蛋白质的进一步分析显示,多聚ADP-核糖聚合酶（PARP）被水解为长85 ku的片段,此片段提示胱天蛋白酶-3的特异切割活性.此外,神经元凋亡抑制蛋白（NAIP）也被切割,产生长约40 ku的小片段,但是它的出现不被胱天蛋白酶-3特异性抑制剂Ac-DEVD-CHO阻断,因此可能是granzyme B直接作用于NAIP所致.以上结果提示,凋亡相关酶切反应可在成年猕猴脑组织提取物中得到重现;NAIP可能是granzyme B而非胱天蛋白酶-3的作用靶点.     

Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis

Biochemical characterization of a trehalase, detected in the mid-exponential growth phase of Candida utilis NCIM Y500, has indicated that it was a neutral trehalase and possibly the only trehalase present in this strain. Unlike Saccharomyces cerevisiae and other C. utilis strains, this strain without acid trehalase grew quite well in minimal or complete medium containing trehalose as the sole source of carbon. Both these observations were contradictory to the findings reported for acid trehalase mutants of S. cerevisiae and C. utilis. The trehalase system of the strain is suggested to be similar to that of fungi.     

Overproduction of glutathione by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.     

Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.     

Continuous culture of Candida utilis: influence of medium nitrogen concentration

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h^-1), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.     

Microbial assimilation of alkyl nitro compounds and formation of nitrite

66 representative strains of bacteria, yeasts and fungi were tested for their ability to grow in a semidefined medium containing 0.5% nitroethane as a nitrogen source. About half of them were found capable of growing in the medium. Hansenula beijerinckii, Candida utilis, and Penicillium chrysogenum were most active in assimilating nitroethane. 2-Nitropropane inhibited growth of most of the microorganisms tested in a medium containing 0.2% peptone and 0.2% glycerol. Hansenula mrakii was found to grow rapidly in the nitroethane-peptone medium after a lag phase. Nitrite was accumulated in the culture fluid after the phase of logarithmic multiplication, and increased with increase of the growth, followed by a decline after the maximum growth. The alkyl nitro compounds were oxidatively denitrified to form nitrite by the crude enzyme from Hansenula mrakii. Nitroethane was generally a poor substrate, but was the best inducer to produce the nitro compounds oxidizing enzyme. 2-Nitropropane and nitroethane were enzymatically oxidized to and acetone and acetaldehyde, respectively, which were isolated as 2,4-dinitrophenylhydrazones and identified. Nitrite formed was found to be reduced into ammonia by the intact cells and also the crude enzyme.     

Functional Purification of the Monocarboxylate Transporter of the Yeast Candida utilis

Plasma membranes of the yeast, Candida utilis, were solubilized with octyl-β-d-glucopyranoside and a fraction enriched in the lactate carrier was obtained with DEAE-Sepharose anion-exchange chromatography, after elution with 0.4 M NaCl. The uptake of lactic acid into proteoliposomes, containing the purified protein fraction and cytochrome c oxidase, was dependent on a proton-motive force and the transport specificity was consistent with the one of C. utilis intact cells. Overall, we have obtained a plasma membrane fraction enriched in the lactate carrier of C. utilis in which the transport properties were preserved. Given the similarities between the lactate transport of C. utilis and the one of mammalian cells, this purified system could be further explored to screen for specific lactate inhibitors, with potential therapeutic applications.     

Russian Article pp. 255–266

The growth of the yeast Candida utilis VKM-Y-2332 was investigated during the cultivation on a mineral medium with addition of ethanol in the regime of the chemostat and the pH-auxostat. The composition of the macroelements of the biomass produced by Candida utilis VKM-Y-2332 was studied. Stoichiometric equations of the growth process of the examined yeast strain on ethanol were presented. The growth of Candida utilis on ethanol and glucose was also characterized by the way of comparision.     

Serine proteinase from Bacillus brevis: lytic action on intact yeast cells

Summary A serine proteinase which showed lytic acitivity against either intact cell or cell wall preparations of Candida utilis has been isolated from Bacillus brevis culture filtrate by affinity chromatography on bacitracin-silochrome and phenylboronale-Sepharose. Both its proteolytic and lytic activities were completely abolished by inhibitors of serine proteinases, including phenylmethylsul-phonylfluoride, the inhibitor from Actinomyces janthinus, and duck ovomucoid. The optimum pH range for the enzyme is 7.5–9.0, the optimum temperature 40°–50°C, its pI value 8.6 and motecular weight 28000. The amino acid composition of this proteinase is similar to that of serine proteinase from B. amyloliquefaciens (subtilisin BPN), its N-terminal amino acid sequence being identical to that of BPN through 21 residues. The enzyme cleaves chromogenous substrates for subtilisins but shows no activity on a substrate for trypsin. By means of both turbidimetry and electron microscopy the enzyme studied was shown to cause yeast cell lysis.     

Flocculation of lag phase yeast cells ofCandida albicans

Flocculation of the yeast form ofCandida albicans occurs during the early or lag phase of growth. Once a developing culture has entered the logarithmic phase of growth, the cells are no longer capable of flocculating. The flocculation during these early stages in the growth of the culture was studied and appeared to be similar in many physical respects to agglutination of mating strains ofHansenula wingei. Variations in the carbohydrate content, nitrogen content, pH, and temperature of the growth medium did not alter the amount of flocculation. Studies with electrical fields indicated that an electrostatic charge is not involved. Treatment of the surface of the cells with solvents showed that lipid solvents do not inhibit flocculation, but phenol, which removes both carbohydrate and protein material, does inhibit flocculation. Lipases also do not affect flocculation but enzymes that break down carbohydrates or protein such as trypsin, papain, and pancreatin do affect flocculation. Phenol extracts from sonicated, washed cell walls were analyzed by electrophoresis and paper chromatography for proteins, amino acids, and polysaccharides. These analyses confirm earlier reports of constituents of the cell wall but do not show a difference in the types of compounds present in the cell walls of flocculant cells as compared with the cell walls of older non-flocculant cells.     

Isolation of aCandida utilis variant by using a nitrogen-limited continuous culture

During continuous culture ofCandida utilis the appearance of a morphologic variant yeast was detected. The new microorganism developed systematically whenever it was changed from normal to stressed propagation conditions. A simple system was used for the isolation of the yeast variant, which was defective in cellular division and showed improved kinetic parameters and oxygen uptake rate. An asynchronic nitrogen-limited continuous culture ofCandida utilis allowed us to enrich the population in the chemostat with the modified yeast and isolate it in a defined medium. Assimilation and fermentation tests indicated it to be a variant ofCandida utilis that showed stable morphologic and physiologic differences with the parental yeast.Candida utilis growing in this nitrogen-limited continuous culture also showed a high mutation rate.