蓝光诱导促进黑曲霉产糖化酶的研究

考察了蓝光对黑曲霉产糖化酶的影响并采用扫描电镜观察蓝光下黑曲霉形态发育过程,结果表明,与黑暗对照组相比,蓝光处理使菌丝粗壮,孢囊增大,分生孢子发育提前,黑曲霉糖化酶活力增加,孢子发育和产糖化酶的进程有一定的对应性。黑曲霉在黑暗下生长至36h时,经蓝光诱导糖化酶产量提高更为明显,提示了黑曲霉存在一个对蓝光反应产生最适光感应的发育阶段,对于光调节黑曲霉产糖化酶来说,蓝光诱导的光强由弱到强,比持续蓝光培养或采用较高光强诱导效果更好,表明黑曲霉产糖化酶存在一种光适应机制,能够感应和适应光强度变化,调节其自身代谢。从抑制性扣除杂交实验和蓝光光强变化对差异基因表达的分析来看,糖化酶基因以及呼吸链中部分氧化还原酶基因在蓝光诱导下表达皆有增强,蓝光信号转导影响了核基因编码的线粒体呼吸链相关酶基因表达水平,交替氧化酶可能参与了蓝光信号途径,影响了黑曲霉产糖化酶和孢子发育。研究结果可为在现有水平上应用蓝光调节提高糖化酶产量找到新的技术突破口和提供新思路。     

黑曲霉TRK基因家族的全基因组鉴定与分析

TRK是一种与钾离子吸收转运相关的基因,在调节生物体生命活动方面起着重要的作用。本研究建立在黑曲霉(Aspergillus niger)基因组数据库基础上,利用生物信息学手段鉴定黑曲霉TRK基因家族成员,分析其基因结构特征和系统进化关系;并利用MEGA 6.06软件,采用最大似然法(MLT)构建系统发育树;使用PAL2NAL软件进行da/ds估算,利用GSDS 2.0软件进行基因结构分析,MEME程序进行Motif分析。在黑曲霉基因组中共鉴定TRK基因家族4个成员:An Trk1、An Trk2、An Trk3、An Trk4,发现均含有TRK特征结构域和保守的Motif基序,不同基因间结构差异较大,但聚类关系较近的基因其结构相似。黑曲霉中TRK基因家族成员在基因结构特征等方面与酵母存在着显著差异,亲缘关系和蛋白互作网络分析进一步验证了这个结论,因此推断黑曲霉中TRK基因还存在其他的调节途径,这个结论为进一步研究黑曲霉TRK渗透胁迫相关蛋白功能、作用机制提供理论依据,从而为了解黑曲霉的渗透调节机理奠定基础。     

黑曲霉作为细胞工厂:知识准备与技术基础

黑曲霉是一种重要工业微生物,在酶制剂、异源蛋白、有机酸等领域应用广泛。2007年黑曲霉基因组的公布将黑曲霉的研究引入后基因组时代,各种组学数据如雨后春笋般涌现,人们对黑曲霉高效生产机制的理解上升到系统、分子层次;与此同时,黑曲霉遗传操作系统也不断成熟,为系统地研究和改造黑曲霉、将黑曲霉打造成通用细胞工厂奠定了基础。     

黑曲霉S1生淀粉糖化酶生物合成的调节研究

本文对黑曲霉S_1(Aspergillus niger S_1)生淀粉糖化酶生物合成调节进行了初步研究,认为该菌生淀粉糖化酶的合成与菌体生长呈负相关,即酶的合成过程是典型的选择性合成过程。该菌生淀粉糖化酶的合成受降解物阻遏调控,缓慢供给低浓度易利用碳源和添加环腺苷磷酸(cAMP)可使酶的合成消阻遏,通过研究放线菌素C_1(Actinomycin C_1)等抑制剂对酶合成的影响而推断黑曲霉S_1生淀粉糖化酶合成的阻遏调控发生在转录水平上。     

柠檬酸发酵前期污染的挽救

用黑曲霉发酵甘薯生产柠檬酸,容易感染酵母菌。感染后,轻则造成产酸的下降和发酵周期的延长,严重时会导致倒罐。本文介绍某些挽救污染的方法。(一)污染的培养液重新灭菌接种取罐内污染的培养液,用CaCO_3调节pH     

柠檬酸发酵菌黑曲霉的菌种改良述评

柠檬酸是微生物发酵法生产的重要有机酸,用途极为广泛。柠檬酸发酵菌黑曲霉的育种在柠檬酸工业中占有重要地位。该文论述了柠檬酸发酵菌黑曲霉的发酵机制、代谢调控及柠檬酸积累的机理,柠檬酸发酵菌黑曲霉的物理和化学手段诱变育种取得的成果,细胞工程和基因工程在柠檬酸发酵菌黑曲霉育种中的应用,分析了柠檬酸发酵菌黑曲霉育种的发展方向。     

黑曲霉及其与普洱茶品质关系研究进展

近年来,黑曲霉菌的研究受到了国内外大量学者的重视,并取得了一系列新进展,这些进展主要集中在:黑曲霉的分离鉴定方法;黑曲霉发酵生产多酚氧化酶、果胶酶和纤维素酶等酶类的机理;黑曲霉对普洱茶色泽、滋味和香气的影响等方面。文章集中对近年来黑曲霉及其与普洱茶品质形成相关的研究进展作简要综述,以期为黑曲霉在普洱茶中研究利用提供一定的参考。     

黑曲霉脂肪酶分子结构预测

根据克隆到的黑曲霉脂肪酶基因序列,概念性翻译出其编码的氨基酸序列.利用BioEdit、PSIPRED和SwissModel等软件及服务器对黑曲霉脂肪酶的一级结构、二级结构和三级结构进行分子结构模型的预测与模拟.预测结果显示,黑曲霉脂肪酶分子的一级结构、二级结构及三级结构的结构特点与丝氨酸水解酶高度吻合.预测的黑曲霉脂肪酶各种二级结构成分含量与实际测定的重组黑曲霉脂肪酶二级结构成分相符.在三级结构水平上,黑曲霉脂肪酶"盖子"结构域所在的位置与已解析的黑曲霉阿魏酸酯酶对应结构域所在的位置存在显著差异.盖子结构域位置的差异预示一种开盖型脂肪酶分子设计和构建的可能.     

肉桂醛、柠檬醛抑制黑曲霉生长的比较研究

黑曲霉是一类极易通过饲料、食品、粮油霉变而具致病性的有害真菌。与物理和化学方法抑制黑曲霉生长相比,生物抑菌剂抗黑曲霉生长具有药效久、无抗药性并安全健康的优点。本实验采用天然肉桂醛、柠檬醛作为抑菌剂,以正常生长的黑曲霉为对照,分别采用牛津杯法、气体扩散法比较对黑曲霉生长效果的影响。结果表明,柠檬醛作用所形成的抑菌圈显著大于肉桂醛作用所形成的抑菌圈,且在同一浓度下柠檬醛对菌丝体形态和孢子囊形态的抑制比肉桂醛显著,而气体扩散法抗黑曲霉效果优于牛津杯法。     

不同银杏内酯对黑曲霉线粒体结构及功能的影响

真菌感染作为威胁人类健康的传染性疾病之一,是普遍的全球性问题。本研究通过在黑曲霉的孢子悬浮液中添加不同浓度的银杏内酯,收集黑曲霉菌丝体,评估黑曲霉线粒体的结构和功能,考察不同银杏内酯B和C对黑曲霉的抗真菌效果。透射电子显微镜扫描发现经银杏内酯B处理后的黑曲霉线粒体表现出明显的空泡化,基质结构被破坏;线粒体三羧酸循环中关键酶琥珀酸脱氢酶和苹果酸脱氢酶活性下降,线粒体膜电位降低,丙二醛和活性氧水平显著升高。研究结果初步揭示了银杏内酯以剂量依赖的方式损坏线粒体膜的完整性,破坏线粒体结构,进一步损害线粒体功能;银杏内酯B的抗黑曲霉效果要显著高于银杏内酯C。     

Presence of angiotensin-converting enzyme in follicular fluids of porcine ovaries and its possible involvement in the intrafollicular breakdown of bradykinin

The follicular fluid of porcine ovaries contains a metalloenzyme capable of hydrolyzing the synthetic substrate, benzyloxycarbonyl-Val-Lys-Met-MCA. This enzyme was purified by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose, CM-cellulose, Zn(2+)-chelating Cellulofine, and Diol-300 gel-filtration columns. The molecular weight of the purified enzyme was estimated to be 170,000 by SDS-PAGE and 400,000 by gel-filtration analysis, suggesting that the native enzyme is a dimer of the 170-kDa subunit polypeptide. The enzyme activity was drastically enhanced by the presence of chloride ion, and strongly inhibited by captopril and bradykinin potentiator B. A 9-residue peptide containing a processing site of human amyloid precursor protein was degraded by its dipeptidyl carboxypeptidase activity. Furthermore, the purified protein was recognized by specific antibody raised against human angiotensin-converting enzyme. The enzyme rapidly degraded bradykinin in vitro. These results indicate that benzyloxycarbonyl-Val-Lys-Met-MCA-hydrolyzing enzyme is a porcine angiotensin-converting enzyme, and that the enzyme may play a role in bradykinin turnover within the follicles of porcine ovaries.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.     

Amylase of the thermophilic actinomycete Thermomonospora vulgaris.

alpha-Amylase of the thermophilic actinomycete Thermomonospora vulgaris was partially purified. Maximal enzyme activity was obtained at 60degreeC and pH 6.0. KM value was l.4%. The effect of some metal salts on enzyme activity was studied. Enzyme activity was inhibited by by KCN, EDTA, and iodoacetate. Inhibition by EDTA was completely nullified by CaCl2, but the inhibition by iodoacetate was not overcome by 2-mercaptoethanol. Exposure of the enzyme to pH 7.0 and 9.0 for 2 hr. did not affect the enzyme, but exposure to pH 3.0 for few minutes completely inactivated the enzyme. Exposure of the enzyme to 60degreeC resulted in an appreciable inactivation and exposure to 80degreeC completely inactivated the enzyme. Addition of CaCl2, 2-mercaptoethanol, or enzyme substrate the 60degreeC exposed enzyme. However, bovine serym albumin had a protective effect when the enzyme was exposed to 60degreeC but not to 80degreeC. The enzyme was stable in the presence of 8 M urea.     

Regulation of the electrogenic (Na+ + K+)-pump of Ehrlich cells by intracellular cation levels.

Dihydrofolate reductase and its complexes have been studied by fluorescence and circular dichroism. NADPH, trimethoprim, pyrimethamine, or Methotrexate binding causes small changes in the enzyme far ultraviolet CD which possibly arise from alterations in polypeptide backbone of the enzyme; however, their effects on enzyme far ultraviolet CD are also explained as the result of ligand interactions with enzyme aromatic groups. In ternary complexes of the enzyme, fluorescence properties of bound NADPH are surprisingly sensitive to the type of inhibitor bound nearby. The effect of temperature on the enzyme and its complexes is clearly shown by changes in enzyme fluorescence and CD. At temperatures near 45 degrees C, the enzyme undergoes an irreversible denaturation, as shown by major alterations in enzyme far ultraviolet CD and by an increased rate of fluorescence quenching. Binary complexes with NADPH or Methotrexate stabilize the enzyme towards this heat denaturation, whereas bound trimethoprim and pyrimethamine do not. Ternary complexes with NADPH and any of the ligands are more stable than the enzyme itself toward heat denaturation. Fluorescence-temperature and fluorescence polarization studies show that near 30 degrees C the enzyme undergoes a reversible transition that is modified by NADPH or methotrexate.     

Effect of nutritional deficiencies on synthesis of the inducible malic enzyme of lactobacillus plantarum

Summary The reduction of synthesis of the inducible malic enzyme by cell suspensions of biotin-deficient Lactobacillus plantarum 17-5 is also shared by cells deficient in nicotinic acid, thiamine, and pyridoxine. Addition of the deficient vitamin at the start of enzyme synthesis increases the amount of enzyme formed.Suspensions of riboflavin-deficient cells also synthesize a reduced amount of enzyme but addition of riboflavin does not increase enzyme synthesis. Suspensions of pantothenate-deficient cells either show a small reduction or a small stimulation of malic enzyme synthesis. Suspensions of p-amino benzoic acid (PAB)-deficient cells synthesize greater than normal amounts of malic enzyme.A more detailed comparison of differences between malic enzyme synthesis by normal and by PAB-deficient cells show that chloramphenicol is more inhibitive to enzyme synthesis by normal cells and that chlorpromazine is more inhibitive to enzyme synthesis by PAB-deficient cells. Possible explanations of the results with inhibitors are discussed.Cells deficient in adenine, act similarly to PAB-deficient cells with respect to amount of enzyme synthesized and effect of inhibitors. The amount of enzyme synthesized and the effect of inhibitors on the adenine-deficient cells is changed to a pattern resembling that of normal cells when adenine is added at the beginning of enzyme synthesis. An interpretation of these results is offered.I thank Professor W. W. Umbreit for his continued interest during these studies and Park-Davis, Inc. and Smith, Kline and French respectively for chloramphenicol and chlorpromazine.     

Activities in Crude Porcine Pancreatic Lipase: Enantioselectivity in Hydrolysis of the Diacetate of 2-Phenylpropane-1,3-Diol

The hydrolysis of a prochiral diacetate by porcine pancreatic lipase is catalysed by the purified enzyme, not by an enzyme present in the crude enzyme but absent from the purified enzyme, as previously reported.     

Radioiodinated antibodies,a tool in studies on the presence and role of inactive enzyme forms: Regulation of chalcone synthase in parsley cell suspension cultures

A new technique, the quantitative determination of total enzyme concentrations by specific immunoprecipitation with purified, radioiodinated antibodies, was used to investigate the presence and possible roles of inactive enzyme in the regulation of chalcone synthase. Dark-grown cell suspension cultures from parsley (Petroselinum hortense) contained neither catalytically active nor detectable amounts of immunoprecipitable chalcone synthase. Irradiation induced large increases and subsequent decreases of both. Significant differences in the peak positions and in the half-lives of active and total chalcone synthase indicated that induced cells contained inactive as well as active enzyme forms. The presence of inactive enzyme could be explained by two different modes of regulation, (i) simultaneous de novo synthesis of active and inactive enzyme (“Simultaneous Model”), or (ii) de novo synthesis of active enzyme only, with sequential steps of inactivation and degradation (“Sequential Model”). Both models were compatible with experimental results, as analyzed mathematically by investigating the relations between curves for rate of enzyme synthesis, enzyme activity, total enzyme, and half-lives of active and total enzyme. However, the “Simultaneous Model” postulated that de novo synthesis of inactive enzyme represented always the vast majority of total enzyme synthesis, while the Sequential Model integrated inactive enzyme with facility in a sequence of irreversible inactivation and degradation of active enzyme. Experiments with repeated induction indicated that cells containing large amounts of inactive enzyme increased enzyme activity by de novo synthesis rather than by activation of preexisting inactive enzyme.     

Change of activity and substrate specificity of human glucose 6-phosphate dehydrogenase by oxidation

Human glucose 6-phosphate dehydrogenase contains about 18 sulfhydryl groups per active dimer (MW = 110,000, and it does not contain S–S bridges. Chloromercuribenzoate stoichinmetrically and reversibly inactivates the enzyme. Oxidation of the enzyme by hydrogen peroxide induces a reduction of enzyme activity, an alteration of the substrate specificity, and an increased anodal electrophoretic mobility. The oxidized enzyme can use 2-deoxyglucose 6-phosphate, deamino NADP, and NAD far more effectively than the native enzyme. Oxidation of the enzyme by air at pH 8.0 does not induce a significant loss of enzyme activity or an alteration of the substrate specificity, although about 70% of the sulfhydryl groups of the enzyme are oxidized by the treatment.     

Inhibition of 5-amino levulinic acid dehydratase activity by arsenic in excised etiolated maize leaf segments during greening

In vivo as well as in vitro supply of sodium arsenate inhibited the 5-Amino levulinic acid dehydratase (5-aminolevulinate-hydrolyase EC 4.2.1.24, ALAD) activity in excised etiolated maize leaf segments during greening. The percent inhibition of enzyme activity by arsenate (As) was reduced by the supply of KNO3, but it was increased by the glutamine and GSH. Various inhibitors, such as, chloramphenicol, cycloheximide and LA, decreased the % inhibition of enzyme activity by As. The % inhibition of enzyme activity was also reduced by in vivo supply of DTNB. The enzyme activity was reduced substantially by in vitro inclusion of LA, both in the absence and presence of As. In vitro inclusion of DTNB and GSH inhibited the enzyme activity extracted from leaf segments treated without arsenate (-As enzyme) and caused respectively no effect and stimulatory effect on arsenate treated enzyme (+As enzyme). Increasing concentration of ALA during assay increased the activity of -As enzyme and +As enzyme to different extent, but double reciprocal plots for both the enzymes were biphasic and yielded distinct S0.5 values for the two enzymes (-As enzyme, 40 micromol/L and +As enzyme, 145 micromol/L) at lower concentration range of ALA only. It is suggested that As inhibits ALAD activity in greening maize leaf segments by affecting its thiol groups and/or binding of ALA to the enzyme.     

Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.