0．23T稳恒磁场对不同温度离体过氧化氢酶的磁效应研究

研究了 0 .2 3T稳恒磁场对不同温度下的离体牛肝过氧化氢酶 (CAT)构象及活力的影响 ,并从分子水平讨论了磁场对不同温度的过氧化氢酶产生不同生物学效应的可能机制。将不同温度的天然酶液置于磁感应强度为0 .2 3T的磁场中分别处理一定的时间 ,处理过程中保持环境温度与酶液温度一致 ,撤离磁场后立即在相同实验条件下对其进行光谱分析及量热分析 ,并用Beers&Sizers法 (改良型 )测定酶活力。结果表明 ,磁场使 2 5℃过氧化氢酶的构象发生明显变化 ,表现为荧光偏振度增加、出现明显的差示扫描量热曲线、产生λ2 10nm～ 310nm的紫外差光谱以及λ330nm荧光发射峰的荧光强度改变 (荧光发射峰的峰位未移动 ) ,构象变化的同时酶活力增加 ;15℃过氧化氢酶的构象及活力变化规律与 2 5℃过氧化氢酶类似 ,但强度均弱于 2 5℃酶 ;而 4℃过氧化氢酶的构象及活力没有发生变化 ,表现出未受磁场处理的影响。相同实验条件下 ,磁场对不同温度的酶分子影响不同 ,随温度的增加 ,影响效应趋于显著。由于不同温度的酶分子之间的差异在于构象状态的不同 ,这表明酶分子自身的构象状态对磁场处理效果有极其重要的影响。不同温度的过氧化氢酶磁效应差异显著可能是由磁致酶构象变化的特殊机制所引起。磁场对酶分子构象的影响可能是通     

水溶性有机溶剂对Fe3O4磁性纳米粒子过氧化物酶样活性的影响

采用化学共沉淀法合成10nm的Fe3O4磁性纳米粒子(MNPs)。以辣根过氧化物酶(HRP)为对照,研究了四氢呋喃、1,4-二氧六环、丙酮、N,N-二甲基酰胺、甲醇和二甲亚砜等6种水溶性有机溶剂对Fe3O4MNPs过氧化物酶样活性的影响。结果表明,在有机溶剂浓度(V/V)为30%～75%时,Fe3O4MNPs相对酶活力迅速下降至近于完全失活。在15%有机溶液中,Fe3O4MNPs的最适反应温度多为50oC,最适反应pH在3.6左右。经15%有机溶液处理后的水相反应酶活显示,Fe3O4MNPs表现出对有机溶剂较强的热稳定性和pH稳定性,且对75%有机溶液也具有良好的稳定性。以上多数性质均优于相同条件下的HRP组,表明Fe3O4MNPs是一种比HRP对水溶性有机溶剂更稳定的过氧化物酶。由于Fe3O4MNPs具有易制备、成本低、易于磁分离和可循环使用的特点,因此其具有替代HRP用于有机催化的应用潜力。     

溶液介电常数对铜锌超氧化物歧化酶活性的影响

溶液介电常数对天然酶和修饰酶的活性影响不同,天然酶随介电常数增加而酶活性下降,修饰酶则反之,这表明静电相互作用在铜锌超氧化物歧化酶(Cu·Zn-SOD)与超氧阴离子(-O_2~(·-))反应过程中起着重要作用,酶分子活性中心附近ε-NH_3~+为O_2~(·-)进入活性中心提供静电吸引力。在有机溶剂中,SOD的构象会发生变化,从而导致酶活性降低。实验还表明,Cl~-对SOD有明显的抑制作用。     

人小肠三叶因子的理化性质及光谱学行为

酵母表达的重组人小肠三叶因子(rh-ITF)飞行质谱测定二聚体的分子量为13154,等电点约为4.5～4.75.紫外和荧光光谱表明rh-ITF在pH2.7～8.4和pH2.7～7.7时,吸收值增加。随pH进一步增加,吸收值降低。推测色氨酸和酪氨酸所处微环境发生了一定的变化。园二色谱表明在不同pH下,rh-ITF所含二级结构百分数有所变化,但仍保留有一定的二级结构,即含有一定数量的α-helix,β-sheet或β-turn,其三级结构基本不变。光电滴定和有机溶剂微扰法表明rh-ITF分子中有两个酪氨酸,一个处于分子表面,另一个参与氢键的形成或存在于一个非极性的环境中。rh-ITF中的色氨酸处于分子内部。另外,质谱测定rh-ITF在体外对酸和蛋白酶有一定的抗性     

菓菠萝蛋白酶的分子构象与活力变化的研究

发现CBZ-Lys·pNP能有效地被菓菠萝蛋白酶(Fruit Bromelain E.C.3.4.22.5)作用,测得Km为4.167×10~(-4)mol/L,k_(cat)为742min~(-1)。以荧光和紫外差示光谱为监测手段,对酶分子构象变化进行研究。酶的荧光强度随胍浓度增大而逐渐下降,4mol/L胍变性时,发射峰自332nm红移到353nm,并在310nm处出现新的发射峰。酶的荧光强度都因SDS存在而下降,SDS浓度大于3.47mmol/L有所回升,并出现红移,同时在315nm处出现新的发射肩;紫外差示光谱显示在236nm有一个较显著的员峰,此峰与β-螺旋结构变化有关,278、286和295nm出现三个负峰,260nm有较小正峰,说明酶分子中Tyr、Trp和Phe的微环境发生了明显的变化。测定酶在不同浓度胍和SDS中的变性和失活速度常数,对酶构象变化及催化活力的关系作了比较研究,酶的失活速度均大于变性速度。     

肉类腐败性假单胞菌群体感应信号分子的研究

【目的】研究两株假单胞菌的标准菌株荧光假单胞菌(Pseudomonas fluorescens)和铜绿假单胞菌(Pseudomonas aeruginosa)在纯培养条件下所释放的AHLs类信号分子种类、量和变化规律。【方法】利用乙酸乙酯等有机溶剂萃取菌种纯培养液的AHLs类信号分子, 检测手段利用HPLC-MS-MS。【结果】荧光假单胞菌释放信号分子的种类为: C4-HSL、C6-HSL、C8-HSL、3-oxo-C10-HSL、3-oxo-C12-HSL、3-oxo-C14-HSL。铜绿假单胞菌释放信号分子的种类为: C4-HSL、C6-HSL、C8-HSL、C10-SL、C12-HSL、C14-HSL、3-oxo-C8-HSL、3-oxo-C10-HSL、3-oxo-C12-HSL、3-oxo-C14-HSL。【结论】两株菌所释放各类信号分子的量均随时间变化, 当菌落数达到109?1010时信号分子的量达到峰值, 两株菌所释放各类信号分子含量差异较大。     

竹叶马兜铃化学成分的研究(Ⅱ)

<正> 竹叶马兜铃Aristolochia bambusifolia的化学成分,我们在前文曾报导,从其块根分得结晶性单体,已鉴定为D—甘露醇、马兜铃酸,马兜铃酸C、朱砂莲内酰胺,β—谷甾醇外,现本文通过光谱方法,报导其另一个化合物晶V1的鉴定。 晶V1:浅黄色针状结晶,溶于氯仿时呈蓝色荧光,熔点207～208℃。其红外光谱有C=O及CH_3吸收峰1710,1225,1060cm~(-1),无NO_2吸收峰。质谱m/z:340(M~+),     

铜离子对钝顶螺旋藻完整细胞中光系统Ⅱ活性和藻胆体能量传递的影响

Cu2 抑制钝顶螺旋藻 ( Spirulina platensis)完整细胞中电子传递活性 ( H2 O→ MV) ,并抑制 PS 的放氧活性。低浓度的 Cu2 处理 ,使钝顶螺旋藻细胞中藻蓝蛋白荧光发射峰位置蓝移、发射强度改变 ,表明藻胆体中能量传递发生了变化。Cu2 处理纯化的藻蓝蛋白 ,使其在长波区 ( 61 6— 62 0 nm)吸收减小、荧光发射峰蓝移 ( 64 7nm→ 64 3nm) ;而变藻蓝蛋白不受影响 ,说明 Cu2 选择性作用于藻蓝蛋白。     

过氧化氢对铜锌超氧化物歧化酶活性及某些理化性质的影响

用H_2O_2作用于牦牛红细胞铜锌超氧化物歧化酶。观察到酶活性随H_2O_2浓度升高及作用时间增加而下降;酶分子连接的铜和锌有所丢失;PAGE图谱中三条酶活性带成为四条酶活性带;等电点下降;680nm处表征二价铜光学性质的可见光吸收减弱;紫外吸收增加,表现为增色效应;内源性荧光减弱;在含有3.0mol/LKCl的PH3.8—5.4琥珀酸缓冲液中溶解度下降;酶对胰蛋白酶水解的敏感性增加。     

无花果蛋白酶在胍溶液中的分子折叠与活力变化研究

本文研究无花果蛋白酶（ＥＣ．３．４．４．１２）在不同浓度盐酸胍溶液中分子构象与活力变化关系。酶的内源荧光光谱,圆二色光谱与酶活力的变化表明：荧光光谱呈现二个明显的变化区域,低浓度胍（低于２ｍｏｌ／Ｌ）中,荧光发射峰基本不变,但荧光强度随胍浓度上升,随胍浓度断续增大（高于２ｍｏｌ／Ｌ）,酶的最大发射波长明显红移。当胍浓度低于１ｍｏｌ／Ｌ时,不仅不会使酶失活,反而使酶激活,当胍浓度高于１ｍｏｌ／Ｌ以上     

Cloning,heterologous expression,and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii

The gene encoding a thermostable glucoamylase from Talaromyces emersonii was cloned and, subsequently, heterologously expressed in Aspergillus niger. This glucoamylase gene encodes a 618 amino acid long protein with a calculated molecular weight of 62,827Da. T. emersonii glucoamylase fall into glucoside hydrolase family 15, showing approximately 60% sequence similarity to glucoamylase from A. niger. The expressed enzyme shows high specific activity towards maltose, isomaltose, and maltoheptaose, having 3-6-fold elevated k(cat) compared to A. niger glucoamylase. T. emersonii glucoamylase showed significantly improved thermostability with a half life of 48h at 65 degrees C in 30% (w/v) glucose, compared to 10h for glucoamylase from A. niger. The ability of the glucoamylase to hydrolyse amylopectin at 65 degrees C is improved compared to A. niger glucoamylase, giving a significant higher final glucose yield at elevated temperatures. The increased thermal stability is thus reflected in the industrial performance, allowing T. emersonii glucoamylase to operate at a temperature higher than the A. niger enzyme.     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

AIMS: Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. METHODS AND RESULTS: First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4.5-5.0 and 65 degrees C. The enzyme was stable at 60 degrees C, the enzyme activity kept 80% after 60 min incubation at 70 degrees C. The half-life was 40 and 10 min under incubation at 80 and 90 degrees C respectively. CONCLUSIONS: A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.     

Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus

The glucoamylase from the thermophilic fungus Thermomyces lanuginosus has a molecular weight of 66 kDa and was characterized with isoelectric point, pH and temperature optimum of 3.8-4.0, 5.0 and 70 degrees C, respectively. In addition, the activation energy is 60.4 kJ/mol, Km is 3.5 mM and kcat is 25.3 s(-1). The glucoamylase was partially sequenced on the protein level, and the complete glucoamylase gene including its promoter (but excluding its terminator region) was cloned and sequenced. The glucoamylase protein comprises 617 amino acid residues and shows 60% identity with the glucoamylase from the thermophilic fungus Talaromyces emersonii. cDNA encoding Thermomyces lanuginosus glucoamylase was expression cloned into Pichia pastoris, producing approximately 7.4 U/ml. It was concluded that alternative mRNA splicing as it might occur in Aspergillus niger glucoamylase is not responsible for the occurrence of different glucoamylase isoforms in Thermomyces lanuginosus.     

Comparison of two glucoamylases from Hormoconis resinae

Two extracellular glucoamylases (EC 3.2.1.3), glucoamylase P and glucoamylase S, were purified to homogeneity from the culture medium of Hormoconis resinae (ATCC 20495; formerly Cladosporium resinae) by a new method. Their apparent molecular masses (71 kDa glucoamylase P; 78 kDa glucoamylase S) and catalytic properties agreed well with those previously reported in the literature. Heat inactivation studies suggested that the high debranching (1,6-glycosidic) activity of glucoamylase P preparations (measured with pullulan) may reside in the same protein molecule as its 1,4-glycosidic activity (measured with soluble starch). Although glucoamylase S had virtually no debranching activity, it cross-reacted with polyclonal antibodies raised against glucoamylase P, and the two enzymes had very similar amino acid compositions. However, peptide mapping and amino-terminal sequencing studies of the peptides showed that the two enzymes have different sequences and must be encoded by different genes.     

Effect of maltose on glucoamylase formation by Aspergillus niger

Low levels of glucoamylase are produced when Aspergillus niger is grown on sorbitol, but substitution of the latter by glucose, maltose, or starch results in greater formation of glucoamylase as measured by enzymatic activity. Both glucoamylase I and glucoamylase II are formed in a yeast extract medium; however, glucoamylase I appears to be the only form produced when ammonium chloride is the nitrogen source. Maltose or isomaltose (1.4 x 10(-4)m), but no other disaccharides or monosaccharides, dextrins, dextrans, or starches, stimulated glucoamylase formation when added to mycelia pregrown on sorbitol-ammonium salts. The induction of glucoamylase by maltose was independent of sulfate concentration but showed a dependency on low pH and the absence of utilizable carbon sources.     

TiO2膜吸附固定糖化酶特性的研究

分别以醋酸纤维素TiO2膜(AC.TiO2膜)、羧甲基纤维TiO2膜(CMC.TiO2膜)和聚丙烯TiO2膜(PP.TiO2膜)为载体吸附固定糖化酶,并与醋酸纤维素、羧甲基纤维素和聚丙烯固定糖化酶的性能进行了比较,得出以AC.TiO2膜和PP.TiO2膜对糖化酶的吸附性能及稳定性能均较好,PP.TiO2膜固定的糖化酶使用8次后其剩余酶活仍能保持在72%.     

生淀粉糖化酶高产菌的选育

从土壤及霉变淀粉质等样品中分离出对生淀粉具有降解作用的菌种约 6 0株 ,其中生淀粉糖化酶最高的一株根霉OR-1 ,其酶活为 90U/mL ,通过一次紫外和亚硝基胍诱变 ,酶活分别达到 200U/mL、325U/mL ,RDA值分别为 70 %、 65%。诱变株是一株产生淀粉糖化酶较高的根霉。     

蓝光促进黑曲霉分生孢子发育和产糖化酶的研究

以黑暗为对照 ,研究了不同光质对黑曲霉产糖化酶及生长发育的影响。持续蓝光作用下 ,孢子萌发后菌丝较粗 ,菌丝细胞顶端膨大显著 ,菌丝细胞膜的通透性增加 ,残糖消耗快 ,孢子和孢子穗增大。在 3(4d时 ,蓝光下菌丝产糖化酶活力最高达 6 6 0 (30U mL ,比黑暗高出了 15. 4 % ,生物量增加了 4 9. 4 8% ,菌丝细胞可溶性蛋白含量提高了10 0. 5 6 % ,尤其是在开始产孢子的阶段 ,蓝光下黑曲霉产糖化酶活力、生物量有很大提高。研究表明 ,蓝光明显促进黑曲霉分生孢子发育和产孢阶段包括糖化酶在内的多种淀粉酶活力的迅速增加。