高效溶栓酶——纳豆激酶的纯化及酶学性质研究

采用硫酸铵分步盐析,Sepharose CM FF离子交换层析和Superdex 75凝胶色谱,对纳豆激酶发酵液进行分离纯化,得到电泳纯的纳豆激酶。并研究了纳豆激酶的酶学性质,实验结果表明,纳豆激酶的最适作用温度为37℃,温度对酶稳定性影响显著;NK的最适作用pH为7.4,pH6~8范围内酶活相对稳定;EDTA、pepstatin、aprotin ine和PMSF对酶有抑制作用,而SBTI和TPCK对酶有激活作用,其中以EDTA的作用最为明显;Zn2 对酶活有较大的抑制作用;A l3 、Cu2 对酶也有一定程度的抑制;Mg2 、Ca2 、是较好的酶活稳定剂和促进剂。     

低温、嗜盐α-淀粉酶Amy3的克隆、表达及重组酶性质

【目的】从分离自北极海底沉积物Pseudoalteromonas sp.K8菌株克隆、重组表达α-淀粉酶Amy3,并研究其酶学性质。【方法】基于Pseudoalteromonas haloplanktis TAC125基因组分析,从亲缘关系较近的Pseudoalteromonas sp.K8克隆获得α-淀粉酶基因amy3,以大肠杆菌为宿主进行重组表达,经Ni-NTA亲和层析柱纯化获得重组蛋白Amy3。以可溶性淀粉等为底物,研究Amy3的酶学性质。【结果】Amy3最适催化pH为8.5,在pH 6.5–10.0范围内酶活力维持在40%以上;其在pH 7.5–8.5范围的稳定性较好,pH 8.0条件下的半衰期可达4 h。Amy3在低温下较稳定,25℃半衰期为5 h;最适反应温度为25℃,并且在0℃可以保持50%以上酶活力,显示良好的低温催化特性。NaCl能够有效提升Amy3的酶活力及稳定性;荧光光谱分析表明,NaCl并未引起Amy3酶蛋白三级结构的改变。动力学分析显示,NaCl影响了酶催化的K_m及k_(cat),进而提升了酶的催化效率。底物特异性分析表明,Amy3对支链淀粉的水解能力优于直链淀粉,并能够有效地水解小麦淀粉、玉米淀粉和木薯淀粉。【结论】来源于Pseudoalteromonassp.K8菌株的α-淀粉酶Amy3具有良好的低温催化及嗜盐性,在洗涤、食品、污水处理等行业中有潜在的应用前景。     

黑曲霉CICIM F0410中α-葡萄糖苷酶的酶学性质研究

研究了黑曲霉(Aspergillus niger)CICIMF0410发酵粗酶液中α-葡萄糖苷酶的酶学性质,发现该酶最适反应温度为55℃,最适反应pH值5.0,在65℃以下保持酶活力相对稳定,且能在pH值(4.0～6.5)范围内保持相对较高的酶活力。以该菌株的染色体DNA为模板,PCR扩增出大小为3.1kb的片段,经序列比对发现与NCBI上已公布的黑曲霉(Aspergillus niger)CBS513.88(登录号XP_001402053)序列有2个碱基的区别:2272(A→G),3098(T→G),导致2个氨基酸残基的变化:687(Asn→Ser)、983(Ser→Arg)。     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·     

长野芽孢杆菌普鲁兰酶基因在枯草芽孢杆菌中的表达及酶学性质研究

根据NCBI上报道的基因序列设计引物,以长野芽孢杆菌(Bacillus naganoensis)ATCC53909的染色体DNA为模板,PCR扩增普鲁兰酶编码基因pulB。将此基因与表达载体pWB980连接构建重组质粒pWB-pulB,并转化枯草芽孢杆菌WB600。SDS-PAGE结果显示,在100 kD处有特异性条带,经测定重组转化子粗酶液酶活力达10.94 U/mL。酶学性质分析表明,其最适反应温度为60℃,最适反应pH为5.0,且在温度30-60℃及pH4.0-6.0范围内稳定,适合淀粉加工行业的应用。     

植物乳杆菌源胺氧化酶的异源表达及功能结构分析

【目的】生物胺是一类广泛存在发酵食品中潜在有害物质,可被胺氧化酶分解。本研究对来源于乳酸菌的胺氧化酶的酶学性质及其降生物胺能力进行了探究。【方法】本研究在大肠杆菌中异源表达了植物乳杆菌中多铜氧化酶基因SufI,经过优化表达条件与纯化重组酶后,分析了该酶的最适反应条件、酶稳定性、降生物胺能力、光谱与结构特性。【结果】重组酶的最适pH值为3.5,最适温度为20℃,在pH 4.0–10.0或15–65℃的条件下,相对酶活力保持在70%以上。重组酶具有较好的稳定性,酶活不受乙醇等抑制剂的影响。在8种生物胺的混合体系内,重组酶对生物胺总量的降解量可达403.23μg/mL,其中对酪胺的降解量最多,超过70μg/mL (34.99%)。在单一生物胺体系内,重组酶对酪的底物亲和性较高,酶活可达18.33U/mL。紫外-可见扫描光谱显示酶蛋白在600 nm处有多铜氧化酶家族特征吸收峰,傅里叶红外光谱解析酰胺Ⅰ带中α螺旋、β折叠、β转角和无规则卷曲的相对含量分别为21.52%、20.72%、33.80%和23.97%。同源建模预测该酶具有3个铜结合域,且含有组氨酸、半胱氨酸、甲硫氨酸和谷氨酸等铜配体结合...     

热稳定α-半乳糖苷酶基因在毕赤酵母中的分泌表达及酶学性质研究

克隆前期筛得分枝犁头霉Absidia ramosa WL511的热稳定α-半乳糖苷酶cDNA基因aga(GenBank No.DQ234280),将aga基因插入表达载体pPICZαA并电转化整合到毕赤酵母P.pastoris GS115的染色体上。30℃、甲醇流加量0.5%(V/V)时,酵母发酵上清液中酶活达32U/ml。纯化后酶的比活力为137U/mg,SDS-PAGE显示单一条带,凝胶过滤和SDS-PAGE估算其分子量分别为348kDa和87kDa,该酶为四聚体结构,糖基化导致重组蛋白的分子量比原酶大6kDa。该酶等电点为5.2,最适反应温度73℃,最适pH6.8,60℃以下及pH5.5～9.0范围内活性稳定。75℃时保温2小时保留54%的酶活,85℃时酶活完全消失。以对硝基苯酚-α-D-半乳糖苷为底物,该酶Km值为0.42mmol/Lol/L;Vmax为413U/mg;kcat为64531/min。     

黑曲霉产木聚糖酶固态发酵条件及部分酶学性质的研究

实验以棉粕和玉米秆为主要原料,采用单因素和正交实验方法对黑曲霉固态发酵产木聚糖酶的培养条件进行了优化,为了获得高酶活产品的发酵条件。结果表明,最适培养基组分为棉粕和玉米秆的比例为3∶2,固水比为1∶1.2,尿素的最适添加量为2%(以干重计),KH2PO4的最适添加量为0.2%。在此条件下,菌株产酶活性可达6 529U/g干曲。该酶的最适反应温度为55℃,最适pH为5.0,pH稳定范围较宽,在30℃、pH 3.5~6.0范围内处理100min,酶活保持在85%以上,但耐热性不是很理想,在60℃保温30min残余酶活只有17%。     

解淀粉芽孢杆菌中温α-淀粉酶基因的克隆、表达与酶学性质分析

以中温α-淀粉酶生产菌株Bacillus amyloliquefaciens M23基因组DNA为模板。PCR扩增得到了2．0kb α-淀粉酶基因全长序列。该基因由上游启动子220bp,结构基因1544bp和终止序列320bp构成。将无信号肽的α-淀粉酶结构基因amyQ,克隆入表达载体pET28a,转化E．coli BL21（DE3）,经诱导,测定α-淀粉酶活性。结果表明：α-淀粉酶基因amyQ获得了活性表达,酶活力为2．297U／mL,SDS-PAGE电泳结果显示出分子量约为58kDa特异性蛋白质条带。酶学性质分析表明,重组α-淀粉酶的最适反应温度为60℃,最适反应pH为6．5,在60℃保温15min保持85％以上活性,超过15min,酶迅速失活,在pH5．5～10．0环境下稳定。水解产物分析表明：淀粉水解终产物主要为麦芽寡糖和糊精和少量葡萄糖。     

植酸酶YiAPPA与生淀粉结合域SBD融合酶的构建及酶学性质分析北大核心CSCD

旨在获得酶学性质改良的植酸酶YiAPPA与生淀粉结合域SBD的融合酶。通过在植酸酶YiAPPA的C末端融合嗜热酸性α-淀粉酶GTamy的生淀粉结合域SBD,获得融合酶YiAPPA-SBD。酶学性质分析表明,YiAPPA-SBD的高温活性和热稳定性得到了提高,并获得了对生玉米淀粉的结合能力。其中YiAPPA-SBD于55-90℃范围内的相对酶活均高于YiAPPA的相对酶活;于80℃的半衰期提高约2倍;在生玉米淀粉浓度大于8%的条件下,YiAPPA-SBD对其结合率达到80%以上。并且YiAPPA-SBD保留有YiAPPA的其它优良酶学性质,最适反应pH为4.5,37℃的绝对酶活高达3 900 U/mg,具有优良的pH稳定性和蛋白酶抗性。     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

（鱼句）亚科花（鱼骨）型鱼类骨骼系统的比较

对我国花型Hemibarbuspattern鱼类作了骨骼系统比较,结果表明,此类型鱼类脑颅较长,副蝶骨平直或稍弯曲,眶蝶骨腹纵嵴发达(铜鱼Coreius septentrionalis例外),下颞窝和咽突中等大,基枕骨后突发达;脑颅中的上筛骨的后突、侧筛骨的外筛突,蝶耳骨的外突、上耳骨的后突、围眶骨和后颞窝等均有明显的差异;咽颅中的舌颌骨、尾舌骨、鳃盖骨和下咽齿的列数等又有显著的区别;附肢骨骼中的腰带骨、脊椎骨中的复合神经骨和第4椎骨腹侧的悬器等也有不同之处。据此,这些差异和区别可作为属间或种间的分类依据。     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.