黑曲霉S4生淀粉糖化酶的分离纯化及其特性

用酒精沉淀、Sephadex G—100凝胶过滤和离子交换柱层析等步骤对黑曲霉(Aspergillusniger)S4的生淀粉糖化酶进行了分离,纯化出三个电泳纯组分(GI、GII和GIII),纯化倍数分别为5.4、6.0和1.9;酶活收率分别为10．2％、10．7％和1．9％。三个组分均为酸性糖蛋白,其糖含量分别为11．68％、8．6％和3．6％;pl值分别为4．0、3．9和3．6;分手量分别为1380 00、62000和57500。 通过对这三个组分的性质和它们对生淀粉作用副Vmax／K。值的测定,探讨了生淀粉糖化酶的作用机理。     

一株曲霉的一种中温糖化酶的纯化及其部分酶学特性

本研究从广西花坪自然保护区采集的土壤中筛选获得了一株产糖化酶丝状真菌菌株57-45,通过形态学观察和真菌内转录间隔区(internal transcribed spacer,ITS)序列比对分析,将其初步鉴定为曲霉属(Aspergillus sp.)的一个种。纯化真菌57-45所产的一种胞外糖化酶经过硫酸铵分级沉淀、疏水层析和阴离子交换层析三步蛋白质纯化步骤,得到在SDS-PAGE上约60kD的单一蛋白质条带,薄层层析分析表明该纯化的蛋白质水解可溶性淀粉的产物只有葡萄糖,证明该纯化的蛋白质为糖化酶。纯化的糖化酶Km值为1.9mg/mL,Vmax为4148μmol/(min·mg),最适作用pH5.5,最适作用温度50℃,在同步糖化发酵中有应用的潜力。金属离子Fe3+、Zn2+、Cu2+对酶活有较强的抑制作用,EDTA对酶活有较强的促进作用。本文结果将为进一步研究曲霉糖化酶的酶学特性提供新的材料。     

黑曲霉木聚糖酶的底物特异性和低聚木糖生产

对黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ）木聚糖酶进行了纯化研究,结果表明,经Ｓｅｐｈａｄｅｘ Ｇ－１００和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０分离后,获得三个组分,称为Ｘ１、Ｘ２、Ｘ３。它们经ＰＡＧＥ电泳分析均为单一组分。对Ｘ１、Ｘ２、Ｘ３的相关性质,特别是底物特异性也作了研究,纯酶Ｘ３组分或部分纯化的酶可用于生产低聚木糖,产品得率为１０％。     

黑曲霉3.795glaA基因及其5'调控区的克隆和序列分析

黑曲霉Ｔ２１是由黑曲霉３．７９５经诱变育种获得的糖化酶高产菌株,为阐明其高产的分子机制,由黑曲霉３．７９５克隆了糖化酶结构基因及其５′旁侧序列,并与黑曲霉Ｔ２１的相应序列进行了比较．由黑曲霉３．７９５菌丝体分离染色体ＤＮＡ,Ｓｏｕｔｈｅｒｎ杂交分析表明,糖化酶结构基因位于～２．５ｋｂ的ＥｃｏＲⅠ－ＥｃｏＲⅤ染色体ＤＮＡ片段上,在此ＥｃｏＲⅠ位点上游约１．０ｋｂ处有一ＳａｌⅠ位点．为构建糖化酶结构基因及其５′旁侧序列的基因组文库,该染色体ＤＮＡ分别用ＥｃｏＲⅠ＋ＥｃｏＲⅤ和ＥｃｏＲ＋ＳａｌⅠ消化,琼脂糖凝胶电泳分离并回收长度在１．０ｋｂ左右和２．５ｋｂ左右的ＤＮＡ片段,分别与ｐＵＣ１９载体连接后转化入Ｅ．ｃｏｌｉＤＨ５．用原位杂交方法筛选到了携带糖化酶基因编码区及其１５０５ｂｐ５′旁侧序列的阳性克隆．对克隆片段的ＤＮＡ序列进行了测定并与黑曲霉Ｔ２１的相应序列进行了比较,结果表明,在糖化酶基因编码区及其１５０ｂｐ３′非编码区内,未发现碱基差异,但在－３４０～－１５０５的５′上游区内发生了９个位置的碱基变化,包括缺失、插入和替换．这些结果表明,黑曲霉Ｔ２１与３．７９５的糖化酶产量的差异与其结构基因无关,但可能与其     

核磁共振波谱方法在顺磁金属蛋白-紫色震颤菌血红蛋白中的应用

金属蛋白由于其所带金属离子具有未配对电子而具有顺磁性,致使弛豫时间缩短、谱线加宽（～５０Ｈｚ）以及谱范围变大（～１００ｐｐｍ）,因而具有与抗磁蛋白不同的谱特征。这时一些常用的２ＤＮＭＲ实验方法将失效。本文探索顺磁金属蛋白的几种ＮＭＲ实验方法,如溶剂峰抑制、２ＤＭＣＯＳＹ、ＮＯＥＳＹ、１ＤＮＯＥ差谱及ＳＵＰＥＲＷＥＦＴ等,并精心设计实验条件使之适用于顺磁金属蛋白的结构研究。这些实验方法成功地应用于紫色震颤菌血红蛋白（ｍｅｔ－ＶｔＨｂ－ＣＮ）。通过这些实验找到了与血红素Ｆｅ３＋配位的组氨酸,并完成了该金属蛋白的顺磁位移的共振峰的识别、活性中心质子间距离的测量、抗磁性化学位移及电离常数ｐＫａ值的确定。     

枯盘斑多毛孢Pf-毒素组分的研究I.活性组分I的结构分析

以正丁醇:水:甲醇(4:2:1)作洗脱剂,通过硅胶H60型柱反复柱层析,将3种有致病活性的物质充分纯化,在-40℃下冷冻干燥后,它们为褐色深浅不一的蓬松状物质,且极易吸潮。活性组分I(Rf0.83)、活性组分II(Rf0.79)和活性组分III(Rf0.80)对马尾松切根幼苗和湿地松切根幼苗针叶都有致萎作用,通过质谱(MS)、核磁共振谱(1HNMR、)和红外光谱(IR)等分析手段确定出所分离的活性组分I的化学组成为C5H11O5N(M=165)。     

北青龙衣抗肿瘤谱效关系研究初探

目的:对北青龙衣抗肿瘤谱效关系进行初步研究。方法:采用乙醇冷浸提取,大孔树脂进行初步分离,所得到的各组分用MTT法进行体外细胞毒试验,测定各组分对人胃癌细胞BGC803的抑制活性,并用薄层色谱进行初步谱效关系研究。结果:大孔树脂经30%乙醇洗脱的组分显示较强的细胞毒活性,薄层色谱显示该组分为极性大和极性小的成分的复杂组合。结论:大孔树脂经30%乙醇洗脱的组分在北青龙衣抗肿瘤活性中起重要作用。     

条斑紫菜中一种琼胶多糖的分离纯化及鉴定

条斑紫菜（Ｐｏｒｐｈｙｒａ ｙｅｚｏｅｎｓｉｓ）的热水提取物,经ＤＥ－２３和Ｓｅｐｈａｄｅｘ Ｇ－２００柱层析纯化,得到一种含微量硫酸基的琼胶精品（ＰＹ１）。此多糖经Ｓｅｐｈａｒｏｓｅ ６Ｂ柱层析鉴定为单一组分,分子量为２２０ｋＤ。紫外光谱显示它不含蛋白质、多肽及核酸。红外光谱揭示它含３,６－内醚－半乳糖的特征吸收以及微量的硫酸基。该多糖的水解产物经气相色谱分析,主要由半乳糖及其衍生物组成,有少量     

极低频率（ELF）磁场对细胞生长周期分布谱的影响及其机理的计算机模拟分析

测定在各种温度条件下和在ＥＬＦ（极低频率）磁场作用下细胞生长周期分布谱的变化。实验结果表明温度不仅能使周期分布谱的离散性发生改变,而且也能使谱的峰值产生位移,而ＥＬＦ磁场只能使周期分布谱的离散性发生变化,对谱的峰值没有显著影响。这一差异是由于温度和ＥＬＦ磁场对细胞生长产生影响的机理不同。用计算模拟不同机理对细胞生长周期分布谱的影响与我们早先提出的机理相吻合,即温度影响细胞内各种生长因子、生物离子的     

新疆甜瓜疫霉菌毒素的分离及其致病效应（简报）

从瓜类疫霉菌液体培养液中分离到一种对新疆甜瓜幼苗有明显致萎效应的毒素，用ＤＥＡＥ－５２柱纯化得到两个组分，其中组分Ⅰ蛋白质含量低于组分Ⅱ，而糖含量则高于组分Ⅱ，组分Ⅱ的致萎效应大于部分Ⅰ。     

氨基酰化酶中金属锌离子的功能作用

 氨基酰化酶是含锌金属酶。该酶每摩尔蛋白中含2摩尔Zn(Ⅱ)离子。金属鳌合剂与酶作用,通过竞争螯合Zn(Ⅱ)离子使酶活力下降。残余活力与残留金属含量呈正相关。竞争螯合的结果,生成不含金属的脱辅基酶蛋白,并导致酶活力的丧失。脱辅基酶由于加入Zn(Ⅱ)离子而恢复其活力。实验表明金属锌离子是氨基酰化酶催化活力所必需。与Zn(Ⅱ)离子相似,Co(Ⅱ)离子也可与脱辅基酶相结合并使之复活。 在190—240nm区域内对比了天然酶、脱辅基酶蛋白与Co(Ⅱ)置换氨基酰化酶的圆二色谱。远紫外圆二色谱表明,与天然酶相比,在脱辅基酶中由于金属离子的丧失导致主链构象发生变化,其中α螺旋增加约7％。因而锌离子(钴离子)对蛋白主链的反应最适构象有一定的稳定作用。脱辅基酶与Co(Ⅱ)离子结合,酶的主链构象恢复至与天然酶几近相同。可认为这是促使酶复活的内在因素。     

Spectroscopic characterisation of n-octanohydroxamic acid and potassium hydrogen n-octanohydroxamate

The crystal structure and spectroscopic characteristics of n-octanohydroxamic acid and the potassium compound of that acid have been investigated by XRD, XPS, FTIR and Raman spectroscopy. XRD revealed that the acid is in the keto Z conformation with the alkyl chains oriented along the z-direction and hydrogen bonding between hydroxamate moieties. Vibrational spectra confirm this conclusion. Chemical analysis, XRD and XPS established that the potassium compound is the acid salt KH(C₇H₉CONO)₂. The crystal structure showed that the hydroxamate groups are also in the keto Z conformation and this is supported by vibrational spectra. In the acid salt, the two hydroxamate moieties are connected by a symmetrical O-H-O short hydrogen bonded linkage between the two hydroxamate oxygen atoms and this explains the absence of a discernible O-H stretch band in the vibrational spectra. Identification of the vibrational bands displayed is supported by deuteration and ¹⁵N substitution.     

Spectroscopic characterisation of copper acetohydroxamate and copper n-octanohydroxamate

The nature of the bonding in acetohydroxamic acid, copper acetohydroxamate and copper n-octanohydroxamate has been investigated by chemical analysis, XPS, FTIR and Raman spectroscopy. Vibrational spectra show the acid to be in the keto Z conformation as was previously established for the n-octano homologue. Chemical analysis established that the copper compounds have a copper:hydroxamate stoichiometry of 1:1. XPS confirms that they are Cu^II compounds. The absence of vibrational spectral bands that were previously identified with N-H vibrations for n-octanohydroxamic acid and its potassium compound, together with the presence of a CN stretch band that shifts when the nitrogen is labelled with ¹⁵N, confirms that the hydroxamate moieties in the Cu^II compounds are in the enol configuration. Some interaction between Cu and N is indicated by the spectra and could explain the 1:1 stoichiometry of the Cu^II hydroxamates investigated.     

Vibrational circular dichroism of gramicidin D in vesicles and micelles

The vibrational circular dichroism (VCD) and absorption spectra of gramicidin D in three model membranes (dioctadecyldimethylammonium chloride vesicles, dimyristoyl-phosphatidylcholine vesicles, and sodium dodecyl sulfate micelles) are presented. The absorption and VCD spectra suggest that the stable gramicidin D conformation in the model membranes is different from those in organic solvents. The presence of cations does not change the membrane-bound conformation of gramicidin D.     

用光谱技术鉴定植物色素突变体

1983年我国报道了从γ-射线处理的“矮杆齐”大麦中得到了一株黄绿色的突变体1832C[1]。本文用光谱技术对该突变体的光合色素成分进行了鉴定。1　材料和方法　　材料为六棱裸大麦“矮杆齐”和由该品种大麦诱变形成的黄绿色突变体1832C(Mb1832C),以及作为对照的缺失Chlb的突变体大麦Chlorina-f2[2]都于3月初播种于实验田中。　　每个样品取30g新鲜的叶片,先用自来水后用蒸馏水冲洗干净。把洗净的叶片摊放在干净的纱布上吸干表面水分,剪碎,加入100mL预冷的含有0.4mol/L山梨醇、0.1mol/LTris-HCl(pH7.6)的缓冲液,用组织捣碎机先慢速捣碎1…     

Polyproline II Confirmation in the Protein Component of Arabinogalactan-Protein from Lolium multiflorum

Circular dichroism spectra of the arabinogalactan-protein from suspension-cultured Lolium multiflorum (ryegrass) endosperm cells demonstrate the presence of polyproline II conformation in the protein moiety of the proteoglycan. Subject to a number of theoretical and practical constraints of the method, it can be estimated that at least 30% of the protein component is in this conformation.     

心磷脂-细胞色素C体系CD谱的研究

 线粒体内膜中含有特异的心磷脂是细胞色素C氧化酶活性的必需脂。本工作测定了心磷脂脂质体对细胞色素C溶液园二色(CD)谱的影响,发现心磷脂可引起血色素铁的氧化,并使其轴向配位场强的对称性下降。提示心磷脂可能参与酶和底物之间的电子转移过程。     

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin.

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

华山松吸收光谱及叶绿素荧光特性的地理变异

利用分离的叶绿体作实验材料,发现华山松(Pinus armandi Franch.)南方种源的4阶导数吸收光谱在680nm处峰值较大,在670nm处峰值较小,而北方种源中出现了在680nm处峰值较在670nm峰值小的类群,推断北方种群反应中心活力有下降趋势。南、北种源之间的低温(77K)荧光发射光谱有明显差异,PSⅠ、PSⅡ发射峰位置出现地理变动。低温荧光激发光谱分析表明,地理变异主要发生在叶绿素a的分子状态上。研究还表明,完整的针叶因为有角质层、松脂等物质干扰,检测不出光谱的差异,不是理想的实验材料。     

锌离子对氨基酰化酶构象及其稳定性的影响

天然氨基酰化酶和脱谷氨基酰化酶无论在二级结构（用ＣＤ和ＦＴＩＲ监测）还是三级结构上（以荧光发射光谱监测）都有明显的差异,表明了脱锌后酶的有序度降低;当比较天然和脱锌氨基酸化酶对去圬剂的稳定性时,结果表明脱锌后酶的构象的稳定性明显降低．因此可以认为锌离子对维持酶分子活性部位的特定构象以及构象的稳定性具有重要的作用．