固定化酶微观活性定位的X射线微区分析

利用X射线微区分析,对共价法得到的固定化L—天门冬酰胺酶的活性进行了分析。L—天门冬酰胺作为底物,MgCl2作为捕捉剂,底物经L-天门冬酰胺酶催化分解产生氨,后者和捕捉剂反应产生沉淀,可以确定固定化L-天门冬酰胺酶的催化活性部位。结果表明：大孔树脂载体,酶活较高,活性L-天门冬酰胺酶分布较均匀。并得到了固定化L-天门冬酰胺酶的活性定位的最佳条件。并对不同结构载体固定化酶活性进行了研究。     

壳聚糖固定化脲酶活性的X射线微区分析

利用X射线微区分析方法,对壳聚糖固定化脲酶活性进行了定位分析。筛选出了能捕捉固定化脲酶活性部位的捕捉剂BaCl₂,以尿素作为底物,在Tris-HCl缓冲溶液中,底物经固定化脲酶催化产生NH₃和CO₂,后者和捕捉剂反应生成BaCO₃沉淀, 沉积在固定化脲酶的催化活性部位,借此确定其催化活性部位。结果表明BaCl₂可用做固定化脲酶活性定位的捕捉剂。同时得到了壳聚糖固定化脲酶活性定位的最佳实验条件。     

酶电极上葡萄糖氧化酶的活性的X射线微区分析

利用X射线微区分析, 对二氧化硅溶胶-凝胶包埋于普鲁士蓝修饰玻碳电极上的葡萄糖氧化酶的活性进行了分析; 以Ce(NO₃)₃为捕捉剂, 底物葡萄糖经葡萄糖氧化酶作用产生过氧化氢,后者与捕捉剂反应生成沉淀于酶的活性部位。从X射线微区分析结果表明： 酶电极表面固定化酶的分布均匀,且保存较高的酶活,从微观的角度说明了酶电极的性能与酶电极表面酶活分布的关系。此法制备的葡萄糖氧化酶电极具有较高的灵敏度,稳定性,这与电化学测试结果是一致的。     

磁性纳米颗粒Fe3O4固定化纤维素酶的光谱学研究

以氨水作沉淀剂,用共沉淀法制备了磁性纳米颗粒Fe3O4,并以此为载体,通过碳化二亚胺的活化作用将纤维素酶固定化,通过傅里叶红外和重复多次催化实验证实纤维素酶在磁性纳米颗粒上的固定,透射电镜表征了固定化酶微粒的形貌.用DNS分光光度法测定固定化纤维素酶的活性,研究表明磁性固定化酶的催化作用的最适温度为60℃和pH值为3.94～5.50.结果表明,磁性固定化纤维素酶具有比自由酶更好的热稳定性,贮存稳定性和更广泛的pH值适用范围,为纤维素的转化和利用效率的提高提供了理论基础.     

用荧光方法检测HIV-1整合酶的体外活性

HIV-1整合酶是抗艾滋病药物研究的一个重要靶点。整合酶催化两步反应使病毒cDNA整合到宿主细胞基因组中, 该反应过程可以在体外利用重组蛋白和DNA底物实现。本研究构建了HIV-1整合酶表达载体, 在大肠杆菌中诱导表达, 亲和纯化得到重组整合酶蛋白, 利用荧光染料标记DNA底物分子和荧光标记抗体, 通过荧光信号检测整合酶对DNA的切割和连接两步反应, 达到检测整合酶体外活性的效果。该方法具有灵敏度高、特异性好等优点, 便于开展以整合酶为靶点的药物筛选等工作。     

一种可用于酚类化合物检测的酶传感器

利用溶胶-凝胶法将辣根过氧化物酶(HRP)固定化于二氧化硅凝胶网络中构建了可用于酚类化合物检测的酶传感器。对二氧化硅载体材料进行了结构表怔。二氧化硅多孔材料的平均孔径为2.95 nm。孔径小于5 nm占总数的84.068%。由于辣根过氧化物酶的分子尺寸远远大于二氧化硅的凝胶网络的平均孔径,因此不会泄露到溶液中去,而尺寸较小的底物可以发生反应。包埋的辣根过氧化物酶在H₂O₂的存在下,能够催化氧化苯酚与4-氨基安替比林反应生成醌亚胺有色化合物。通过紫外-可见光谱测定醌亚胺有色化合物的吸光度,即可以确立苯酚的含量。对于象含氯酚类的重要污染物, 如邻氯酚、间氯酚、2,4-二氯酚,这种方法也同样适用。此外,对多次测定以后的酶的活性下降的问题进行了讨论,结果表明酶传感器可以重复使用10次以上。但响应时间会变长。     

脉冲电场对木瓜蛋白酶影响的荧光光谱分析

木瓜蛋白酶溶液在电场强度为50 kV·cm-1,频率1 500 Hz,脉冲宽度40 μs的脉冲电场下接受19 800个脉冲处理后,其活性降低了56.5％。文章采用荧光偏光光谱对处理前后的样品进行了分析。 处理后酶样的荧光强度明显大于处理前,在峰值位置其荧光强度增加超过50％,峰位从342 nm移到了346 nm左右,其荧光偏振幅度明显减小。由此推断出木瓜蛋白酶经脉冲电场处理后酶蛋白的α-螺旋结构松散拉伸,分子内部的氨基酸残基暴露于分子表面,并有部分发生离解游离于溶液中,导致活性部位的结构发生变化,最终导致酶失活。     

新型饱和烷基类自由基捕捉剂的合成及ESR研究

设计合成了新的饱和烷基类线型硝酮捕捉剂N-（亚乙基）-t-丁胺-N-氧化物(EBN)和N-（亚乙基）-1-二乙氧基磷酰基-1-甲基乙基胺N-氧化物(EPN),并运用ESR、MS、IR、UV等一系列手段对其结构进行了表征,同时对Fenton体系中产生的羟基及不同类型的氧中心、碳中心和硫中心自由基的捕获能力进行了系统的研究. 结果表明,这两种捕捉剂合成方法比较简单,产率较高,对羟基等自由基有比较强的捕捉能力. 期望本文的工作能为自由基捕捉剂的研究提供一个新的思路.     

动态高压微射流技术对木瓜蛋白酶活性的影响(英文)

研究了动态高压微射流技术对木瓜蛋白酶活性的影响,并以荧光光谱为检测手段对木瓜蛋白酶的分子构象进行表征。结果显示,动态高压微射流处理(120~180 MPa)后,木瓜蛋白酶酶活降低。经180 MPa处理1次,木瓜蛋白酶相对酶活降至90.04%。随着处理压力的增加,木瓜蛋白酶分子、酪氨酸残基、色氨酸残基的荧光发射峰位置分别从对照组的334、285、277.5 nm红移至140 MPa处理组的335.5、285.5、278.5 nm,然后回移至180 MPa处理组的334、285、278 nm。在0~4℃放置24 h后,酶活进一步降低,木瓜蛋白酶和酪氨酸残基的荧光强度出现波动(降低、上升然后再降低),表明动态高压微射流处理(120~180 MPa)改变木瓜蛋白酶分子构象的效果较为明显,形成的新构象稳定性低。     

超声波对木瓜蛋白酶催化活性影响的机理研究

木瓜蛋白酶经适当参数的超声波处理后酶活力提高。超声处理后酶的米氏常数Km变小,最大反应速率Vm也减小。超声处理后酶的紫外吸收光谱不变,荧光发射光谱也不改变,而差示光谱出现明显的正峰和负峰。研究结果表明,超声波处理后,木瓜蛋白酶的构型没有改变,而构象发生了变化。本文讨论了超声波影响木瓜蛋白酶活性的可能机理。     

十二烷基硫酸钠对木瓜酶酶活性和构象的影响

采用紫外、荧光和圆二色等光谱技术研究了阴离子表面活性剂十二烷基硫酸钠（SDS）对木瓜酶酶活力及其构象变化的影响.结果表明：（1）SDS对木瓜酶酶活力的影响与pH有关,pH=8.00时,SDS对木瓜酶酶活力有增强作用,pH=6.30时,除了在SDS的临界胶束浓度（cmc）时木瓜酶酶活力得到增强外,其他浓度下SDS对木瓜酶酶活力没有影响,pH=9.50时,SDS对木瓜酶酶活力产生抑制作用.（2）SDS对木瓜酶的荧光产生淬灭,0.1cmc的SDS木瓜酶荧光峰红移,而SDS浓度≥1cmc后则使之蓝移;（3）当[SDS]＜cmc时,木瓜酶的α-螺旋、β-折叠、回转等有序结构的含量减少而无定形结构含量增加,当[SDS]＞cmc后,木瓜酶的α-螺旋、β-折叠构型含量有所增加,无定形构型含量则有所减少.有序结构的含量越大,酶活力越强.     

Influence of sodium alginate pretreated by ultrasound on papain properties: Activity,structure, conformation and molecular weight and distribution

The aim of the study was to investigate the impact of sodium alginate (ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and molecular weight and distribution of papain. ALG solutions were pretreated with ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45 W/cm²), 135 kHz, 50 °C for 20 min. The maximum relative activity of papain increased by 10.53% when mixed with ALG pretreated by ultrasound at 0.25 W/cm², compared with the untreated ALG. The influence of ultrasound pretreated ALG on the conformation and secondary structure of papain were assessed by fluorescence spectroscopy and circular dichroism spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG increased the number of tryptophan on papain surface, especially at 0.25 W/cm². It indicated that ultrasound pretreatment induced molecular unfolding, causing the exposure of more hydrophobic groups and regions from inside to the outside of the papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes in the secondary structure of the papain. The content of α-helix was slightly increased after ultrasound pretreatment and no significant change was observed at different ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the secondary structure of papain, especially at 0.25 W/cm². The free sulfhydryl (SH) content of papain was slightly increased and then decreased with the increase of ultrasonic power. The maximum content of free SH was observed at 0.25 W/cm², under which the content of the free SH increased by 6.36% compared with the untreated ALG. Dynamic light scattering showed that the effect of ultrasound treatment was mainly the homogenization of the ALG particles in the mixed dispersion. The gel permeation chromatography coupled with the multi-angle laser light scattering photometer analysis showed that the molecular weight (M_w) of papain/ALG was decreased and then increased with the ultrasonic pretreatment. Results demonstrated that the activity of immobilized papain improved by ultrasonic pretreatment was mainly caused by the variation of the conformation of papain and the effect of interactions between papain and ALG. This study is important to explain the intermolecular interactions of biopolymers and the mechanism of enzyme immobilization treated by ultrasound in improving the enzymatic activity. As expected, ALG pretreated by appropriate ultrasound is promising as a bioactive compound carrier in the field of immobilized enzyme.     

光谱法研究CTAB对木瓜酶酶活和构象的影响

采用荧光光谱、圆二色谱(CD)和紫外光谱研究了十六烷基三甲基溴化铵(CTAB)在NaHPO4-NaH2PO4缓冲溶液中对木瓜酶构象变化及其酶活的影响,并探讨了CTAB与木瓜酶的作用机理.荧光光谱表明,在CTAB存在下,木瓜酶的荧光强度增强,荧光峰发生红移,CTAB使木瓜酶分子链趋于伸展,酪氨酸等发色基团更多地暴露在水中.CD谱表明,当CTAB浓度(CCTAB)为0.5 mmol/L时,木瓜酶的a-螺旋构象和β-折叠构象含量比纯木瓜酶(Papain)体系分别增加了1.3％和0.1％,回转构象和无定型构象含量则分别减少了0.3％和1.2％.酶活测定结果表明,CTAB对木瓜酶的酶活有较大影响,当CCTAB由0增加到 1.5mmol/L时,木瓜酶酶活由11.2×105U/g增大到16.3×105U/g,当CCTAB由1.5mmol/L增加到3.5mmol/L时,木瓜酶的酶活又由峰值(16.3×105U/g)降到11.7×105U/g.CTAB使木瓜酶酶活增强与木瓜酶的构象有序度提高有关.     

大孔吸附树脂吸附法结合氯仿溶解法分离混合二元酸

建立利用大孔吸附树脂吸附法结合氯仿溶解法分离混合二元酸中丁二酸、戊二酸和己二酸的方法并使其分别达到一定的纯度和收率。首先利用LC-ESI-MS方法测定了混合二元酸中丁二酸、戊二酸和己二酸含量,然后通过对3种大孔吸附树脂AB-8、NKA-Ⅱ和D-380吸附性能的综合比较,筛选出AB-8大孔吸附树脂作为吸附剂,以水、10%、30%和95%乙醇水溶液作为洗脱剂对混合二元酸进行初步的分离,得到A、B、C和D4个分离部位,再利用氯仿对二元酸溶解度的不同,将该4个分离部位在55℃水浴恒温分别用氯仿进行溶解、过滤,通过LC-ESI-MS方法测定确定相应部位的组成情况后进行适当合并,最终达到将混合二元酸分离的目的。丁二酸纯度:70.3%,收率:92.7%;戊二酸纯度:94.1%,收率89.2%;己二酸纯度:90.7%,收率:88.7%。     

Magnetic enzyme reactors for isolation and study of heterogeneous glycoproteins

The newly developed magnetic micro- and nanoparticles with defined hydrophobicity and porosity were used for the preparation of magnetic enzyme reactors. Magnetic particles with immobilized proteolytic enzymes trypsin, chymotrypsin and papain and with enzyme neuraminidase were used to study the structure of heterogeneous glycoproteins. Factors such as the type of carrier, immobilization procedure, operational and storage stability, and experimental conditions were optimized.     

Synthesis of bi-metallic Au–Ag nanoparticles loaded on functionalized MCM-41 for immobilization of alkaline protease and study of its biocatalytic activity

In this work, Au–Ag nanoparticles (Au–Ag-bi-MNPs) have been prepared on amine functionalized Si-MCM-41 (NH₂–Si-MCM-41) particles through a reduction of AgNO₃ and HAuCl₄ by NaBH₄ at ambient conditions. Au–Ag-bi-MNPs loaded on the NH2–Si-MCM-41, provide a good biocompatible surface for immobilization of the enzyme alkaline protease. This immobilization, presumably due to bonding between core shell nanoparticles and OH in serine 183 in alkaline protease seems to be of an ionic exchange nature. We found that the alkaline protease immobilized on the Au–Ag-bi-MNPs/Si-MCM-41 is an active biocatalyst, stable at different pH and temperature. The bio catalytic activity of free alkaline protease in solution was 64 U/mg (Units per milligram), whereas that of the alkaline protease immobilized on Au–Ag-bi-MNPs/Si-MCM-41 was 75 U/mg. This improvement of the biocatalytic activity may be due to a really increased activity per molecule of immobilized enzyme or to a purification of the enzyme. The alkaline protease molecules immobilized on the (Au–Ag)/ NH₂-MCM-41 surface retained as much as 80% of the catalytic activity recorded at pH=8, and showed significant catalytic activity of alkaline protease in the bioconjugate material. The biocatalytic materials were easily separated from the reaction medium by mild centrifugation and exhibits excellent reuse and stability characteristics over four successive cycles. The optimum temperature ranged from 35 ^°C–55 ^°C and pH=8 for bioactivity of the alkaline protease in the assembly system was observed to be higher than that of the free enzyme in solution. The enzyme biocatalytic activity was monitored by UV-visible spectroscopy. Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and dispersive analysis of X-RAY (EDAX) were used to characterize the size and morphology of the prepared materials.