Access of the substrate to the active site of yeast oxidosqualene cyclase: an inhibition and site-directed mutagenesis approach

A structural model of Saccharomyces cerevisiae oxidosqualene cyclase (SceOSC) suggests that some residues of the conserved sequence Pro-Ala-Glu-Val-Phe-Gly (residues 524-529) belong to a channel constriction that gives access to the active-site cavity. Starting from the SceOSC C457D mutant, which lacks the cysteine residue next to the catalytic Asp456 residue Cys457 has been replaced but Asp456 is still there, we prepared two further mutants where the wild-type residues Ala525 and Glu526 were individually replaced by cysteine. These mutants, especially E526C, were very sensitive to the thiol-reacting agent dodecyl-maleimide. Moreover, both the specific activity and the thermal stability of E526C were severely reduced. A similar decrease of the enzyme functionality was obtained by replacing Glu526 with alanine, while substitution with the conservative residues aspartate or glutamine did not alter catalytic activity. Molecular modeling of the yeast wild-type OSC and mutants on the template structure of human OSC confirms that the channel constriction is an important aspect of the protein structure and suggests a critical structural role for Glu526.     

抗氧剂3114合成反应热力学和动力学的研究

研究了常温溶剂法合成抗氧剂3114反应热力学和动力学过程。控制温度在117±2℃,研究结果表明,反应是零级反应,反应速率常数k=1.91×10-3/min,反应的平衡常数K=138.4(mol/L)-6,反应活化能Ea=1366.2J/mol。     

Novel stabilization system for polypropylene via the Ziegler–Natta catalyzed polymerization in the presence of aluminum aryloxide

The aluminum aryloxide was prepared via the reaction of phenolic antioxidant, 3‐(3,5‐di‐t‐butyl‐4‐hydroxyphenyl)‐N‐octadecylpropionamide, with triethyl aluminum. Propylene polymerization using supported Ziegler–Natta catalyst systems was carried out in the presence of the antioxidant or its aluminum aryloxide. Although the antioxidant gave rise to decrease in catalyst yield and change in hydrogen response, the aluminum aryloxide had no influence on the catalytic polymerization behavior, and thus the obtained polymer characteristics such as molecular weight, polydispersity, and meso pentad as a stereoregularity were comparable to that polymerized without the antioxidant and the aluminum aryloxide. Polypropylene obtained in the presence of the aluminum aryloxide was well stabilized for oxidation and its stability was over 1000 h at 100°C (estimated to be over 30 years at room temperature). © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 1350–1358, 2006     

Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR

In an effort to improve the properties of cyclodextrin glucanotransferase (CGTase) as an antistaling enzyme, error-prone PCR was used to introduce random mutations into a CGTase cloned from alkalophilic Bacillus sp. I-5 (CGTase I-5). A mutant CGTase[3-18] with the three mutations M234T, F259I and V591A was selected by agar plate assay. Sequence alignment of various CGTases indicated that M234 and F259 are located in the vicinity of the catalytic sites of the enzyme and V591 in the starch binding domain E. The cyclization activity of CGTase[3-18] was dramatically decreased by 10-fold, while the hydrolyzing activity was increased by up to 15-fold. These mutations near subsite +1 (M234T) and at subsite +2 (F259I) are likely to alter the enzyme activity in a concerted manner, promoting hydrolysis of substrate while retarding cyclization. The addition of CGTase[3-18] reduced the retrogradation rate of bread by as much as did the commercial antistaling enzyme Novamyl during 7-day storage at 4 degrees C. No cyclodextrin (CD) was detected in bread treated with CGTase[3-18], whereas 21 mg of CD per 10 g of bread was produced in bread treated with wild-type CGTase.     

茶叶活性成分综合提取过程研究

以低档绿茶为原料,研究了茶多酚、咖啡碱和茶多糖的提取、分离、纯化等工艺条件,探索出一条综合提取新工艺。研究表明,以纤维素酶和果胶酶为主体的复合酶解提取,可提高茶叶中各有效成分的提取率,并可降低其活性损失;通过超滤膜过滤酶提液不但精制了料液而且利用膜浓缩液可方便地制备茶多糖;采用H-600树脂吸附阶段洗脱层析法分离茶多酚咖啡碱,解决了传统工艺中使用有毒溶剂脱除咖啡碱的难题。文章计算了吸附柱的传质区高度和总传质系数,为放大设计奠定了基础。     

削弱或抑制抗氧剂、光稳定剂作用的有关因素

介绍了削弱或抑制抗氧剂、光稳定剂作用的常见因素及实例，分析了防老化塑料制品发生提前老化现象的可能性和原因。     

现代生物工程技术简介

对组成现代生物工程的六大技术系统：基因工程、蛋白质工程、酶工程、细胞工程、微生物工程、生化工程的历史和现状，工作内容及应用前景作了简明的介绍。     

杂色云芝漆酶的分离、纯化和酶学特性研究

杂色云芝发酵液,经超滤、DEAE-Sephadex A250柱层析两步分离、纯化出两型漆酶。以愈创木酚与酶带在适当条件下反应呈棕红色谱带鉴定出漆酶谱带。两型漆酶分别命名为LacA和LacB。经测定两漆酶亚基的分子量分别为66.0kDa和30.7kDa;等电点pI分别为4.79和5.18;LacA的最适作用温度是40℃,LacB为50℃;保温2h, LacA的半失活温度为60℃,LacB为90℃;LacA和LacB催化愈创木酚的酶活最适pH值均为4.0; 二者的稳定pH值也都在2.5~5.5之间。LacA催化氧化愈创木酚的Km和Vmax分别为286.5molL-1和292.4nmol mL-1 min-1;LacB催化氧化愈创木酚的Km和Vmax分别为813.0mol L-1和127.4nmol mL-1 min-1。     

Oil and fat hydrolysis with lipase fromAspergillus sp.

Hydrolysis of olive oil, soybean oil, mink fat, lard, palm oil, coconut oil, and a hydrogenated, hardened oil with lipase from anAspergillus sp. has been studied. The lipase had high specific activity (60,000 U/g) and did not show any positional specificity. The lipase proved to be a more effective catalyst than Lipolase fromA. oryzae, with an optimal activity at 37°C and pH 6.5–7.0. It was activated by Ca²⁺ but inactivated by organic solvents such as isopropanol and propanone. All substrates examined could be hydrolyzed to corresponding fatty acids with this enzyme at concentrations of 5–30 U/meq with yields of 90–99% in 2–24 h. The degree of hydrolysis was almost logarithmically linear with reaction time and occurred in two stages. The lipase was stable and could be repeatedly recycled for hydrolysis.     

对羟基苯甲酸-壳寡糖改性水性聚氨酯

用对羟基苯甲酸(p-HBzA)对壳寡糖(OCS)进行接枝改性,得到了对羟基苯甲酸接枝壳寡糖(p-HBz A-g-OCS)。将p-HBz A-g-OCS作为交联剂,与水性聚氨酯预聚体(PPU)聚合,合成了对羟基苯甲酸-壳寡糖改性水性聚氨酯(HOCS-WPU)。通过FTIR和UV-Vis证实了样品的结构;分别用DLS、力学性能分析、XRD、TGA、ABTS和DPPH自由基清除率对HOCS-WPU的性能进行了考察。结果显示:当加入质量分数为2%的p-HBz A-g-OCS(以合成PPU原料的总质量计)时,HOCS-WPU乳液粒径较小,为57.85nm,相比未改性WPU,HOCS-WPU胶膜的拉伸强度从14.03 MPa提高到16.85 MPa,结晶度从3.57%下降到3.45%;初始分解温度从204.6℃提高到216.7℃。自由基清除率测定结果显示:当HOCS-WPU的质量浓度为2.5 g/L时,该聚合物的ABTS和DPPH自由基的清除率分别达到了62%和35%,抗氧化活性相比未改性WPU明显提高。