有机相酶促合成阿魏酸糖酯

利用酶促方法合成阿魏酸葡糖酯,对酶和有机溶剂进行了比较和筛选,并对影响阿魏酸葡糖酯产率的因素(酶量、反应时间、反应温度、底物比)进行了研究。结果表明,葡萄糖和阿魏酸乙烯酯(摩尔比4∶1)分别加入到体积比2∶1的无水吡啶和叔丁醇中,20 g/L Novozym 435脂肪酶,在220 r/min的空气振荡器中50℃反应72 h,产率能达到80.9%。     

盖子环替换及定点突变提高菜豆环氧化物水解酶对邻甲基苯基缩水甘油醚的催化性能

菜豆环氧化物水解酶1和2（PvEH1、PvEH2）能够动力学拆分外消旋邻甲基苯基缩水甘油醚（rac-oMGE）,从而保留(R)-oMGE。基于对PvEH1和PvEH2结构的同源模拟和分析,发现二者分子中的盖子环差异较大,故本文选择盖子环作为研究目标。经融合聚合酶链式反应（FPCR）,获得了PvEH2的盖子环区域被PvEH1对应区域替换的杂合酶Pv2Pv1。用全细胞酶E. coli/pv2pv1催化rac-oMGE,当(S)-oMGE刚好水解完全时,产物(S)-3-邻甲苯基-1,2-丙二醇（(S)-oTPD）的ee_p由PvEH2的58.3%提高至75.5%。为进一步提高酶的性质,在Pv2Pv1中选取11个氨基酸位点进行丙氨酸(A)突变,获得最优突变子E. coli/pv2pv1^K176A,活性为E. coli/pv2pv1（4.2U/g）的2.1倍,且当S构型的底物刚好完全水解时,(S)-oTPD的ee_p进一步提高为80.3%。分子对接分析发现,盖子环替换和K176位点突变为A,均使(R)-oMGE环氧环中的C_α更易受到酶中D101位点的攻击。利用E. coli/pv2pv1^K176A催化150mmol/L rac-oMGE水解制备(R)-oMGE（ee_s＞99%）和(S)-oTPD（ee_p=80.4%）,二者的产率Y_S和Y_P分别为32.7%和60.1%,时空产率STY_S和STY_P为1.6g/(L·h)和3.3g/(L·h)。本实验为改善EH的催化性质提供了一种有效策略。     

Liquid chromatography–electrospray ionisation mass spectrometry method for the rapid identification of citrus limonoid glucosides in citrus juices and extracts

A rapid and selective liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS) method to screen citrus samples for limonoid glucosides and estimate their relative concentrations has been developed. This method utilises a phenyl stationary phase, whereas previous methods have relied on C-18. Samples may be analysed directly without treatment other than dilution. Peak areas from the extracted deprotonated molecular ion mass signals for individual limonoid glucosides were normalised against the sum of the areas to establish their relative concentrations. The method was successfully applied to the analysis of various juice, extracts, and liquid samples of partially purified limonoid glucosides.     

微波和脱皮处理菜籽对油中活性成分的影响

以高、中、低芥酸和硫甙含量菜籽为原料,微波和脱皮预处理后分别冷榨制油。结果表明,不同处理方式对同一品种菜籽的基本品质并无影响,但油中植物甾醇和维生素E含量均存在明显差异,且含量大小依次为低芥酸低硫甙菜籽〉中芥酸中硫甙菜籽〉高芥酸高硫甙菜籽。利用SPSS（统计产品与服务解决方案,Statistical Productand Service Solutions）方差分析,探讨4种不同处理方式与3种不同原料对菜籽油中维生素E和植物甾醇的影响。结果表明,不同处理方式对双低菜籽油中α、γ维生素E和△5-燕麦甾醇影响差异显著,对菜籽甾醇、菜油甾醇和β-谷甾醇影响不显著;同一种处理方式下,不同原料对维生素E和植物甾醇都存在显著差异。微波处理后油中维生素E和植物甾醇含量最高,且低芥酸低硫甙菜籽更适宜通过微波处理提高油的品质。     

Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress

The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are beta-glucosyl ceramides consisting of a saturated alpha-hydroxy or non-hydroxy fatty acid and a Delta4,8-diunsaturated, C9-methyl-branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol-beta-D-glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae. We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach.     

大孔树脂纯化何首乌中二苯乙烯苷的工艺研究

本论文探讨了何首乌中二苯乙烯苷的提取纯化工艺。选择大孔吸附树脂对何首乌乙醇粗提物进行纯化，通过选型，选定Ⅱ型树脂进行纯化和富集，采用不同浓度的乙醇溶液作为洗脱液，得到二苯乙烯苷适宜洗脱的乙醇浓度为40％。纯化后的产物经高效液相色谱分析，其中二苯乙烯苷的含量达到84．47％，得率为75．12％。     

复合膳食纤维固体饮品的研制

以豆腐渣和海带为原料水解制备水溶性膳食纤维(SDF)和多糖,并制备复合膳食纤维固体饮品.用碱法和酶法结合提取大豆不溶性膳食纤维(IDF)和SDF,采用复合酶法制备海带多糖.结果表明:大豆IDF的提取条件为氢氧化钠浓度5%,浸泡时间60min、浸泡温度80℃;纤维素酶添加量为IDF重量的1%,纤维素与水的比例为1:12(W/V),pH为4.5,水解时间为12h,水解温度为40℃,在以上条件下,SDF的产率为36.01%.提取海带多糖条件为加入海带粉重1%的复合酶,复合酶(纤维素酶、果胶酶和蛋白酶)的最佳配比为2:1:2,在pH6.0、50℃酶解6h总多糖得率最高,可达10.71%.     

干酪乳杆菌GL-B体外降胆固醇的作用机理

研究了干酪乳杆菌GL-B所产胆盐水解酶的分离纯化及其体外降解胆固醇的作用机理,通过硫酸铵沉淀、DEAE-Sepharose FastFlow层析、Sephadex G-100层析分离胆盐水解酶。结果表明,胆盐水解酶纯度为原来的27倍,纯化后电泳谱图显示一条带,分子量约为50 ku;干酪乳杆菌GL-B对胆固醇的降解是吸收和共沉淀共同作用的结果,且共沉淀作用大于吸收作用。