Purification and Characterization of a Novel Alginate Lyase from a Marine Streptomyces Species Isolated from Seaweed

Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn²⁺, Co²⁺, and Fe²⁺ increased the alginate degrading activity, but it was inhibited in the presence of Zn²⁺ and Cu²⁺. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.     

麦根腐长蠕孢毒素对小麦苯丙氨酸氨裂解酶活性的影响

在根腐毒素浸黄化苗根和离体苗试验中，感病品种ＰＡＬ比活力显著高于对照，在一定范围内酶活性与毒素浓度成正相关，而抗病品种ＰＡＬ比活力变化不大。在旗叶圆片试验中，感病品种处理后１２ｈ，ＰＡＬ比活力明显低于对照，２４ｈ后回升，接近对照的水平；抗病品种ＰＡＬ活性除个别场合外变化不大。在酶波中直接加毒素波几乎可以完全抑制感病品种ＰＡＬ活性，对抗病品种ＰＡＬ活性也有强烈抑制作用。认为在浸根试验中未直接接触毒素的叶组织中ＰＡＬ活性变化的差异能反映品种的抗感差异，ＰＡＬ活性的相对稳定性与小麦抗根腐病有关。     

不同方法提取鳗鲡病原茵DNA模板的差异分析

利用吸光值、琼脂糖电泳及PCR差异扩增等指标，对酚一氯仿法、水煮法、碱裂解法3种方法制备的鳗鲡病原菌DNA模板进行了差异分析．结果表明：1）不同方法提取的病原菌模板DNA的浓度、纯度及分子质量大小存在差异，但以这3种方法提取的病原菌DNA为模板，均能成功扩增到细菌16SrD．NA和外膜蛋白的保守序列．其中，酚一氯仿提取的模板DNA浓度较低、纯度较高；水煮法和碱裂解法提取的模板DNA浓度较高、纯度较低．2）电泳显示，3种方法获得的模板DNA扩增出的16SrDNA保守序列条带均一，没有显著差异，但在外膜蛋白保守序列的扩增中却因菌种和模板提取方法的不同而存在差异．     

蒎烷空气氧化反应的研究

研究了蒎烷的氧化反应，实现了用空气代替纯氧气进行氧化以制备蒎烷氢过氧化物（PHP）的设想。蒎烷的空气氧化反应需要用催化剂；反应时间约在25h左右，比用纯氧氧化延长了3-5h；反应温度一般控制在110℃以内，与纯氧氧化温度类似。在空气氧化蒎烷得到的产物中，PHP含量一般可达45%-50%，与用纯氧氧化的结果相当。氧化产物进行常压催化加氢所得还原产物粗蒎醇用GC分析的结果表明，其蒎醇含量（超过38%）及主要产物组成与用纯氧氧化所得产物基本一致。在回收蒎烷中含较多反式蒎烷。实验结果表明，空气氧化工艺有可能代替纯氧氧化工艺应用于合成PHP的工业生产。     

九孔鲍褐藻酸酶降解褐藻胶的反应条件与酶解产物的分析

以九孔鲍(Haliotis diversicoloeaquatilis)为原料制备一定纯度的褐藻酸酶,并对影响其降解褐藻胶的条件和产物的性质进行分析。该酶的最适温度和pH分别为45℃和7.0;与磷酸盐缓冲液相比,其在Tris-HCl缓冲液中与底物的亲和力相对较高。动力学曲线表明酶解反应主要发生在1 h之内,在2 h之内达到平衡,酶的剂量和底物的浓度对该平衡起到一定的影响作用。反应2 h之后,添加同数量的酶或者底物发现,产物的反馈抑制只是反应达到平衡的一个影响因素,而酶的变性失活也起到了重要的作用。金属离子对酶的活性具有明显的影响,Co2 、Mg2 对酶的活性具有较强的促进作用,而Cu2 、Ag 和Zn2 则具有较强的抑制作用。产物特性黏度的变化主要是在1 h内,2 h后基本不再改变。1HNMR图谱分析发现随着聚甘露糖片段,特别是M-M二聚体的降解,MG二聚体、GGM和MGM三聚体的量相应增多,表明该酶属于甘露糖裂解酶(EC 4.2.2.3)。     

黄瓜不同抗病品种感染镰刀菌枯萎病菌后几种酶活性的变化

 镰刀菌枯萎病的侵染使寄主黄瓜根部的苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶活性增加,抗病、感病各品种的3种酶活性曲线均呈现两个峰值,抗病各品种酶活性峰Ⅰ值比感病品种高。其中,感病品种的3种酶活性峰Ⅱ值大于峰Ⅰ值,而抗病品种酶活性峰Ⅰ值、Ⅱ值则在不同酶里有不同变化。在未感染情况下,抗病品种根部的苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶活性比感病品种高。     

Effects of food colorants on photooxidation of lipids added to Alaska pollack <Emphasis Type="Italic">Theragra chalcogramma</Emphasis> surimi

Food colorants including rose bengal, erythrosine B, phloxine B and cochineal extracts were added to Alaska pollack Theragra chalcogramma surimi which was previously added with methyl linoleate (MeLe) or ethyl eicosapentaenoate (EPA Et). All the surimi were exposed to a 100-watt light tungsten bulb (4500 lux) in a cold room at 5°C. Four isomeric hydroperoxides, including 13-cis, trans-, 13-trans, trans-, 9-trans,cis- and 9-trans,trans-MeLe hydroperoxides, were generated by oxidation of MeLe added to surimi with and without cochineal extracts after exposure to light for several hours. For the surimi containing rose bengal, erythrosine B and phloxine B, extracted lipids contained not only the above four hydroperoxide isomers but also 12-cis,trans-as well as 10-trans,cis- MeLe hydroperoxides. EPA Et added to surimi which contained rose bengal, erythrosine B and phloxine B generated characteristic isomeric hydroperoxides, including 6-cis,trans- and 17-cis,trans-EPA Et hydroperoxides due to ¹O₂ mediated oxidation. The pro-oxidative potentials of four food colorants in surimi against added EPA Et or MeLe increased as the order of cochineal extracts < phloxine B<erythrosine B<rose bengal. These results clearly demonstrated that the addition of certain food colorants to surimi accelerated ¹O₂ mediated lipid oxidation to result in the generation of characteristic hydroperoxide isomers.     

枣疯病过氧化物酶及苯丙氨酸解氨酶的研究

采用分光光度计和不连续聚丙烯酰胺凝胶电泳,比较分析了不同枣树品种及株系接入病原植原体后,其过氧化物酶及同工酶和苯丙氨酸解氨酶的变化。结果表明:同一枣树品种,不同发病程度的株系间过氧化物酶活性和同工酶有一定差异,抗病品种苯丙氨酸解氨酶活性高于感病品种。这两项可作为研究枣树抗枣疯病机理的重要生理指标。     

植物激活蛋白对黄瓜的促生诱抗相关酶的影响

为探讨植物激活蛋白对植物的促生、诱抗作用，以30mg．L^-1的激活蛋白处理黄瓜种子、幼根和叶片，测定了激活蛋白对黄瓜幼根生长以及脱氢酶、过氧化物酶、苯丙氨酸解氨酶和脯氨酸含量的影响。结果表明，激活蛋白诱导处理黄瓜种子24、36h后，幼根长度均明显高于对照，二者差异显著；处理幼根12h后脱氢酶活性比对照提高29．62％；处理子叶1．5h后脯氨酸含量较对照提高44．61％，4d后苯丙氨酸解氨酶较对照提高15．36％；处理叶片1～6h过氧化物酶活性均高于对照，1．5h后比对照增加了248．73％。