碳和氮代谢被抑制诱导雨生红球藻细胞内虾青素的合成

为研究雨生红球藻(Haematococcus pluvialis)合成虾青素的机理,文中分析了不同诱导条件下藻细胞内氮和碳代谢的变化。结果表明:强光照(HL)、添加乙酸钠(AA)、缺氮(NF)和缺磷(PF)都直接或间接地影响了雨生红球藻细胞内1,5-二磷酸核酮糖羧化酶(Rub isco)和硝酸还原酶(NR)的活性,导致2种酶的活性大幅度下降。只有当Rub isco和NR的活性降到非常低的水平时,藻细胞才开始合成虾青素。与此相反,对照(CK)中这2种酶的活性一直较高,但细胞内没有虾青素积累。由于Rub isco和NR是雨生红球藻碳代谢和氮代谢的关键酶,因此碳和氮代谢被抑制是诱导雨生红球藻合成虾青素的原因。     

强光照对雨生红球藻合成虾青素的影响

在不同的光照强度下研究了雨生红球藻细胞内虾青素的合成与初级代谢的关系.在强光(HL)和中等强度(ML)的光照条件下,雨生红球藻细胞内1,5-二磷酸核酮糖羧化酶 (Rubisco)和硝酸还原酶(NR)的活性第1天大幅度提高,2天后又迅速下降.与此同时,硝酸盐浓度也快速降低.当虾青素在第4天(HL)和第6天(ML)开始合成时, HL中Rubisco和NR活性以及NO3-浓度分别下降了75.5%,71.5% 和96.2%,而ML中则下降了76.5%,74.7% 和94.3%.相比之下,在低光照(LL)条件下,实验结束时三个指标仅下降了25.9%,29.8% 和56.8%,细胞中没有虾青素积累.结果表明强光提高了Rubisco 和 NR活性,导致硝酸盐浓度迅速降低而最终又抑制了这两种酶的活性,造成雨生红球藻光合作用效率下降即"碳饥饿".在此状态下,为了生存,细胞内合成虾青素的相关基因被激活,藻细胞开始合成并积累虾青素.     

从雨生红球藻藻泥中选择性提取虾青素

以雨生红球藻湿藻泥为原料,研究了不同有机溶剂对胞内油脂和虾青素选择性提取分离的影响,通过酸解破壁提高虾青素和油脂的提取效率。结果表明,连续乙醇提取可对胞内色素和油脂有效分级提取,先提取出极性组分(叶绿素和极性脂),再提取中性组分(类胡萝卜素和中性脂)。中等极性溶剂或溶剂体系对类胡萝卜素的选择性和提取率较好;乙醇/乙酸乙酯混合溶剂提取类胡萝卜素的总得率(干重)达25.31 mg/g,提取率为69.35%。对雨生红球藻湿细胞进行酸解破壁处理有助于提高虾青素和油脂的提取率。在最优酸解破壁条件(盐酸浓度1 mol/L,温度60℃,时间60 min)下,含水80%的雨生红球藻藻泥的油脂总得率(干重)达418 mg/g,总脂提取率达97%。     

雨生红球藻合成虾青素过程中分泌铵离子的研究

雨生红球藻在在缺氮(NF)的条件下,随着虾青素的合成,肽链内切酶(EP)的活性上升而蛋白质含量下降,天冬酰氨酸含量在前4天增加然后又减少,铵离子浓度则持续上升。相比之下,在氮充足(NR)的培养液中,藻细胞不合成虾青素,蛋白质和天冬酰氨酸的含量以及EP的活性基本稳定,培养液中也没有检测到铵离子。上述结果表明:①降解的蛋白质为虾青素的合成提供了碳源,②EP参与了蛋白质的降解反应,③为避免铵离子的毒害作用,蛋白质降解所产生的部分氨临时贮存在天冬酰氨酸中,而其余的则分泌到细胞外。     

雨生红球藻的新繁殖方式及其藻蓝蛋白的合成

雨生红球藻(Haematococcus pluvialis)是一种能够合成强抗氧化剂-虾青素的单细胞绿藻,然而由于其生长速率缓慢,制约了虾青素产量的提高。在对雨生红球藻细胞周期的研究中,首次发现了雨生红球藻在高温和低光照条件下的一种特殊繁殖方式,该繁殖方式比已知的营养繁殖和无性繁殖速度快。繁殖过程中厚壁孢子萌发产生大量的小细胞,并且这些小细胞中含有以前在绿藻中从未发现的捕光色素蛋白—藻蓝蛋白(最大吸收波长λmax=621 nm,最大荧光发射波长λmFax=643 nm)。这一发现对于提高天然虾青素的产量和藻蓝蛋白的生产以及藻类的进化有着重要的意义。     

转光膜在雨生红球藻养殖上的应用

通过对雨生红球藻在不同光质条件下生长的比较,确定了红色光有利于藻生长,进而用2.5 L气升式光照反应器在转光膜及普通PE膜下培养藻进行对比,结果显示雨生红球藻生物量、色素、光合活性等几项生物指标在转光膜条件下明显高于普通PE膜. 在气升式反应器内培养的藻细胞,接种9 d,虾青素含量可达3.57 mg/L,叶绿素浓度达到12.42 mg/L,干重提高8.8%以上.     

雨生红球藻合成虾青素过程中分泌铵离子

The present study is focused on protein degradation during astaxanthin synthesis in Haematococcus plu- vialis under high irradiance and nitrogen deficient conditions. It was found that with the onset of astaxanthin synthesis in the cultures of high light and nitrogen-free （HF）, high light and nitrogen-repletion （HR）, and low light and nitrogen-free （LF）, （1） endopeptidase （EP） activities increased along with decrease in protein content, （2） asparagine in HF and HR rose significantly before the first 4 and 5 day, but fell after that time. While, it increased slowly and continuously in LF, （3） ammonium increased continuously in HF and HR, whereas in LF, it was detected on the sixth day, and increased slowly on the following days. By contrast, in low light and nitrogen-repletion culture, （LR）, the contents of protein and asparagine as well as EP activity were maintained relatively constant, no astaxanthin and ammonium were detected. Furthermore, when HF was sealed and bubbled with CO2-free gas （02 and N2）, astaxan- thin content increased as the protein level decreased. These results strongly suggest that （1） the degraded protein served as a substitutive carbon source, to some extent, for the biosynthesis of astaxanthin, （2） endopeptidase was involved in the degradative process, （3） for detoxification, part of the ammonium generated by protein degradation was transiently stored in asparagine, whereas the rest of it was expelled into the culture broth.     

一种快速测定虾青素含量的方法

建立一种用分光光度法快速测定雨生红球藻粉和含雨生红球藻粉的复配产品中虾青素含量的方法。利用单因素实验,对雨生红球藻粉中虾青素的提取试剂进行了选择,确定最佳提取试剂为二甲基亚砜。结果表明,吸光度值与虾青素质量浓度在0.2～0.8μg/mL范围内有良好线性关系,R²=0.9997。与GB/T 31520—2015比较,测定方法的误差为0.02%～0.09%,测定含雨生红球藻粉的复配产品中虾青素含量的误差为0.003%～0.011%;平均加标回收率分别为98.7%、98.3%。所建立的方法可以快速、准确测定雨生红球藻粉和含雨生红球藻粉的复配产品中虾青素的含量。     

超声辅助提取虾青素的工艺优化及动力学研究

以乙醇为溶剂,采用超声辅助方法从新鲜雨生红球藻中提取虾青素,优化工艺参数。首先采用单因素方法研究提取时间、超声功率、料液比对提取得率的影响,然后通过响应面方法优化工艺参数。结果表明提取时间28 min,超声功率87 W,料液比1∶25时,虾青素得率最高,达到(35.93±0.37)mg/g。该最佳条件下的虾青素提取符合二级动力学规律(R²=0.998 9),提取速率与浓度差的平方成正比。该方法制备的虾青素提取液对DPPH和OH的半抑制浓度分别为(16.14±1.58)μg/mL和(0.93±0.18)μg/mL。     

Concomitant NH_4~+ Secretion During Astaxanthin Synthesis in Haematococcus pluvialis Under High Irradiance and Nitrogen Defi-cient Conditions

The present study is focused on protein degradation during astaxanthin synthesis in Haematococcus plu- vialis under high irradiance and nitrogen deficient conditions. It was found that with the onset of astaxanthin syn-thesis in the cultures of high light and nitrogen-free (HF), high light and nitrogen-repletion (HR), and low light and nitrogen-free (LF), (1) endopeptidase (EP) activities increased along with decrease in protein content, (2) aspar-agine in HF and HR rose significantly before the first 4 and 5 day, but fell after that time. While, it increased slowly and continuously in LF, (3) ammonium increased continuously in HF and HR, whereas in LF, it was detected on the sixth day, and increased slowly on the following days. By contrast, in low light and nitrogen-repletion culture, (LR), the contents of protein and asparagine as well as EP activity were maintained relatively constant, no astaxanthin and ammonium were detected. Furthermore, when HF was sealed and bubbled with CO2-free gas (O2 and N2), astaxan-thin content increased as the protein level decreased. These results strongly suggest that (1) the degraded protein served as a substitutive carbon source, to some extent, for the biosynthesis of astaxanthin, (2) endopeptidase was involved in the degradative process, (3) for detoxification, part of the ammonium generated by protein degradation was transiently stored in asparagine, whereas the rest of it was expelled into the culture broth.     

3,5-双三氟甲基苯甲酸的合成研究

3,5-双三氟甲基苯甲酸[CAS:725-89-3]的合成是先通过1,3-二溴-5,5-二甲基乙内酰脲,与1,3-双三氟甲基苯生成3,5-双三氟甲基溴苯,收率98%,选择性99%.然后由3,5-双三氟甲基溴苯作原料来合成3,5-双三氟甲基苯甲酸,收率72%以上.本文讨论了不同反应摩尔比,反应温度等条件,并得到最优化条件.     

5-苄巯基-1H-四氮唑(BTT)的合成工艺研究

以水和乙醇为溶剂,在相转移催化剂十六烷基三甲基溴化铵的作用下合成了标题化合物。通过对影响收率的关键因素进行研究,找到了最佳反应条件:物料比为1∶1.2∶1.2,95℃下回流16 h,收率达到85.2%。合成工艺操作方便,收率高,污染小,适合于工业化生产,有广阔的市场应用前景。     

5-(4-氯丁基)-1-环己基-1H-四氮唑的合成研究

5-（4-氯丁基）-1-环己基-1H-四氮唑为合成抗血小板聚集药物西洛他唑的重要中间体。以δ-环戊内酯和环己胺为原料,生成N-环己基-5-羟基戊酰胺,之后与PCl₅反应,以吡啶为相转移催化剂,中间体不分离,直接与叠氮化钠发生反应,生成目标产物,总收率76.7%。对反应条件进行了探索和优化。     

帕罗西汀对慢性不可预见温和刺激的抑郁症大鼠抑郁行为的影响及其机制

目的探讨帕罗西汀对慢性不可预见温和刺激(Chronic unpredicted mild stress,CUMS)的抑郁症大鼠抑郁行为的影响及其机制。方法将SD大鼠随机分为正常对照组(NG)、模型组(MG)、帕罗西汀模型组(PMG)及帕罗西汀对照组(PNG),MG组和PMG组采用孤养结合CUMS方式复制大鼠抑郁模型,每天PMG组和PNG组灌胃盐酸帕罗西汀1.8 mg/kg,NG组和MG组灌胃等体积的5%CMC-Na溶液。通过高架十字迷宫试验和糖水偏好试验评价大鼠的抑郁行为;采用常规生物化学方法测定大鼠大脑皮层丙二醛(Malonaldehyde,MDA)含量及超氧化物歧化酶(Superoxide dismutase,SOD)和过氧化氢酶(Catalase,CAT)活性;RT-PCR检测海马5-羟色胺转运体(5-Hydroxy-tryptamine transporter,5-HTT)和去甲肾上腺素转运体(Noradrenaline transporter,NET)基因mRNA的转录水平。结果与NG组相比,MG组大鼠进入开放臂的次数和停留时间显著减少(P<0.01或P<0.001),而在封闭臂停留的时间明显延长(P<0.01);糖水偏好百分率显著下降(P<0.001);大脑皮层MDA含量显著升高(P<0.01),SOD和CAT活性显著降低(P<0.05);海马5-HTT和脑桥NET基因mRNA的转录水平均显著降低(P<0.001)。帕罗西汀能明显抑制CUMS诱导的上述改变(P<0.05),而对正常大鼠无显著影响(P<0.05)。结论帕罗西汀能明显改善CUMS所致的大鼠抑郁行为,其机制与增强机体抗氧化应激能力、上调海马5-HTT和脑桥NET基因mRNA的转录水平有关。     

橙皮苷对小鼠急性肝衰竭的保护作用及其机制

目的观察橙皮苷对脂多糖(Lipopolysaccharide,LPS)联合D-氨基半乳糖(D-Galactosamine,D-GalN)所致小鼠急性肝衰竭(Acute hepatic failure,AHF)的保护作用及其机制。方法小鼠腹腔注射LPS(50μg/kg)和D-GalN(800 mg/kg),复制急性肝衰竭模型,分别用橙皮苷(200 mg/kg)或橙皮苷(200 mg/kg)联合锌原卟啉IX(Zinc protoporphyrin IX,ZnPP)(45 mg/kg)干预。造模后6 h检测肝损伤程度和炎症反应强度,48 h统计小鼠死亡率。结果橙皮苷使AHF小鼠血清转氨酶(ALT和AST)水平下降,肝损伤减轻,生存率提高;血清白细胞介素-10(Interleukin-10,IL-10)水平和肝内血红素加氧酶-1(Heme oxygenase-1,HO-1)的表达较AHF小鼠明显增加,肝内HO-1酶活性明显增高;同时,使血清肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)水平、肝组织中Caspase-3蛋白酶和髓过氧物酶(Myeloperoxidase,MPO)活性较AHF小鼠明显降低。ZnPP不影响橙皮苷促进肝内HO-1酶表达,但通过抑制HO-1酶活性,使橙皮苷抗炎和肝损伤保护作用显著降低。结论橙皮苷主要通过诱导HO-1蛋白表达,使HO-1酶活性增强,从而使炎症反应和肝损伤减轻,对LPS联合D-GalN诱导的AHF产生保护作用。