Sortase A介导的(S)-羰基还原酶Ⅱ寡聚体高效立体选择性转化(S)-苯基乙二醇

【目的】以金黄色葡萄球菌(Staphylococcus aureus)的sortase A为"分子订书机",用于(S)-羰基还原酶Ⅱ分子之间的连接,获得催化功能与稳定性增强的氧化还原酶寡聚体,高效催化2-羟基苯乙酮,合成(S)-苯基乙二醇。【方法】从S.aureus基因组中克隆sortase A基因,在大肠杆菌中表达,通过镍柱和凝胶层析纯化重组酶,获得纯酶sortase A。通过基因工程手段在(S)-羰基还原酶Ⅱ的C末端添加GGGGSLPETGG序列,蛋白纯化获得(S)-羰基还原酶Ⅱ-GGGGSLPETGG,摸索了sortase A催化(S)-羰基还原酶Ⅱ-GGGGSLPETGG的分子连接,形成(S)-羰基还原酶Ⅱ寡聚体的最佳条件,并研究了寡聚体酶学性质及生物转化(S)-苯基乙二醇的效率。【结果】(S)-羰基还原酶Ⅱ寡聚体比酶活力为38.5 U/mg,比原始型(S)-羰基还原酶Ⅱ提高了6倍,最适反应温度为50°C,最适pH为6.0,在50°C放置1 h后酶活仍旧保持90%以上;蛋白质变性实验结果显示,(S)-羰基还原酶Ⅱ寡聚体的变性温度为60.1°C,比原始酶提高了10°C;生物转化结果显示(S)-羰基还原酶Ⅱ寡聚体在3 h内完全转化5 g/L 2-羟基苯乙酮,产生光学纯度为100%的(S)-苯基乙二醇,相比于重组大肠杆菌(S)-羰基还原酶Ⅱ全细胞催化时间缩短了16倍。【结论】本研究首次将sortase A应用于氧化还原酶的分子连接,显著提高了酶的催化效率和热稳定性,表明sortase在手性催化中有很大的潜在应用价值。     

Conversion for Avicel and AFEX pretreated corn stover by Clostridium thermocellum and simultaneous saccharification and fermentation: insights into microbial conversion of pretreated cellulosic biomass

In this study, efforts were taken to compare solubilization of Avicel and AFEX pretreated corn stover (AFEX CS) by SSF and Clostridium thermocellum fermentation, with an aim to gain insights into microbial conversion of pretreated cellulosic biomass. Solubilization rates for AFEX CS are comparable for the two systems while solubilization of Avicel is much faster by C. thermocellum. Initial catalyst loading impacts final cellulose conversion for SSF but not for C. thermocellum. Hydrolysis of the two substrates using cell-free C. thermocellum fermentation broth revealed much smaller difference in cellulose conversion than the difference observed for growing cultures. Tests on hemicellulose removal and particle size reduction for AFEX CS indicated that substrate accessibility is very important for enhanced solubilization by C. thermocellum.     

Gefitinib Synergizes with Irinotecan to Suppress Hepatocellular Carcinoma via Antagonizing Rad51-Mediated DNA-Repair

Chemotherapy is the only choice for most of the advanced hepatocellular carcinoma (HCC) patients, while few agents were available, making it an urgent need to develop new chemotherapy strategies. A phase II clinical trial suggested that the efficacy of irinotecan in HCC was limited due to dose-dependent toxicities. Here, we found that gefitinib exhibited synergistic activity in combination with SN-38, an active metabolite of irinotecan, in HCC cell lines. And the enhanced apoptosis induced by gefitinib plus SN-38 was a result from caspase pathway activation. Mechanistically, gefitinib dramatically promoted the ubiquitin–proteasome-dependent degradation of Rad51 protein, suppressed the DNA repair, gave rise to more DNA damages, and ultimately resulted in the synergism of these two agents. In addition, the increased antitumor efficacy of gefitinib combined with irinotecan was further validated in a HepG2 xenograft mice model. Taken together, our data demonstrated for the first time that the combination of irinotecan and gefitinib showed potential benefit in HCC, which suggests that Rad51 is a promising target and provides a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and gefitinib in HCC.     

霍乱弧菌脂多糖基因在大肠杆菌中的克隆

利用柯斯质粒pHC 79为载体,构建了霍乱弧菌178(埃尔托生物型,小川血清型)染色体基因文库。经血清凝集试验及菌落固相ELISA检测,从基因文库中筛选到13株能够表达霍乱弧菌脂多糖O抗原的阳性克隆。经热酚水法从转化于中提取并纯化的脂多糖能与霍乱弧菌抗血清发生特异性结合。针对重组柯斯质粒PMM—VO 38进行了多种酶切分析,测定其分子量为46kb。     

嗜热脂肪芽孢杆菌β—半乳糖苷酶的性质

利用硫酸铵分级沉淀、离子交换层析(DEAE-22)、SephadexG-75凝胶过滤从嗜热脂肪芽孢杆菌胞内提纯得B半乳糖甘酶。研究表明,该酶最适表观反应曙度和最适PH分别为60OCT6.4D50OC该酶具有良好的热稳定性.碱金属和碱土金属盐对酶有激活作用,重金属Zn     

HL-60细胞诱导分化期间细胞表面ConA受体结合量变化的研究

 <正> 在用二甲亚砜(DMSO)诱导人急性早幼粒细胞性白血病细胞系(HL-60 Cell)沿粒系统分化成熟的实验基础上,我们用受体荧光标记技术和荧光分光光度法,进一步观察了HL-60细胞诱导分化期间,细胞膜流动性动态降低对膜上伴刀豆球蛋白(ConA)受体结合量的影响。     

黄雀发声核团与部分听觉中枢内P物质的分布和性双态比较

用免疫组织化学方法研究P物质在雌雄黄雀发声控制核团和听觉中枢内的分布,结合计算机图像分析仪检测SP免疫阳性细胞和末梢的灰度值,并作雌雄比较。结果如下：1．在发声学习中枢嗅叶X区有大量的SP阳性神经末梢和一些神经细胞。2．在发声控制核团前脑高级发声中枢(HVc)、古纹状体栎核、发声学习中枢新纹状体巨细胞核和丘脑背内侧核外侧部内有许多的SP免疫阳性细胞。3．在发声控制中枢中脑背内侧核和延髓舌下神经核气管鸣管部、听觉中枢丘脑卵圆核的壳区、中脑背外侧核壳区及中脑丘间核等有密集的SP免疫阳性神经末梢和纤维分布;雄性发声中枢内SP的分布比雌性丰富,两者有显著的差异。结果表明：SP的分布在雌雄发声中枢之间存在显著的性双态;SP广泛分布于黄雀发声控制核团和部分听觉中枢内,提示SP可能在发声控制及听觉中枢内具有重要的生理功能。     

高压力制备柯萨奇病毒疫苗

以HeLa细胞和BALB／c小鼠为模型,研究了高压力对柯萨B组病毒（CVB）感染活性和免疫原性的影响,发现在230MPa压力下,结合其他相应的物理条件,CVB的感染活性可完全消失。该CVB仍具有抗原性,可诱导小鼠产生CVB特异性抗性,效价可达1：1500。用高压力处理的CVB免疫小鼠,再用正常CVB攻击,其生存率为67％,具有疫苗的特性。这些结果表明,高压力处理的CVB具有免疫保护作用,可作为一种具有潜在应用前景的病毒疫苗。     

虎耳草石油醚提取物的化学成分分析

目的:对虎耳草微波辅助-石油醚提取物的化学成分进行研究。方法:采用气相色谱-质谱-数据系统联用技术对虎耳草微波辅助-石油醚提取物的化学成分进行分析鉴定。结果:石油醚提取物共鉴定出102种成分。结论:采用气相色谱-质谱-数据系统联用技术能够对低极性部位的化学成分快捷、简便、准确分析。