Development and hormonal control of thioredoxin and the thioredoxin-reductase system in the rat liver during the perinatal period

Summary The development and hormonal regulation of thioredoxin and of the thioredoxin-reductase system were investigated during the perinatal period in rat liver. An immunological procedure was developed in order to quantify thioredoxin in fetal and neonatal hepatocytes. Both immunoreactive thioredoxin and thioredoxin-reductase activity appeared on day 16.5 of pregnancy. The level of immunoreactive thioredoxin increased during the late fetal period, and its level was the same 24 h after birth. Moreover, its development was not subjected to hormonal regulation by corticosteroids and glucagon. In contrast, thioredoxin-reductase activity increased 3 times during the late fetal period and presented a marked increase 24 h after birth. In the absence of glucocorticoids there was no increase in the level of thioredoxin reductase, while administration of hydrocortisone acetate and glucagon to fetuses prematurely evoked its activity. This study suggests that if thioredoxin acts physiologically, this activity is related to the state of reduction of the molecule rather than to the total concentration in the liver.     

Site-directed mutagenesis reveals new and essential elements for iron-coordination of the sulfur oxygenase reductase from the acidothermophilic Acidianus tengchongensis

Previous study on refolding of sulfur oxygenase reductase (SOR) inclusion bodies from recombinant Escherichia coli showed that iron was critical to the activity of the SOR from Acidianus ambivalens. In this study, enzymatic assays showed that 2,2′-Dipyridyl, Tiron and 8-hydroxyquinoline, which are specific for chelating ferrous or ferric ions, strongly inhibited the activity of SOR from A. tengchongensis, suggesting that iron atom is essential for SOR activity. Alignment of several functionally identified S...     

采用低浓度三氟乙酸去除溶解浆中木聚糖

采用低浓度三氟乙酸(TFA)与木浆反应,并通过响应优化器和正交试验分析得出最优工艺.结果表明:在TFA质量分数为0.15%,温度为116.8℃,反应时间为9 h条件下,可以去除所有残存木糖,而葡萄糖损失仅为3.2%.该工艺为除去溶解浆中少量的半纤维素提供了新方法.     

3-酮基嘌呤还原酶(TSC10)表达载体的构建及其在毕赤酵母中的表达

目的：Tsc10基因所编码的3-酮基嘌呤还原酶是酵母中神经酰胺合成的重要因子,本文研究从酵母中提取神经酰胺经济、便捷、高效的方法。方法：1.利用构建含有tsc10基因的毕赤酵母GS115表达载体pPIC3.5K-tsc10。2.电转化法将该表达质粒转化到GS115感受态细胞中。3.用G418筛选以及PCR鉴定。4.使用qRT-PCR和SDS-PAGE进行检测。结果：经过G418筛选以及PCR鉴定后确定获得了包含tsc10基因的毕赤酵母转化子,经过qRT-PCR和SDS-PAGE进行检测后发现tsc10基因在20个阳性菌株中均可以稳定、高效表达。结论：本方法成功构建了3-酮基嘌呤还原酶高表达的毕赤酵母菌株,并为后续获得高收率神经酰胺奠定了基础。     

指纹频域内木糖拉曼光谱的研究

为深入了解木糖分子在指纹区（1000～400 cm-1）的特征振动,利用拉曼光谱仪,检测木糖得到该频域内的拉曼光谱,并用密度泛函理论选用B3LYP/6-311＋G＊＊基组,计算了木糖分子在该频域内的拉曼光谱。结果表明：检测值和理论值有很好的一致性。并根据势能分布进行分析,对振动进行归属和指认,该频域内的振动为基本上可以归属于木糖分子的即弯曲振动、剪式振动、扭曲转动以及伸缩振动,其他振动类型所占比例较小。这些振动可以作为木糖指纹区的特征振动。     

从木糖生产酒精发酵工艺的研究

本研究应用酵母Saccharomyces Cereviciae AM12菌株,在木糖异构酶存在下,以木糖为基质发酵生产酒精。重点研究了基质浓度对酒精发酵速率的影响及菌体的接种量、增殖与乙醇生成的关系。实验结果表明,无论是氧限制或厌气发酵过程,在木糖异构酶的存在下,该菌株均可利用木糖发酵生成酒精。但是,在乙醇积累的同时,伴有副产物木糖醇的生成。     

管囊酵母D-21木糖乙醇发酵条件的优化

对一株能利用木糖产乙醇的管囊酵母D-21进行了发酵条件的优化,研究了接种量、氮源、碳源和维生素对乙醇产量和还原糖利用率的影响。结果表明:接种量过高会影响乙醇产量,以6%(体积分数)为宜;当木糖质量浓度为50 g/L,碳氮质量比为20∶1,使用酵母膏和尿素组成的复合氮源时(质量比为2∶1),发酵72 h后所得乙醇质量浓度为12.5 g/L,达到理论产量的55%;当木糖与葡萄糖的质量比为3∶1时,乙醇质量浓度为15.72 g/L,为理论产量的69%;在发酵培养基中添加质量分数为2×10-5维生素B6可使乙醇产量提高18.3%。     

吡喃糖氧化酶电极对葡萄糖和木糖的响应特征

采用戊二醛交联方法,将葡萄糖氧化酶(GOD)和吡喃糖氧化酶分别固定在醋酸纤维素载体膜上,制备电流型电化学酶电极。在SBA双电极分析仪器上安装葡萄糖氧化酶电极和吡喃氧化糖氧化酶电极,分别对不同浓度的葡萄糖、木糖及葡萄糖木糖混合样品进行测试,以葡萄糖氧化酶电极为对照,研究吡喃糖氧化酶电极对葡萄糖、木糖的响应特征。结果表明,吡喃糖酶电极对葡萄糖和木糖都具有良好的响应特征。葡萄糖测定的精密度（RSD）为1.10%（n=10）,在10~1 000 mg/L范围内有良好的线性相关性（R=0.999 0）;木糖测定的RSD为0.80%（n=10）,在10~1 600 mg/L范围内有较好的线性相关性（R=0.999 8）;在葡萄糖木糖混合液测定时,吡喃糖氧化酶电极呈现明显的双底物催化特征,在一定的浓度范围内,对葡萄糖和木糖的响应具有线性相关性。     

Structure and function of eukaryotic NAD(P)H:nitrate reductase

Pyridine nucleotide-dependent nitrate reductases (NRs; EC 1.6.6.1–3) are molybdenum-containing enzymes found in eukaryotic organisms which assimilate nitrate. NR is a homodimer with an ∼100 kDa polypeptide which folds into stable domains housing each of the enzyme's redox cofactors—FAD, heme-Fe molybdopterin (Mo-MPT) and the electron donor NAD(P)H—and there is also a domain for the dimer interface. NR has two active sites: the nitrate-reducing Mo-containing active site and the pyridine nucleotide active site formed between the FAD and NAD(P)H domains. The major barriers to defining the mechanism of catalysis for NR are obtaining the detailed three-dimensional structures for oxidized and reduced enzyme and more in-depth analysis of electron transfer rates in holo-NR. Recombinant expression of holo-NR and its fragments, including site-directed mutagenesis of key acative site and domain interface residues, are expected to make large contributions to this effort to understand the catalytic mechanism of NR.     

硫酸盐还原菌产亚硫酸盐还原酶的发酵工艺研究

对硫酸盐还原菌产亚硫酸盐还原酶的发酵工艺条件进行了研究,主要内容包括碳源、氮源、温度、起始pH、装液量及发酵周期等.通过单因素实验研究表明,以可溶性淀粉为碳源,以尿素作为氮源,选C∶N比为1∶3,在初始pH 7.0的条件下,接种量为5%,装液量为1∶3,35℃摇床培养42h,酶活力可达到16.2U/mL.