蛋清中核黄素结合蛋白的提纯和鉴定

应用层析方法从蛋清中提纯得到核黄素结合蛋白（RBP）。用紫外分光光度法、SDS—聚丙烯酰胺凝胶电泳法（SDS—PAGE）及荧光分光光度法测定其核黄素结合力为275μg／mg，分子量为3420～3450Da。可用于制备抗—RBP抗体。     

核黄素超声结晶工艺优化

核黄素作为药品、色素、营养及动物饲料添加剂被广泛应用,目前生产及销售的核黄素基本上都是针状晶习,其堆密度小、分散性和流动性差,影响压片等加工性能。本文对酸溶法、碱溶法等5种核黄素结晶纯化方法进行了筛选,发现酸溶法收率和纯度的综合效益最佳;在此基础上采用超声辅助结晶,对功率、时间及温度进行优化,通过透射电镜（TEM）及X射线衍射分析法（XRD）等表征方法对结果进行分析。最终确定超声结晶的最佳实验条件为：超声功率为400W,超声时间为40min,超声温度为50℃,核黄素的收率可达到95%,纯度超过96%。核黄素晶习和结晶度有所改善,便于加工应用。     

纳米银与核黄素相互作用的光谱分析

制备了纳米颗粒的银溶胶,研究了核黄素水溶液中加入纳米银后荧光光谱和吸收光谱的变化.纳米银可以使核黄素的荧光发生猝灭,它们之间的相互作用属于静态猝灭机制,求出了猝灭常数Ksv=1.04×104L/mol,Kq=1.5×1013L·mol-1·s-1.纳米银对核黄素荧光的这种静态猝灭引起吸收光谱发生变化,随着纳米银浓度的增...     

单壁碳纳米管修饰玻碳电极测定核黄素含量

应用循环伏安法研究了核黄素在单壁碳纳米管修饰玻碳电极上的电化学行为,实验发现核黄素在p H值为3.2的磷酸氢二钠—柠檬酸底液中产生良好的循环伏安曲线,氧化峰、还原峰峰电位分别为-0.106 3、-0.276 2V,核黄素在修饰电极上的峰电流明显高于玻碳裸电极,建立了测定核黄素的电化学新方法,方法应用与维生素片中核黄素测定,样品回收率在96%~104.8%,结果令人满意。     

氮掺杂碳纳米粒子荧光共振能量转移测定核黄素

以柠檬酸和乙二胺为前驱体,在油酸介质中通过微波辅助法一步合成了氮掺杂荧光碳纳米粒子,量子产率达41.5%。碳纳米粒子与核黄素可发生有效的荧光共振能量转移,基于此建立了一种定量测定核黄素含量的新方法。测定核黄素含量的线性范围为0~20μg/m L,检出限为68.5 ng/m L。该方法简单、快速、灵敏度高,可用于实际样品中核黄素含量的测定。     

p(N-MAM-co-DMAA)智能水凝胶的制备及其药物缓释作用

以N-羟甲基丙烯酰胺(N-MAM),N,N-二甲基丙烯酰胺(DMAA)为聚合单体,过硫酸钾为引发剂,N,N-亚甲基双丙烯酰胺(MBA)为交联剂,通过自由基水溶液聚合法制备了p(N-MAM-co-DMAA)智能水凝胶。研究了凝胶对温度、p H值、离子浓度敏感性能和对核黄素药物的缓释性能。实验表明,凝胶的溶胀度随温度升高和盐浓度增加而逐渐降低,随溶液p H值的增大先增大后逐渐减小,在中性、酸性和碱性环境中的核黄素累积释放量分别是68%,51%和42%。     

复合维生素B片剂中核黄素的直接测定

对核黄素的示波极谱特性进行了研究,发现在0.1mol/L NaOH 溶液中,核黄素在-0.62V(vs.SCE)处产生—灵敏的二阶导数阴极波,波高与5.0×10~(-9)～6000×10~(-9)范围内的核黄素浓度成线性关系。该法已成功地应用于复合维生素B 片剂中核黄素的直接测定。     

大肠杆菌中核黄素的生物合成途径改造及生产

利用PCR技术从枯草芽孢杆菌(Bacillus subtilis 168)中扩增出3.5 kb的核黄素操纵子,将其分别连接到不同拷贝数的表达载体pSC101、p15A、pBR322,得到重组载体pSC101-BSrib、p15A-BSrib和pBR322-BSrib,并分别转化到大肠杆菌(Escherichia coli K-12 MG1655)。对含有核黄素操纵子的重组大肠杆菌进行摇瓶发酵,结果表明其核黄素合成能力随着质粒拷贝数的增加而增强。随后对E. coli K-12 MG1655 ECX3菌株的诱导剂IPTG浓度和发酵温度进行了优化。结果显示,0.1 mmol·L^-1 IPTG和42 ℃为核黄素生产的最适宜条件。在此条件下,菌株ECX3在LB培养基中核黄素产量达到251.4 mg·L^-1。最后,通过无痕基因操作技术,减弱了工程菌株ECX3核黄素激酶/黄素腺嘌呤二核苷酸氨酰转移酶(ribF)的表达以减少核黄素转化为FMN和FAD,摇瓶中工程菌株ECX4的核黄素产量提高到292.3 mg·L^-1。     

核黄素合成途径关键酶：新型生防制剂筛选靶标

植物和微生物可以自身合成核黄素,动物必须完全从食物中摄取,因此核黄素合成途径中关键酶抑制剂可开发为杀菌剂或除草剂,对人畜安全、无副毒作用.论述了有关核黄素合成途径中抑制作用靶标位点、筛选模型和抑制剂种类的研究进展,探讨了今后研究思路.以催化核黄素合成途径最后3步反应的羟基磷酸丁酮合成酶DHBPS、二氧四氢喋啶合成酶LS和核黄素合成酶RS作为抑制作用靶标位点,离体和活体筛选抑制剂.新筛选靶标和模型的建立有助于开发新型生防制剂.     

医药兽药

广济药业第三代高产菌株制核黄素高技术形成2500t/a产能河南广济药业(孟州)有限公司采用目前国际最先进的"清汤发酵法技术"制核黄素的第三代菌种发酵生产工艺,形成食品医药级核黄素2500t/a生产能力,投资总额     

Improvement of the riboflavin production by engineering the precursor biosynthesis pathways in Escherichia coli

3,4-Dihydroxy-2-butanone 4-phosphate (DHBP) and GTP are the precursors for riboflavin biosynthesis. In this research, improving the precursor supply for riboflavin production was attempted by overexpressing ribB and engineering purine pathway in a riboflavin-producing Escherichia coli strain. Initially, ribB gene was overexpressed to increase the flux from ribulose 5-phosphate (Ru-5-P) to DHBP. Then ndk and gmk genes were overexpressed to enhance GTP supply. Subsequently, a R419L mutation was introduced into purA to reduce the flux from IMP to AMP. Finally, co-overexpression of mutant purF and prs genes further increased riboflavin production. The final strain RF18S produced 387.6 mg riboflavin · L?1 with a yield of 44.8 mg riboflavin per gram glucose in shake-flask fermentations. The final titer and yield were 72.2%and 55.6%higher than those of RF01S, respectively. It was concluded that simultaneously engineering the DHBP synthase and GTP biosynthetic pathway by rational metabolic engineering can efficiently boost riboflavin production in E. coli.     

Base pairing at the abasic site in DNA duplexes and its application in adenosine aptasensors

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K(d) =14.1 μM for adenosine and 41.8 μM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors-a limit of detection of 0.7 μM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.     

To prepare the collagen‐based artificial cornea with improved mechanical and biological property by ultraviolet‐A/riboflavin crosslinking

In the present study, we optimized the ultraviolet‐A (UVA)/riboflavin crosslinking for the collagen‐based artificial cornea for the first time to resolve the lack of donor cornea for corneal disease. We found that the crosslinking conditions, including irradiation time and irradiation intensity, played important roles on the property of the sample. At the optimal condition (with the irradiation time of 30 min and the irradiation intensity of 5 mW/cm²), the sample exhibited excellent crosslink degree and mechanical property while keeping good light transmittance. Meanwhile, the sample showed improved enzyme tolerance capacity and thermal stability. This developed artificial cornea also had good biocompatibility to human corneal epithelial cells in vitro . In vivo lamellar keratoplasty results showed that the developed sample could promote complete epithelialization in about 4 weeks, and the transparency is restored quickly in the first 6 weeks. Corneal rejection reaction and keratoconus are not observed. This optimized UVA/riboflavin crosslinking method would have great potential for collagen‐based artificial cornea in clinic. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134 , 45226.     

Biosynthesis of vitamin B2: a unique way to assemble a xylene ring

The biosynthesis of one riboflavin (vitamin B(2)) molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. In the final step, the tricyclic isoalloxazine chromophore, which is the hallmark of flavocoenzymes, arises from a highly unusual dismutation of bicyclic 6,7-dimethyl-8-ribityllumazine that is catalyzed by riboflavin synthase but can also proceed without catalysis. The reaction proceeds via a pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazine molecules, whose cleavage into riboflavin and a pyrimidine derivative (by a sequence of two elimination steps) is mechanistically straightforward. Recently, the formation of the pentacyclic adduct has been proposed to involve a hydride transfer step followed by a [4+2] cycloaddition. Surprisingly, two different classes of riboflavin synthases utilize different diastereomers of the pentacyclic adduct, but the newly generated chiral centers are lost upon the intermediates' subsequent fragmentation.     

Characterization of a Novel Mesophilic CTP-Dependent Riboflavin Kinase and Rational Engineering to Create Its Thermostable Homologues**

Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.     

Study on Riboflavin Recovery from Wastewater by a Batch Foam Separation Process

Abstract

A batch recovery of riboflavin via foam separation from industrial simulative wastewater was studied using a cationic surfactant, cetyltrimethyl ammonium bromide (CTAB). The experimental parameters examined were the surfactant concentration, air flow rate, pH, and foam height. Under optimal operating conditions obtained through an orthogonal experiment, the maximum enrichment ratio of 48.7 was achieved for riboflavin along with 99.3% removal efficiency. The optimal operating conditions had the concentration of CTAB at 0.3 g/L, air flow rate at 400 ml/min, foam height at 90 cm, and pH at 12. Therefore foam separation proved to be an effective method to recover the riboflavin in terms of the good enrichment and removal efficiency.     

熊果苷的合成方法及其检测研究进展

熊果苷由于对黑色素合成酶酪氨酸酶有很好抑制效果,对人体肌肤有良好的美白作用,因而在化妆品中得到广泛作用。由于天然提取法工艺复杂,生产成本高,不能大规模生产,人工合成法是熊果苷的主要制备方法。本文介绍了熊果苷的合成与检测方法的研究进展,通过有机合成法,酶合成法和生物转化法等3种合成熊果苷方法的比较,阐述有机合成法是制备熊果苷的主要方法,同时指出需要进一步研究合成熊果苷的检测分析技术,指导和优化熊果苷合成与纯化.此外合成并研究熊果苷的各种衍生物,寻求熊果苷更广阔的制备方法和应用前景也是以后研究的方向之一。