不同供氧水平对L-精氨酸分批发酵过程的影响

以钝齿棒杆菌Corynebacterium crenatum SYPA5-5为研究菌株,通过不同摇瓶装液量试验,考察发酵过程中供氧对L-精氨酸合成的影响。作者分别采用发酵动力学模型和代谢流量分析对实验结果进行分析后发现：在发酵前期,高供氧使HMP途径更加活跃,生成大量NADPH促进菌体生长;高供氧控制同样利于L-精氨酸的合成,一方面是由于菌体的高葡萄糖摄入量,另一方面是由于有更多的流量从α-KG流向Arg的前体物质Glu。     

齿叶黄皮中香豆素类化学成分研究

综合运用硅胶柱层析、ODS柱层析、Sephadex LH-20凝胶柱层析以及制备型HPLC等现代色谱分离技术,对芸香科黄皮属植物齿叶黄皮(Clausena dunniana)枝叶的化学成分进行系统分离与纯化。结合理化性质和多种现代波谱鉴定技术,并通过与文献报道的数据对照,鉴定了从齿叶黄皮枝叶90%乙醇提取物的石油醚萃取部位中分离得到8个香豆素类化合物的化学结构,分别为6-羟基-8-甲氧基-香豆素(1)、秦皮素(2)、异秦皮素(3)、佛手柑内酯(4)、花椒毒素(5)、异欧前胡素(6)、九里香酮(7)和蛇床子素(8)。以上所有化合物均为首次从齿叶黄皮中分离得到。     

沙枣花挥发油的化学成分及其指纹图谱的研究

应用GC /MS -计算机联用技术对沙枣花挥发油的化学成分进行分离、鉴定。共分离出 4 3种化合物 ,鉴定出17种化合物 ,占挥发油总量的 89.3%。应用HPLC -DAD技术建立沙枣花挥发油指纹图谱 ,以相对保留时间和相对峰面积为指标 ,确定了 7个特征峰     

利用SAS软件优化L-精氨酸发酵培养基

利用SAS软件中二水平设计和响应面分析法较系统地研究了谷氨酸棒杆菌(Corynebacterium glutamicum)精氨酸发酵培养基,得到了精氨酸产量在一定条件下随硫酸铵、玉米浆、磷酸二氢钾的变化规律,并根据分析结果优化了发酵培养基,产量可提高近64%.     

石韦的化学成分研究

目的:研究石韦的化学成分。方法:应用硅胶柱色谱、凝胶柱色谱技术对石韦化学成分进行分离纯化,利用NMR、MS和IR等波谱分析方法进行结构鉴定。结果:从石韦石油醚萃取部位分离得到4个化合物,鉴定为:何帕-22(29)-烯(hop-22(29)-ene)(1),β-谷甾醇(β-sitosterol)(2),胡萝卜苷(daucosterol)(3),山奈酚(kaempferol)(4)。化合物1首次从该植物中分离得到。     

气相色谱-质谱法分析歧化松香中性部分的化学组成

用皂化法将歧化松香中的酸性和中性部分分离,并由GC/MS法对中性部分的化学组成进行了分析,为进一步拓宽松脂可再生资源的用途提供了科学依据。实验考察了中性部分化学组分分析的GC和MS条件,检索对照了NIST数据库的标准图谱,结果表明,歧化松香中性部分共分离出55个组分,其中高沸点中性部分共分离出32个组分,鉴定了其中的30个组分(占歧化松香中性物质质量的80.3%,下同),主要化学成分为:脱氢枞酸甲酯、7-二氢海松酸甲酯、8(14)-二氢异海松酸甲酯、8α-四氢异海松酸甲酯、7-二氢枞酸甲酯、8-二氢海松酸甲酯、8α-四氢海松酸甲酯以及4个高沸点化合物;低沸点中性部分分离出23个组分,鉴定了其中的22个组分(占歧化松香中性物质质量的19.7%),主要是倍半萜和少量单萜化合物,系残留的歧化松节油。     

基于质谱技术的大鼠硫化氢中毒血浆蛋白质组学研究

硫化氢中毒机理尚未在蛋白质层面阐明,寻找血液中的蛋白质标识物在临床及法医学上具有重要意义。近年发展起来的液相色谱-质谱技术的定量蛋白质组学(iTRAQ/TMT)技术为筛选生物标识物提供了新的有力工具。采用蛋白质组学方法研究了硫化氢中毒的大鼠模型的结果。大鼠暴露于150mg/m~3浓度受控的硫化氢环境中,时间分别为0、2和4h。收集大鼠血液后,分离血浆并用蛋白质浓缩试剂盒(ProteoMiner)去除高丰度蛋白质,并通过串联质谱标签标记技术和纳流液相色谱-串联质谱(Nano HPLC MS/MS)进行定量蛋白分析。实验采取两组平行重复以确认鉴定及定量结果。在2次重复实验中,共有520个蛋白被鉴定并进行后续生物信息学分析。结果表明:蛋白质组学技术是在蛋白质水平上研究并揭示硫化氢中毒的机理。     

多聚精氨酸蛋白转导域-凋亡素融合蛋白的原核表达及其生物活性

目的原核表达多聚精氨酸蛋白转导域-凋亡素融合蛋白,并检测其生物活性。方法应用PCR法扩增Arg9-VP3序列,与载体pET-43.1a连接后,转化E.coliBL21(DE3),IPTG诱导表达。表达产物经Ni2+-NTA纯化后,进行肠激酶裂解、超滤浓缩,并检测其生物活性。结果重组表达质粒pET-43.1a-Arg9-VP3经酶切鉴定和序列分析,证明构建正确。转化E.coliBL21(DE3)后,重组蛋白获得可溶性表达。纯化的融合蛋白纯度达90%以上,可抑制HeLa细胞增殖。结论已成功地在大肠杆菌中表达了多聚精氨酸蛋白转导域-凋亡素融合蛋白,纯化的融合蛋白具有诱导HeLa细胞凋亡的能力。     

生防芽孢杆菌的分离筛选与初步鉴定

从不同环境中采集7份土壤样品用于芽孢杆菌的分离纯化,通过多次平板划线分离纯化得到30个菌落形态不同的菌株。以黄瓜枯萎病菌为指示菌对得到的菌株进行抑菌活性筛选,其中的4个菌株具有明显的抑菌作用。采用对峙生长法,测定4株芽孢杆菌对其它植物病原真菌的活性,结果显示4个芽孢杆菌均具有广谱抑菌性。进一步试验表明,4株芽孢杆菌的发酵液也具有明显的抑菌作用,说明对病原真菌有抑制作用的物质水溶性较大,大部分释放到发酵液中,并且发酵液可使黄瓜枯萎等植物病原真菌菌丝膨大、褐变、末端钝圆,促进镰刀菌产生小孢子。通过菌落形态特征、系列生化测定对其进行初步鉴定,结果表明其中一株为枯草芽孢杆菌。     

百里香挥发油成分及其对运动损伤易感染细菌的抑制作用

采用气相色谱-质谱联用(GC–MS)对秦岭地区的百里香全草挥发油化学成分进行了分析鉴定。结果分离鉴定出53种化学成分,其主要成分为对伞花烃(13.689%)、香芹酚(13.201%)、松油烯-4-醇(11.225%)等;对运动损伤易感染细菌的抑制作用研究结果显示:对柠檬色葡萄球菌(S.citreus)、普通变形杆菌(P.vulgaris)、产气肠杆菌(E.aerogenes)、绿脓杆菌(P.aeruginosa)、大肠杆菌(E.coli.)有较好地抑制作用。     

Molar mass distributions of polymers from size exclusion chromatography and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: Methods for comparison

Equations are presented for calculating molar mass averages and molar mass distributions from matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) data and from size exclusion chromatography (SEC) data. The utility of polydispersity is examined as an indicator of the expectation of MALDI‐TOF MS mass discrimination effects. Cumulative distributions are found to be rich in information for comparing the two techniques and are easily obtained from both SEC and MALDI‐TOF MS data. Analyses of a series of narrow molar mass distribution poly(methyl methacrylate) (PMMA) standards and one polydisperse sample have been performed with both methods. MALDI‐TOF MS did not detect dimer and trimer in the PMMA samples, and it often indicated lower amounts of high‐molar‐mass polymers than did SEC. The results showed that the distribution breadth, as evidenced by the standard deviation of the distribution (calculated from the polydispersity and number‐average molar mass), correlated well with the molar mass range observed in the MALDI‐TOF MS spectra, whereas the polydispersity alone did not. Ratioing the extremes in the molar mass concentrations measured with the SEC differential refractometer, which were necessary to adequately define molar mass distributions, showed that detector dynamic range values as high as approximately 370,000 were required for the polydisperse samples. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 627–639, 2005     

D(-)-α-(4-乙基-2,3-双氧代哌嗪-1-甲酰胺基)对羟基苯乙酸的合成

用双 三氯甲基碳酸酯(三光气)代替光气和4 乙基 2,3 双氧代哌嗪发生酰氯化反应,制备氧哌嗪酰氯(EOCP),进而和D(-) α 对羟基苯甘氨酸(p HPG)缩合,水解合成了D(-) α (4 乙基 2,3 双氧代哌嗪 1 甲酰胺基)对羟基苯乙酸(OH—EPCP)。酰氯化反应条件为:-15℃,n(Nu)∶n(三光气)∶n(氧哌嗪)=0 08∶0 4∶1 0,反应4h。EOCP收率大于90%。缩合反应温度15～20℃,n(EOCP)∶n(p HPG)=1 1∶1 0,反应3h。然后在室温下水解,得到白色粉末状产品。经精制后,w(OH—EPCP)>98 0%,收率(相对于p HPG用量)达88 0%。用元素分析、IR、1HNMR和MS对产品进行表征,证明了合成方法的可靠性。     

Synthesis,structure, and characterization of glyoxal‐urea‐formaldehyde cocondensed resins

To decrease the formaldehyde emission of urea‐formaldehyde (UF) bonded products at source, monomethylol urea (MMU) was chosen to react with glyoxal (G), a nonvolatile and nontoxic aldehyde, to prepare a novel glyoxal‐urea‐formaldehyde (GUF) cocondensed resin. The GUF resins were synthesized with different MMU/G molar ratios, and the basic properties were tested. The GUF resins were characterized by ultraviolet‐visible spectroscopy, Fourier transform infrared spectroscopy, carbon‐13 nuclear magnetic resonance spectroscopy and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF‐MS). The results show that the synthesized GUF resins remain stable for at least 10 days at ambient temperature. Conjugated structures, and large amounts of ? OH, ? NH? , C? N, and C?O groups with different levels of substitution exist in the GUF resin. There are two repeating motives in the MALDI‐TOF‐MS spectrum of the GUF resin, one of 175 ±1 Da and a second one of 161 ± 1 Da. Moreover, the peaks due to the dehydration condensation reaction of MMU also appear in the spectra. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41009.     

Preparation and characterization of mono‐ and di‐d‐α‐tocopheryl polyethylene glycol 1000 succinate

Mono‐d‐α‐tocopheryl polyethylene glycol 1000 (TPGS 1000) and di‐TPGS 1000 were prepared from the synthesized TPGS 1000 mixture. The key separation step was performed by a Simulating Moving Bed chromatographic process. The chemical structures and molecular weight distrubution were characterized by ¹H‐NMR and MALDI‐TOF mass spectroscopy. NMR and MALDI‐TOF MS data confirmed the occurrence of di‐TPGS. Both NMR and MALDI‐TOF MS results showed the degree of polymerization of the two esters and the molecular mass. The melting temperatures of the two polymers were investigated by DSC and the thermal decomposition temperatures have been determined by TGA. The melting temperatures of the two esters were 33 and 15°C, separately. And the two separated TPGS esters exhibited different thermal decomposition courses. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Physiological comparison of Pseudomonas putida between two growth phases during cometabolism of 4‐chlorophenol in presence of phenol and glutamate: a proteomics approach

BACKGROUND: In cometabolic transformation of 4‐chlorophenol (4‐cp) in the presence of phenol and sodium glutamate (SG), a new biphasic growth pattern has been reported. This study investigates the physiological changes of Pseudomonas putida P8 during the biphasic growth by means of 2‐dimensional gel electrophoresis (2‐DE) and MALDI‐TOF analyses. RESULTS: A total of 49 protein spots were selected and identified in the 2‐DE gels from P. putida P8 grown on a mixed substrate containing 200 mg L^?1 of phenol, 200 mg L^?1 of 4‐cp and 1000 mg L^?1 SG. Among them, 16 protein spots were found differentially expressed in the two exponential growth phases during the biphasic growth, including six catabolic enzymes (DmpC, DmpD, DmpE, DmpF, DmpG and AspA) for substrate utilization. The expression of other proteins involved in detoxification and stress responses, carbohydrate and energy metabolism, and environmental information processing as well as a multifunctional xenobiotic reductase (XenA) was quantitatively analyzed and discussed. CONCLUSION: The expression levels of the identified catabolic enzymes during growth in the two growth phases correlated well with the substrate utilization patterns observed in previous kinetics studies. Furthermore, the results show that cells growing on a mixture of aromatic substrates undergo significant physiological changes. Copyright © 2009 Society of Chemical Industry     

温敏性萃取水凝胶对生物大分子的分离

合成了均聚的聚N 异丙基丙烯酰胺(PNIPAM)水凝胶以及N 异丙基丙烯酰胺与丙烯酰胺共聚的〔P(NIPAM-AM)〕水凝胶,并研究了它们的溶胀性能及其对生物大分子的萃取分离性能。结果表明,两种温敏凝胶具有很好的溶胀性能,其低临界共溶温度(LCST)分别为30 4和31 0℃,它们对蛋白质和酶的分离效率在LCST附近发生突跃,如PNIPAM水凝胶对白蛋白的分离效率在LCST前后从96 2%降至59 8%。当交联剂N,N 次甲基双丙烯酰胺(Bis)的质量分数w(Bis)>4%时,分离效率大于90%(LCST以下)。     

不同碳源及通气条件下lpdA基因敲除对大肠杆菌代谢的影响

lpdA基因编码的lipoamide dehydrogenase是丙酮酸脱氢酶复合体、α-酮戊二酸脱氢酶复合体和甘氨酸裂解多酶体系的组成亚基之一.比较了不同碳源及通气条件下,lpdA基因敲除突变E. coli与野生E. coli的发酵特性,并通过对一些主要的酶活和胞内代谢产物浓度的测量,考察了lpdA基因敲除对E. coli代谢的影响.结果表明：葡萄糖为碳源的有氧条件下,lpdA基因的敲除导致丙酮酸、D-乳酸、L-谷氨酸的积累,TCA循环的抑制,乙醛酸途径和磷酸戊糖途径中的磷酸葡萄糖脱氢酶的激活.乙酸或丙酮酸为碳源的有氧发酵及葡萄糖为碳源的微氧发酵实验补充了以上的结论.     

Characterization of the biocide polyhexamethylene biguanide by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry

The biocide polyhexamethylene biguanide (PHMB) has been characterized by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Previously, no method has been able to provide a detailed structural characterization of PHMB. MALDI‐TOF MS was able to detect PHMB oligomers with n ≤ 6. Six different PHMB product types were identified, which possess combinations of amine, cyanoamine, guanidine, or cyanoguanidine end‐groups. Postsource decay (PSD) fragmentation was used to confirm the correct assignment of PHMB structure for the dominant PHMB molecular ion. MALDI‐TOF MS analysis of a ¹⁵N‐labeled PHMB confirmed the correct assignment of PHMB molecular ions, and also indicated the existence of a polymerization–depolymerization equilibrium during melt polymerization of the polymer. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 4928–4936, 2006     

二维电泳-质谱分析麦拓莱霉素发酵过程中差异蛋白

引言 本课题组从土壤中筛选到一株藤黄灰链霉菌菌株,该菌株能产生一种新型的抗生素--麦拓莱霉素.麦拓莱霉素具有抗HIV蛋白酶活性和抗Coxsackie-virus B6 病毒 [1]活性,有很好的工业化前景.但目前产量还比较低,其生产过程的优化和产量的提高是亟待解决的问题.