壳寡糖-水解麦醇溶肽共聚物的制备和表征

以初步纯化的微生物转谷氨酰胺酶(MTGase)催化合成壳寡糖-水解麦醇溶肽共聚物,研究最佳合成条件并对共聚物进行了表征。 结果表明,合成共聚物的最佳条件是:壳寡糖/水解麦醇溶肽质量比为1:40,在pH值6.00~6.50、50 ℃下搅拌反应50 min,共聚物的生成率达到60%~70%。 红外光谱分析显示,与壳寡糖相比,由于引入的吸电子基团产生诱导效应,共聚物酰胺-C=O基的伸展振动峰向高波数位移动且吸收强度加强。 由DTA分析可知,共聚物在60.91 ℃处失水,387.55 ℃处熔融,665.25 ℃处开始彻底分解,与壳寡糖和麦醇溶肽的差异明显。 XRD分析可知,共聚物的结晶度显著降低,晶胞数据不同于壳寡糖,表明其不易结晶。 HPLC分析表明,共聚物主要由两个组分构成,占共聚物总量的80.6%,其相对分子质量分别为66069和27285。 共聚物不溶于水及多种有机溶剂,微溶于1%NaOH,溶解度为0.184 mg/100 g。 熔程为162~163 ℃。     

小分子玉米肽Leu-Asp-Tyr-Glu保护线粒体抗氧化损伤的研究

线粒体是细胞内氧代谢的主要场所,其膜上富含不饱和脂肪酸,使得线粒体成为自由基进攻的主要靶部位^[1].自由基作用于线粒体膜发生脂质过氧化损伤反应,引起膜的结构与功能改变,使得膜上一系列重要的酶空间排列紊乱,活性降低,膜的通透性改变,诱发多种疾病^[2].目前,抗氧化肽类作为一种非酶类自由基清除剂的研究正在兴起^[3].来源于玉米醇溶蛋白酶降解物中的低分子量肽具有许多重要的生理功能,如促进乙醇代谢、抗疲劳作用以及降血脂等功效^[4].而这些功能的实现主要依赖于其抗氧化活性^[5].最近,我们从玉米蛋白酶解物中获得了一种新的小分子抗氧化肽Leu-Asp-Tyr-Glu(LD YE,LD YE经文献检索为一种新的寡肽)^[6].     

酶解鹿茸肽的制备、纯化及抗氧化活性

利用一种工业用碱性蛋白酶对新鲜梅花鹿鹿茸进行水解制备生物活性肽。首先,考察了酶用量、底物浓度和反应时间对水解反应的影响,确定了最佳水解条件为酶浓度1：150,底物浓度1：13,水解时间60 min。其次,利用硫酸胺分级沉淀法制备了鹿茸多肽粗提液,同时采用缓冲液保护其活性,再经Sephadex G-25凝胶层析柱进行分离纯化,得到了具有最强抗氧化活性的鹿茸肽组分（VAP-B）。最后,利用制备型高效液相色谱柱对VAP-B进一步纯化,得到了分子量在800以内的小肽活性组分（VAP-B1）,其蛋白含量为70.45%。在此基础上,对小肽活性组分VAP-B1进行了理化性质分析,检测其抗氧化活性,包括清除超氧阴离子,羟自由基以及抗脂质过氧化的能力和还原能力。实验结果表明,鹿茸肽VAP-B1浓度为20mg/ml时,对邻苯三酚自氧化的抑制率达到91.9%,有明显的清除超氧阴离子的作用;浓度为10mg/ml时,对羟基自由基的清除作用达到100%,且在一定的浓度范围内呈剂量依赖关系。此外,鹿茸肽VAP-B1具有一定的防止脂质过氧化和还原力作用。     

一种新的质子泵抑制剂苯并咪唑衍生物的合成与表征

以2-巯基苯并咪唑为原料,在碱性条件下与自制的盐酸2-氯甲基-4-(3-甲氧基丙氧基)-3-甲基吡啶缩合得2-{[4-(3-甲氧基丙氧基)-3-甲基吡啶-2-基]甲基硫}-1H-苯并咪唑,再经氧化、成盐反应生成产物2-{[4-(3-甲氧基丙氧基)-3-甲基吡啶-2-基]甲基亚硫酰基}-1H-苯并咪唑钠,总收率66%.此产物通过红外光谱、核磁共振、质谱表征加以确认.     

2709碱性蛋白酶水解BSA蛋白肽谱变化的研究

以2709碱性蛋白酶酶解牛血清白蛋白为研究对象,采用液相色谱-飞行时间质谱联用分析方法和位点非特异性酶切肽谱鉴定方法,分析水解过程中肽谱的动态变化。蛋白电泳分析结果显示,2709碱性蛋白酶在0.1%的添加比例条件下,0.5 h内可将全部蛋白降解,表明其具有极强的水解能力。肽谱分析结果显示,该酶酶解BSA蛋白过程中肽谱变化复杂多样,不同序列多肽具有不同的动态变化特征。酶切位点分析结果显示,2709碱性蛋白酶几乎能在所有种类的氨基酸位点发生酶切,但酶切的频率并不相同,这表明该酶在水解位点上具有宽泛的选择性和一定的倾向性,其中在肽键C端氨基酸种类的选择上具有明显的亮氨酸倾向性。该文可为研究其他工业蛋白酶水解肽谱的规律提供借鉴,并有助于提高我国工业蛋白酶制剂的应用水平和蛋白质加工水平。     

核桃蛋白酶解物的制备及抗氧化活性

采用碱性蛋白酶对核桃蛋白进行酶解, 检测了所得酶解物的抗氧化能力, 包括对1,1-二苯基-2-三硝基苯肼(DPPH)和羟基自由基(·OH)的清除能力; 利用Sephadex G-25 凝胶层析柱和反相柱对核桃蛋白酶解物进行分离纯化; 采用液相色谱-质谱(LC-ESI-Q-TOF)联用方法测得抗氧化能力最强的多肽的序列为Ala-Gly-Gly-Ala, 其还原力和还原型谷胱甘肽相当.     

一种基于肽质量指纹谱技术鉴定蓖麻毒素的新方法

应用基质辅助激光解吸电离飞行时间质谱(MALD I-TOF/MS)法实现了对蓖麻毒素(R ic in)的鉴定。测定蓖麻毒素的分子量为62925Da,实现了蓖麻粗毒的凝胶电泳分离,并通过胶内酶切获得了蓖麻毒素的肽质量指纹谱(PMF);经过数据库检索,在输入检索的22条肽段中有17条获得了匹配。检索结果显示,利用生物质谱技术是鉴定蓖麻毒素的最有效的新方法之一。     

一种鱼肉抗氧化活性肽及其制备方法

<正>申请公布号:CN104177477A申请公布日:2014.12.03申请人:滨州万嘉生物科技有限公司摘要:本发明公开了属于水产食品加工领域的一种鱼肉抗氧化活性肽,其氨基酸序列为Asn-Trp-Asp-Asp-Met。该肽可以人工合成,也可以鱼肉为原料制备。其制备包括将鱼肉制     

低抗凝肝素寡糖的制备、结构表征及免疫活性评价

以猪肠黏膜来源低抗凝肝素为原料,采用苄酯碱水解法制备了寡糖.通过聚丙烯酰胺凝胶电泳(PAGE)、高效凝胶渗透色谱(HPGPC)-多角激光光散射法(MALLs)联用、核磁共振波谱(NMR)和液相色谱-质谱(LC-MS)联用技术对所制备寡糖的分子量分布及化学结构进行了表征.以该寡糖对活化X因子(FXa)及人凝血酶(FⅡa)活性的抑制作用评价了其抗凝血活性,并通过测定其对RAW264.7巨噬细胞吞噬能力及NO释放量的影响评价其免疫活性.结果表明,所制备寡糖的聚合度分布于2~22之间,平均分子质量为5300,无明显的抗凝血活性,但具有显著的免疫增强活性.     

复合酶耦合酶解酸处理后玉米秸秆的研究

以玉米秸秆为研究对象,经过2%硫酸预处理后,利用果胶酶、β-葡萄糖苷酶、纤维素酶三种酶协同酶解,以提高玉米秸秆的酶解产糖量。结果表明:当酶解时间为48h,果胶酶、β-葡萄糖苷酶、纤维素酶分别为45U/mL、30U/mL、60U/mL时,葡萄糖、木糖和酶水解得率分别为67.83%、3.25%、73.65%,相比纤维素酶单一酶解的葡萄糖、木糖和酶水解得率分别提高了65.04%、20.82%、65.06%。分步糖化发酵5天后,相比单一酶解发酵乙醇含量提高了72.5%。说明利用三种酶复合处理,能明显提高酶解产糖量。研究结果为玉米秸秆转化为可发酵糖技术的研究提供重要参考。     

Preparation and Anti-oxidative Effects of Corn Peptides

IntroductionBioactive peptides include the natural peptidesfrom organism itself and the active peptide fromhydrolysate of protein. In the pastfew years,someresearchers have found that small peptides derivedfrom the hydrolysates of food proteins play animportant role in regulating autonomic nervoussystem,activating the cellular immunity function,ameliorating the cardiovascular function,antioxidizing and antiaging,etc.[1— 7] . Smallpeptides prepared from food proteins becomenitrogenous source f…     

MALDI-TOF-MS用于组合化学肽库的鉴定

组合化学是一种快速制备大量相关化合物,并可快速对其进行生物活性筛选的革新性的技术和方法。组合化学法自问世以来在生命科学和医药科学技术等领域获得了飞速发展。组合化学肽库已成为同时制备大量不同序列多肽的方法,并已广泛用于抗原决定簇、生物分子相互作用、亲和抑制剂、新药筛选等研究。在亲和筛选研究中,合成组合肽库的评价以及结构鉴定是研究成败的关键步骤之一。     

Protease-Catalyzed Peptide Synthesis: Basic Principles,New Synthesis Strategies and Medium Engineering

The application of enzymes to perform selective transformations is the central aim of industrial biocatalysis. Since a simple handled C-N ligase is not available, the only alternative of general practical interest for enzymatic peptide bond formation are proteases due to the principle of microscopic reversibility. The stereo- and regiospecifitity of proteases, which guarantee racemization-free segment condensation and require only minimal side-chain protection, are significant for preparative purposes. Unfortunately, proteases are not perfect acyltransferases. Both unwanted proteolytic side reactions and the hydrolysis of the acyl-enzyme in the kinetic approach are the major problems in enzymatic peptide synthesis. The object of this paper was to demonstrate several new synthesis strategies including medium engineering that enables one to bypass unfavorable reaction routes. Planning and optimization of enzymatic peptide synthesis require the S′ subsite mapping of proteases and the knowledge of additional basic parameters that determine the reaction course.     

Expression,Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.     

Identification of the Structural Determinants for Anticancer Activity of a Ruthenium Arene Peptide Conjugate

Organometallic Ru(arene)–peptide bioconjugates with potent in vitro anticancer activity are rare. We have prepared a conjugate of a Ru(arene) complex with the neuropeptide [Leu⁵]‐enkephalin. [Chlorido(η⁶‐p‐cymene)(5‐oxo‐κO‐2‐{(4‐[(N‐tyrosinyl‐glycinyl‐glycinyl‐phenylalanyl‐leucinyl‐NH₂)propanamido]‐1H‐1,2,3‐triazol‐1‐yl)methyl}‐4H‐pyronato‐κO)ruthenium(II)] ( 8 ) shows antiproliferative activity in human ovarian carcinoma cells with an IC₅₀ value as low as 13 μM , whereas the peptide or the Ru moiety alone are hardly cytotoxic. The conjugation strategy for linking the Ru(cym) (cym=η⁶‐p‐cymene) moiety to the peptide involved N‐terminal modification of an alkyne‐[Leu⁵]‐enkephalin with a 2‐(azidomethyl)‐5‐hydroxy‐4H‐pyran‐4‐one linker, using Cu^I‐catalyzed alkyne–azide cycloaddition (CuAAC), and subsequent metallation with the Ru(cym) moiety. The ruthenium‐bioconjugate was characterized by high resolution top‐down electrospray ionization mass spectrometry (ESI‐MS) with regard to peptide sequence, linker modification and metallation site. Notably, complete sequence coverage was obtained and the Ru(cym) moiety was confirmed to be coordinated to the pyronato linker. The ruthenium‐bioconjugate was analyzed with respect to cytotoxicity‐determining constituents, and through the bioconjugate models [{2‐(azidomethyl)‐5‐oxo‐κO‐4H‐pyronato‐κO}chloride (η⁶‐p‐cymene)ruthenium(II)] ( 5 ) and [chlorido(η⁶‐p‐cymene){5‐oxo‐κO‐2‐([(4‐(phenoxymethyl)‐1H‐1,2,3‐triazol‐1‐yl]methyl)‐4H‐pyronato‐κO}ruthenium(II)] ( 6 ) the Ru(cym) fragment with a triazole‐carrying pyronato ligand was identified as the minimal unit required to achieve in vitro anticancer activity.     

Combination of Chromatographic Separation Methods with Mass Spectrometry—a Modern Technique for Studying Metabolism

Powerful separation methods developed during the past few decades, in particular glass capillary gas chromatography (GC) and high performance liquid chromatography (HPLC), can be combined with sensitive methods of detection such as mass spectrometry (MS) to provide new insights into metabolic processes. Research in a field that was long thought to be exhausted has thus been revitalized. New perspective have been opened up for studying the metabolism of foods, spices and flavorings, drugs, and compounds produced in the body as well as for establishing the way in which the organism rids itself of harmful substances or how and when these compounds can interfere with the metabolic processes occurring in the body.