方格星虫多肽的酶解法制备工艺优化与抗氧化作用研究

分别采用胰蛋白酶、木瓜蛋白酶、胃蛋白酶等8种蛋白酶酶解方格星虫制备方格星虫多肽,通过测定酶的活力和羟自由基清除率,确定以木瓜蛋白酶作为方格星虫多肽的酶解最适酶。通过单因素实验研究了加酶量、酶解温度、酶解时间、酶解pH和料水比等因素对羟自由基清除率的影响,并通过正交试验确定了木瓜蛋白酶对方格星虫优化的酶解工艺为:加酶量300U/g,酶解温度50℃,酶解时间60min,酶解液pH7.0,由此条件酶解,获得的羟自由基清除率为95.42%。结果表明,方格星虫多肽具有很强的清除自由基的能力。     

基于响应面法的螺旋藻多肽制备工艺的研究

应用响应面分析法优化木瓜蛋白酶酶解制备螺旋藻多肽的工艺条件。根据中心组合试验设计的原理,在单因素试验的基础上,以多肽得率为响应值,利用响应面法对影响螺旋藻蛋白酶解反应的各种影响因素如温度、pH、酶解时间和加酶量进行了系统研究,得到最佳工艺条件为:反应温度55℃、pH值7、酶底比1.6%、酶解2 h,制得多肽含量得率可达20.61%,与模型预测的肽含量21.25%较接近。     

响应面法优化小球藻抗氧化肽的制备工艺研究

以小球藻藻种为原料,通过实验室培育得到藻粉,再通过碱性蛋白酶制备小球藻抗氧化肽,以DPPH自由基清除率为指标,通过单因素和响应面设计探究加酶量、pH、酶解时间和反应温度对抗氧化肽的抗氧化性的影响,通过超滤离心对多肽进行分离,测定了各组分的DPPH自由基清除率,再通过氨基酸分析仪和凝胶渗透色谱仪测定了DPPH自由基清除率最好的多肽组分的氨基酸组成和分子量,又测定了它的热稳定性和贮藏稳定性。结果表明：小球藻蛋白制备抗氧化肽的最佳条件为：pH为7,反应温度为40℃,反应时间为30 min,加酶量为4 000 U·g^-1,此时DPPH自由基的清除率是58.03%。超滤分离得到五个组分,其中分子质量为5 KD-10 KD的DPPH自由基清除率最佳,多肽中的抗氧化氨基酸含量为49.9%,它的相对分子量为474,抗氧化肽有着良好的热稳定性以及贮藏稳定性。     

改性竹炭脱除贝类酶解液中的重金属镉

以蚶子贝肉酶解液为材料,利用聚天冬氨酸(PAsp)改性的竹炭吸附脱除酶解液中的重金属镉。考察了pH、脱除时间、竹炭投加量和温度对酶解液中镉脱除率的影响,并在单因素试验基础上通过响应面法确定了镉的最佳脱除条件。当pH为5.0、反应时间为25min、竹炭投加量(10m L酶解液)为0.26g,重金属镉的脱除率可达96.59%。研究表明,PAsp改性竹炭能显著提高吸附重金属的效率和能力,可用于脱除蚶子贝肉酶解液中的镉。     

气相色谱-质谱联用鉴定基于水酶法制备的虹鳟鱼骨油组分

采用水酶法制备虹鳟鱼骨油,单因素分析法优化虹鳟鱼骨酶解的工艺条件,考察了料液比、pH值、酶解时间、酶解温度、加酶量5个因素对鱼油提取率的影响,采用气相色谱-质谱联用 (GC-MS) 技术对鱼骨油的脂肪酸组成和含量进行了分析鉴定.结果表明,在55℃、pH 7.5、酶解时间为3 h、料液比为1∶1、加酶量为2000 U/g的条件下,利用碱性蛋白酶提取的虹鳟鱼骨油中的油脂含量最高.GC-MS分析结果表明,虹鳟鱼骨油中主要成分是不饱和脂肪酸,含量为脂肪酸总量的80.4% (w/w),其中单不饱和脂肪酸和多不饱和脂肪酸分别约占不饱和脂肪酸的76.9%和23.1% (w/w), DHA和EPA的总量为3.4% (w/w).本研究优化了虹鳟鱼油的提取技术,对虹鳟鱼油的主要挥发性物质进行了分析鉴定,初步确定了其中对鱼油风味起主要贡献的物质,对鱼油产品的分析与鉴别具有参考价值.     

改性稻壳脱除贝类酶解液中的重金属镉

用聚天冬氨酸(PASP)改性碳化稻壳作为吸附剂脱除贝类酶解液中的镉。通过单因素试验研究了pH、脱除时间、稻壳投加量和温度对镉脱除率的影响。使用响应面分析法获得了镉的最佳脱除条件:pH为4. 5,脱除时间为50 min,稻壳投加量为0. 30g。在此条件下镉的理论脱除率可达到94. 39%。实验测得贝类酶解液试样中镉的脱除率为92. 04%(94. 38%。研究表明,PASP改性碳化稻壳能显著提高吸附镉的效率和能力,可用于脱除贝类酶解液中的重金属镉。     

纤维素酶预处理法提取郁金中姜黄素的研究

提出了纤维素酶预处理法提取郁金中姜黄素的新工艺。探讨了酶的用量、酶解时间、酶解pH值、酶解温度、提取次数、提取时间等因素对姜黄素提取率的影响。筛选出了最佳的单因素工艺条件为:每10g郁金粉纤维素酶的用量为180U、酶解时间120min、pH值3.5、温度50℃、提取次数2次、提取时间90min。与传统提取方法相比,该方法及其新工艺能显著提高姜黄素的提取率。     

褪色光度法测定Fenton反应产生的羟自由基及其应用

建立检测Fenton反应产生羟自由基的新方法。Fenton反应产生的羟自由基与苋菜红反应，颜色发生变化，用分光光度计测定其△A510值的变化，可间接测定羟自由基的生成量。通过对测定条件的研究，确定了体系最佳实验条件。抗氧化药物甘露醇、硫脲与羟自由基清除率具有明显的量效关系。测定了阿魏酸、芦丁等几种中药活性成分清除羟自由基的功能，此法可用于羟自由基清除剂的筛选及抗羟自由基机理研究。     

铁皮石斛中石斛多糖与石斛碱的纤维素酶法提取研究

本文利用纤维素酶法联合提取铁皮石斛中的多糖与生物碱,通过单因素实验研究酶用量、酶解时间、酶解温度和酶解pH对石斛多糖与生物碱收率的影响.实验结果表明,在本实验范围内,最佳提取工艺为:纤维素酶用量为原料的0.6%(质量分数),酶解时间为1.5h,酶解温度为50℃,酶解pH值等于5.0.在此工艺下,石斛碱的收率达19%,石...     

多孔硅球固定化木瓜蛋白酶的制备和性质

用载体交联法制备了多孔硅球固定化木瓜蛋白酶。考察了固定化时间、温度、pH值、给酶量和成二醛浓度对固定化木瓜蛋白酶活力的影响。研究了固定化木瓜蛋白酶的性质，并同溶液酶进行了比较。着重考察了固定化木瓜蛋白酶的热稳定性。所制得的固定化木瓜蛋白酶最适温育温度达到80℃，对底物酪蛋白的水解活力随温度的升高而增加，在90℃达到最高值；在70℃温育12小时后酶活力仍能保持高水平。     

Features of Spontaneous Peroxide Fragmentation of Hemoglobin

The intensity of spontaneous peroxide fragmentation of the polypeptide chain of hemoglobin was studied in relation fo the protein concentration and pH. A chain reaction mechanism with cross chain termination was suggested. The main chain-leading radical in the process is hydroxy radical, which is responsible for peroxide damage of other biopolymers also.     

高效液相色谱–质谱法分析僵蚕蛋白酶解多肽

分析僵蚕蛋白酶解多肽类成分的相对分子质量和氨基酸组成。采用高效液相色谱–质谱联用正离子模式进行分析,以Hola C_(18)色谱柱(100 mm×2.1 mm,2.7μm)为分离色谱柱,以0.05%甲酸水溶液和0.05%甲酸–乙腈溶液为流动相,根据质谱一级、二级碎片离子信息,确定酶解多肽类相对分子质量信息和氨基酸组成。僵蚕样品经酶解后得到相对分子质量在500~1 000之间的多肽,经LC–MS分析,多肽由低于10个的氨基酸组成。高效液相色谱–质谱法分析平台可用于分析多肽化合物的相对分子质量和氨基酸组成,这有利于酶解多肽的生物活性分析。     

Multisensitive polymers based on 2‐vinylpyridine and N‐isopropylacrylamide *

Poly(2‐vinylpyridine) (P2VP) containing functionalized end groups was synthesized using nitroxyl‐mediated radical polymerization with a hydroxy‐functionalized stable free radical. It was shown that P2VP could be synthesized with variable molar masses and low polydispersities. The transformation of the hydroxy groups to an acrylic ester led to the formation of macromonomers. A free‐radical copolymerization of these macromonomers with N‐isopropylacrylamide gave a graft copolymer with a poly(N‐ispopropylacrylamide) backbone and P2VP side chains. Polymers containing different amounts of the monomers were synthesized. It was possible to vary both the amount of P2VP side chains at a constant chain length of the macromonomer and the chain length at a nearly constant chain number. The behavior of the multifunctional macromolecules at different temperatures and pH values was investigated using dynamic light scattering and DSC. The macromolecules were found to retain the specific properties of the homopolymers. The hydrodynamic radii of the synthesized graft copolymers were both dependent on the temperature and pH value. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 3797–3804, 2001     

酶法获得的多肽库用于新型手性固定相的制备

采用胰蛋白酶酶解牛血清白蛋白,将制备的酶解液超滤,得到相对分子质量小于6000的小分子多肽库。应用羰基咪唑法将该多肽库键合至多孔硅胶,得到一种新型手性固定相。应用该新型手性固定相成功地拆分了两种氨基酸对映体。     

Spectroscopic Studies of a Water-soluble Derivative of Hypocrellin A and Its One- and Two-Electron Reduction Products

In this study the spectroscopic characteristics of a water-soluble derivative of hypocrellin A (HA), 14-dehydroxy-15-deacetyl-hypocrellin A-13-sulfonate(13-SO3Na-DDHA),and its one- and two-electron reduction products have been investigated. From the changes in absorbance with pH it was observed that the two phenolic hydroxy groups at C-3 and C-10 positions of 13-SO3Na-DDHA or HA dissociated stepwise with increase of pH values. The pKa values for 13-SO3Na-DDHA and HA were determined using an effective method established in this study. Attempts were also made to use absorption and ESR spectroscopies to study the photoreduction of 13-SO3Na-DDHA. It was found that 13-SO3Na-DDHA was directly reduced to its two-electron reduction product in buffered aqueous solution (pH 7. 7). However, in DMF-buffer (1 :1/ v : v,pH 7. 7), it proceeded with one-electron reduction to generate its semiquinone radical anions. The semiquinone radical anions decayed according to second-order kinetics. indicating that the terminatio     

Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus

The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.     

芬顿试剂对田菁胶的氧化降解

考察了芬顿试剂对田菁胶的氧化降解行为. 系统研究了H_2O_2和Fe~(2+)用量、温度和降解时间对田菁胶粘度的影响. 结果表明,H_2O_2和Fe~(2+)合适的体积比为2:1. 在较低的温度(25 ℃)和较短的时间(20 min)内芬顿试剂就能使田菁胶粘度下降90%以上. 另外,pH值的变化对其降解性能影响不大,显示了较好的降解效果.     

基于特征肽的超高效液相色谱-串联质谱法检测矛头蝮蛇蛇毒种属来源及类凝血酶含量

蛇毒血凝酶类药物是以蝮蛇蛇毒为原料制备的止血药,主要活性成分为蛇毒类凝血酶(svTLEs)。不同蛇种来源的svTLEs结构不同,止血机制不同,药理作用也存在差异,因此准确鉴别蛇毒种属来源和svTLEs含量对于保障该类产品的质量至关重要。研究基于蛋白质组学技术,筛选出了具有种属特异性的矛头蝮蛇svTLE特征肽,并建立了基于特征肽的超高效液相色谱-串联质谱(UHPLC-MS/MS)检测矛头蝮蛇蛇毒种属来源及类凝血酶含量的方法。采用胰蛋白酶对纯化的矛头蝮蛇svTLE进行酶解,利用纳升液相色谱-四极杆/静电场轨道阱高分辨质谱(Nano LC-Q-Exactive-MS)和Proteome Discoverer 2.2软件分别进行多肽的检测和鉴定,通过BLAST搜索与Uniprot数据库对比分析,筛选出具有种属特异性的矛头蝮蛇svTLEs特征肽“EAYNGLPAK”。针对该特征肽对酶解温度、酶解时间和酶用量等样品前处理方法进行了优化,利用超高效液相色谱-串联质谱,以m/z 481.9>315.2和481.9>485.2作为检测离子对,采用ESI⁺模式进行了多反应监测(MRM)定性定量分析。结果显示,特征肽在2.5~30 ng/mL范围内线性关系良好,相关系数(r)大于0.9996,多水平加标回收率范围为95.5%~101.9%,各水平平行测定结果的相对标准偏差(RSD)为1.1%~3.2%,完全能够满足实际样品检测需求。方法简便快捷,灵敏度高,专属性强,可用于矛头蝮蛇蛇毒种属鉴别及svTLE含量测定,从源头保证血凝酶类产品的质量,并可为其他蛇毒类产品的质量控制提供参考。