聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

以苦瓜籽为材料,研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明,柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时,各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点,pH 6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液（pH 4.0）,1%PAA选择性沉淀碱性蛋白（等电点pI为8.65～9.30）的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0,等电点沉淀后的上清液用PAA沉淀碱性蛋白,当PAA（1%）用量为160μL/mL提取液时,pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀;当PAA用量为120μL/mL提取液时,pH 4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液（pH＞9.0）,当溶液NaCl浓度为3.0%时,溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经Sephadex G-75柱层析分离,分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD,pI值约为9.5,主峰Ⅱ的分子量约为10kD,pI值约为9.3。     

枯草杆菌(Bacillus subtilis)产果胶酶的研究Ⅰ.菌种选育、鉴定及其酶学特征分析

分离到1株高产果胶酶菌株,经形态、生理以及生化指标鉴定,确认为枯草杆菌(Bacillus subtilis)并命名为枯草杆菌18.发酵液用90%硫酸铵沉淀,透析后的粗酶经CM-52柱层析,收集酶峰,再过Shephadex G-100得部分纯化酶.该酶最适pH9.0,在pH9～11稳定,最适反应温度60℃,50℃加热50 min保存60%酶活,60℃加热50 min酶活则保存10%.酶的等电点(pI)4.0,分子量31 000.该酶对苎麻脱胶有较好的特异性.     

山蕗菜根酶活性的初步研究

通过蛋白质、多糖和纤维素含量的变化,研究了新鲜山蕗菜根所含内源蛋白酶、多糖水解酶和纤维素酶的活力。结果表明,新鲜山蕗菜根匀浆后1 h蛋白质水解程度为41.58%,2 h多糖水解程度约为26%,3.5 h纤维素水解程度约为3.8%。说明山蕗菜根蛋白酶和多糖水解酶具有较高活力,而纤维素酶活力较小。通过福林法和DNS法分别测得其粗提液蛋白酶和多糖水解酶的活力分别为24 221.57U/g粗酶蛋白和45 018.65U/g粗酶蛋白。     

马槟榔甜味蛋白的研究——Ⅰ.提取、纯化和某些特性

从中药马槟榔(Capparis masaikai Levl.)成熟种子中分离了一种能引起持久甜味的蛋白质,取名为马槟榔甜蛋白(Mabinlin),分子量11,700,等电点pH11.8,在280nm波长有最大吸收峰,其引起甜味感觉的最低浓度为0.1％,种仁含甜蛋白量约4％。     

β-环糊精葡基转移酶的纯化方法及酶学性质的研究

β-环糊精葡基转移酶的粗酶液应用酚酞分光光度法测得该酶的环化活性为11.79U/mL。该粗酶液先经过淀粉-酒精沉淀或淀粉-硫酸铵沉淀初步纯化,然后经Sephacryl S-100凝胶层析后,比活力分别提高了16、21、50倍,由原来的11.44U/mg蛋白质增加到572.67U/mg蛋白质,回收率分别为72.4%、66.4%、40.9%,经SDS-PAGE电泳显示为单一的蛋白带,酶的分子量约为70kDa。酶学性质研究表明该酶的最适pH和最适温度分别为6.0和60℃,在pH6.0~10.0范围内,55℃以下保温30min基本保持稳定。纯化后的β-环糊精葡基转移酶的环化活性每克相当于35727U。     

麻疯树叶中过氧化物酶JCP-1的分离纯化和几种特性的检测

麻疯树叶片蛋白粗提液经硫酸铵分级沉淀,强阴离子琼脂糖、强阳离子琼脂糖和交联葡聚糖层析,得到一个比活为4499U·mg-1（蛋白）过氧化物酶,命名为JCP-1。其分子量为49kDa,等电点为pH3.3,最适pH为5.0-6.0。以H2O2为底物的Km为2.14mmol·L-1。JCP-1具有宽泛的最适保存pH（7.0-11.0）和较高的耐热性（80℃高温处理15min,活性保持在90%以上）。30%PEG6000处理模拟干旱胁迫及50℃高温胁迫麻疯树苗,其叶片中JCP-1活性分别提高121%和155%。     

水稻植硅石沉积硅的有机组分研究

硅是最新确认的植物必需元素,但硅在高等植物中的沉积机理尚未揭示.以新鲜水稻茎、叶为材料,分别采用传统湿法灰化和低温粉碎自然沉降法分离出植硅石,HF溶液溶解、离心后用NaOH等溶液对沉淀物逐级分离、提取,得到一系列碱性有机物质,上清液层析脱盐后得到酸性蛋白质;将各组分分别与硅溶液进行反应.结果表明传统湿法灰化提取水稻植硅石不含有能够沉淀硅的物质,低温粉碎自然沉降法提取的植硅石含有两种组分能够诱导形成硅沉淀.这两种物质分别为来自HF提取液中的酸性蛋白和来自NaOH提取液的碱性多肽与酚类混合物.酸性提取液中含有相对分子质量约为14.4 kD的蛋白质,与硅藻沉淀硅的蛋白相对分子质量近似.不同酸碱度下酸性蛋白对硅的沉积量不同,以pH 5左右诱导量最大.     

响应面法优化苦荞麸皮粉蛋白质提取工艺

目的：分析苦荞麸皮粉中蛋白含量及其蛋白组成。方法：采用碱法提取-等电点沉淀分离蛋白。在单因素试验基础上,选取料液比（w/v）、pH、时间等3个影响因素,采用响应面法 （Box-Behnken 中心组合）优化苦荞麸皮粉蛋白提取工艺。结果：料液比、pH、时间对麸皮蛋白提率有显著影响,影响顺序为料液比> pH>时间。响应面优化得到最佳提取条件为pH 10.5、料液比1∶35、时间3 h 40 min,此条件下苦荞麸皮蛋白提取率为（97.31±4.64）%。结论：本研究为有效开发利用苦荞麸皮粉的蛋白提供科学依据。     

金属螯合亲和层析介质用于六聚组氨酸融合蛋白的纯化研究

以Sepharose CL-6B为载体,环氧氯丙烷为活化剂,羧甲基天冬氨酸（CM-Asp）为螯合配基制备载有Co2+的金属螯合亲和层析介质Co-CM-Asp-Sepharose,并将其用于六聚组氨酸融合蛋白的纯化研究。对纯化200 μL细胞裂解液中靶蛋白所需Co-CM-Asp-Sepharose介质用量,Co-CM-Asp-Sepharose与细胞裂解液的孵育时间,介质清洗条件及靶蛋白洗脱时所需咪唑浓度等进行了优化。比较了Co-CM-Asp-Sepharose与Ni-NTA-Agarose(Qiagen公司)两种螯合介质对融合蛋白的纯化效果,开展了从5mL细胞裂解液中放大规模纯化融合蛋白的研究,并通过Bradford法测定了Co-CM-Asp-Sepharose对CD155D1蛋白的纯化量。结果表明：对200μL细胞裂解液纯化体系, Co-CM-Asp-Sepharose（50%悬浮液）的优选体积为60μL,最佳孵育时间为30min,洗脱液最佳咪唑浓度为200mmol/L,纯化得到融合蛋白的量约为200μg。介质用量放大为1.5mL（50%悬浮液）对CD155D1蛋白的纯化量可达4.6mg。与商品化Ni-NTA-Agarose相比,本介质具有选择性好,清洗条件简单,得到的靶蛋白纯度高等优点。     

二维液相色谱法在羊草地上部总蛋白分离中的应用

取8周龄羊草的地上部分,用三氯乙酸-丙酮法沉淀总蛋白,沉淀裂解后将缓冲液置换为起始缓冲液,进行第一维色谱聚焦分离。将第一维分离收集的pH值为8.5至4.0之间的组分分别进行第二维无孔硅胶反相高效液相色谱分离,利用ProteoVue软件获得羊草植株总蛋白pI/UV图谱,即羊草植株总蛋白质表达谱。文中对二维液相色谱法分离羊草蛋白质进行了方法学的研究,在第二维分离中尝试用3种不同的洗脱梯度条件进行分离,优化二维液相色谱分离条件并与传统凝胶双向电泳进行了比较,另外还对二维液相色谱的重现性和准确性进行了检验。实验建立了利用二维液相色谱分离羊草总蛋白的技术方法。     

淡水珍珠功能成分的水提取工艺

为更好的利用淡水珍珠中的功能成分,减少提取过程中对蛋白等成分的影响。研究了以pH值为3.0的醋酸与醋酸钠水提取珍珠中功能成分的工艺。采用固定床提取方法,以pH值为3.0的醋酸水溶液提取珍珠中水溶性成分,在选定的最适工艺条件下,珍珠中蛋白质提取率达86.3%,水解为氨基酸的蛋白质仅为0.036%。同时提取得到了一种新功能成分,经初步结构确定为黄酮类化合物,提取率达80.8%,为淡水珍珠的利用提供了基础。     

枯草芽孢杆菌B034拮抗蛋白的分离纯化及特性分析

枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）Ｂ０３４分离自水稻叶面,对水稻白叶枯病菌具有较强的拮抗能力。除去菌体培养液以７０％饱和度硫酸铵沉淀所得的拮抗物粗提液对热稳定,对胰蛋白酶不敏感,对蛋白酶Ｋ、链霉蛋白酶Ｅ部分敏感,对氯仿部分敏感,其作用的活性ｐＨ范围低至４,高至１２以上,比较耐碱性。粗提液经ＰｈｅｎｙｌＳｅｐｈａｒｏｓｅＣＬ４Ｂ柱层析、ＤＥＡＥＳｅｐｈａｃｅｌ柱层析和ＨＰＬＣ的Ｓｕｐｅｒｄｅｘ７５ＨＲ１０／３０柱层析,得到二个拮抗活性峰：Ｐ１和Ｐ２。Ｐ２经ＳＤＳＰＡＧＥ和ＰＡＧＥＩＥＦ电泳显示为单一蛋白带,分子量５０３ｋＤ,等电点６２５。自动Ｅｄｍａｎ降解法从Ｐ２的Ｎ端测出残基序列为ＩｌｅＳｅｒＡｓｎＰｒｏＸＩｌｅＡｓｐＶａｌ     

Validation of a HPLC method for the determination of p-nitrophenol hydroxylase activity in rat hepatic microsomes

We report a HPLC-UV method for determination of p-nitrophenol (PNP) hydroxylation to 4-nitrocatechol (4NC) as a marker for CYP2E1 activity in rat hepatic microsomes. Proteins were precipitated by addition of 50 microL phosphoric acid (50%, v/v in water) to 500 microL microsomal suspensions. Following vortex mixing and centrifugation the supernatant (20 microL) was injected onto a Supelcosil C(18) column (150 mm x 4.6 mm, 5 microm), and mobile phase (22% acetonitrile, 0.1% trifluoroacetic acetic acid, 0.5% triethylamine) delivered at 1.0 mL/min produced resolved peaks for internal standard, 4NC, and PNP in < 11 min. Calibration curves were linear (r(2) = 0.999) from 0.1 to 40 microM with intra- and inter-day precision < 12% and accuracy >90%. The method's improved sensitivity (LOQ = 0.1 microM) and minimal sample processing allowed rapid monitoring of PNP hydroxylase activity in fetal, neonatal, juvenile, and adult rat livers.     

Isolation from ascites carcinoma Krebs II cells of an unlinking enzyme hydrolyzing a covalent bond between picornavirus RNA and VPg

A new purification procedure for the isolation of the "unlinking" enzyme, which hydrolyzes the phosphodiester bond between 5;-terminal uridylic acid of the encephalomyocarditis viral RNA and protein VPg has been developed. The enzyme (tyrosine-(5;P-->O)-uridylylpolynucleotide phosphodiesterase, Y-pUpN PDE) was purified from frozen mouse carcinoma Krebs II cells. The purification procedure included ammonium sulfate fractionation of the cell extract, pH fractionation by acidification of the protein solution to pH 4.0, cation-exchange chromatography on CM-52-cellulose, chromatofocusing, and size-exclusion HPLC on a TSK 2000 SW column. The enzyme was shown to exist as several forms characterized by different isoelectric points (ranging from 4.0 to 5. 2) and molecular masses. The pH fractionation and ion-exchange chromatography on CM-cellulose influenced the pI and molecular mass values for each form (pI increased, whereas molecular mass decreased from 30 to 26 kD). The employment of these two stages removed (almost completely) an accompanying proteolytic activity, which co-purified with Y-pUpN PDE and digested free VPg. The molecular mass of 26 kD determined by HPLC for the native form coincided with the molecular mass of the major protein band determined by SDS-PAGE for the denatured form of the enzyme.     

Cellulase Complex of the Fungus Chrysosporium lucknowense: Isolation and Characterization of Endoglucanases and Cellobiohydrolases

Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44.     

High-throughput determination of fudosteine in human plasma by liquid chromatography-tandem mass spectrometry, following protein precipitation in the 96-well plate format.

A 96-well protein precipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated for the determination of fudosteine in human plasma. After protein precipitation of the plasma samples (50 microL) by the methanol (150 microL) containing the internal standard (IS), erdosteine, the 96-well plate was vortexed for 5 min and centrifuged for 15 min. The 100 microL supernatant and 100 microL mobile phase were added to another plate and mixed and then the mixture was directly injected into the LC-MS/MS system in the negative ionization mode. The separation was performed on a XB-CN column for 3.0 min per sample using an eluent of methanol-water (60:40, v/v) containing 0.005% formic acid. Multiple reaction monitoring (MRM) using the precursor-product ion transitions m/z 178-->91 and m/z 284-->91 was performed to quantify fudosteine and erdosteine, respectively. The method was sensitive with a lower limit of quantification (LLOQ) of 0.02 microg mL(-1), with good linearity (r>0.999) over the linear range of 0.02-10 microg mL(-1). The within- and between-run precision was less than 5.5% and accuracy ranged from 94.2 to 106.7% for quality control (QC) samples at three concentrations of 0.05, 1 and 8 microg mL(-1). The method was employed in the clinical pharmacokinetic study of fudosteine formulation product after oral administration to healthy volunteers.     

Cationic polymer precipitation for enhanced impurity removal in downstream processing

Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation–depth filtration and the current platform process for the purification of biologics.     

星天牛幼虫肠道木聚糖酶的纯化和性质

星天牛Anoplophora chinensis (Frster)幼虫肠道匀浆液经80%丙酮沉淀、Q-Sepharose阴离子交换柱层析、PAGE制备电泳等方法纯化后,获得在SDS-PAGE上呈现单一区带的木聚糖酶。该酶的分子量约25 kD,等电点约4.0,最适温度50℃,最适pH 5.4,pH 3.0～7.8对酶活性的恢复无大的影响, 50℃保温2 h仍有60%酶活性。Hg²⁺、M_nO^-4、变性剂SDS完全抑制该酶活性, Cu²⁺、Mn²⁺、Ag⁺、Zn²⁺、Pb⁺、脲对酶活性有强烈的抑制作用。该酶具有水解纤维素的交叉活性,其K_m值为2.47 mg/mL,V_max为0.6 IU/mL。     

模拟酸雨对茄科3种蔬菜种子萌发的影响

采用pH 2.0、3.0、4.0、5.0的模拟酸雨和pH 6.5的中性溶液(ck)处理番茄、茄子、辣椒3种蔬菜种子,研究酸雨胁迫强度对3种蔬菜种子萌发的影响。结果表明：从发芽率、发芽势、发芽指数3个萌发指标来看,番茄种子在pH 2.0～3.0的强酸雨胁迫下,萌发受到轻微抑制;茄子种子在pH 2.0～5.0的酸雨胁迫下,萌发一直受到抑制,尤其是在pH 2.0的强酸雨胁迫时表现出萌发几乎完全受到抑制,而辣椒种子在酸雨胁迫下,萌发几乎没有受到抑制。3个萌发指标均随着酸雨pH值的减小而减少。pH 2.0～5.0酸雨胁迫下,辣椒吸水值的变幅（13.75）﹤番茄（19.91）﹤茄子（20.58）,同等酸雨胁迫强度下,根长抑制指数、蛋白质含量降幅、丙二醛增幅辣椒﹤番茄﹤茄子。这表明抗酸雨胁迫能力：辣椒>番茄>茄子。     

Gibberellic Acid-induced Increase in Activity of a Particular Isozyme of Acid Phosphatase in Wheat Half-seeds

Embryoless half-seeds of Triticum aestivum L. contain at leastnine acid phosphatase isozymes of isoelectric pH ranging from4.0 to 7.2. Treatment with GA₃ resulted in activation of a particularisozyme of pI 4.0. Three major isozymes (pi 4.0, 4.9 and 6.2)differed in their relative specificities. A similar increaseof the pI 4 isozyme was also observed in the endosperm of germinatingwheat seeds. (Received April 7, 1981; Accepted July 1, 1981)