<Emphasis Type="Italic">Penicillium</Emphasis> sp. ZD-Z1 chitosanase immobilized on DEAE cellulose by cross-linking reaction

Chitosanase obtained fromPenicillium sp.ZD-Z1 was immobilized on DEAE cellulose with glutaraldehyde by cross-linking reaction. The optimal conditions of immobilization were as follows: 0.1 g DEAE cellulose was treated with 5 ml 5% glutaraldehyde solution; then 2.3 mg chitosanase was immobilized on the carrier. The optimal temperature and pH was 60 °C and 4.0, and the K _m value was 18.87 g/L. Under optimal conditions, the activity of immobilized enzyme is 1.5 U/g, and the recovery of enzyme activity is 81.3%. After immobilization, the optimal temperature and K _m value increased (from 50 °C to 60 °C, from 2.49 g/L to 18.87 g/L), whereas the optimal pH was reduced (from 5.0 to 4.0). The enzyme activity loss was less than 20% after 10 times batch reaction; the immobilized enzyme showed good operation stability.     

牛乳中主要过敏原组分的分离纯化及鉴定

目的:分离纯化并鉴定牛乳中引起过敏反应的主要致敏组分。方法:采用聚丙烯酰胺凝胶电泳(SDS- PAGE)分析了脱脂乳和乳清的蛋白组分,用饱和硫酸铵分段盐析-DEAE离子交换柱层析纯化过敏原蛋白,脱脂乳和乳清蛋白皮下注射Balb/c小鼠获得高效价特异性IgE抗血清用于免疫印迹分析。结果:免疫印迹分析显示β-乳球蛋白(β-Lg)是引起牛乳过敏的主要过敏原,建立了牛乳过敏小鼠模型。结论:明确了牛乳过敏的主要过敏组分,对牛乳过敏症的诊断治疗以及无过敏原牛乳的制备提供了实验依据。     

一步柱层析纯化B-藻红蛋白及其毛细管电泳结合激光诱导检测

本研究采用硫酸铵沉淀结合DEAE-SepharoseFF离子交换柱层析技术分离纯化紫球藻中的B-藻红蛋白。试验结果表明,藻红蛋白粗提物经65%硫酸铵沉淀2d后过柱层析,B-藻红蛋白的纯度为A545nm/A280nm=3.0;而用65%硫酸铵沉淀2个月后,只需采用一步柱层析,B-藻红蛋白的纯度可提高到5.37,即达到纯品要求(纯度大于4.5)。说明65%硫酸铵对B-藻红蛋白具有高度选择性,其工艺效果对分离纯化有很大影响。特征吸收光谱和荧光光谱证实纯化后的产物符合B-藻红蛋白的性质,Native-PAGE电泳只出现单一染色带,表明获得的B-藻红蛋白是均一的;SDS-PAGE电泳出现2条染色带,一条着色较深带为α和β亚基,分子量为16～18kD左右,另一条较浅为γ亚基,分子量大约为31kD。同时建立了涂层毛细管柱结合激光诱导荧光电泳技术快速检测B-藻红蛋白纯度的分析方法,结果出现单一峰,表明毛细管电泳用于B-藻红蛋白的纯度检测高效可靠。     

葛根抗氧化肽的分离及清除自由基活性研究

目的：分离纯化葛根抗氧化肽并对其进行体外清除自由基活性的研究。方法：以野生葛根为实验材料,经30%的乙醇浸提、透析、冷冻干燥,进一步采用DEAE阴离子交换层析,反相高效液相色谱的方法,对葛根抗氧化肽进行纯化,对不同组分进行还原能力的测定,还原能力最强的命名为PE1。对PE1进行茚三酮反应和分子质量测定。采用邻苯三酚自氧化法、DPPH法、Fenton反应法检测PE1体外对超氧阴离子自由基、DPPH自由基、羟自由基的清除作用。结果：反相高效液相色谱检测PE1纯度,显示为单一峰,PE1与茚三酮反应呈阳性,分子质量约为746D,PE1在体外可以有效清除以上3种自由基。在质量浓度为0.652mg/mL时,羟自由基清除率可达到80.01%;在5mg/mL质量浓度条件下,对超氧阴离子自由基的清除率高达97.35%;在3.5mg/mL质量浓度条件下,对DPPH自由基的清除率达到75.02%,其IC50为2.83mg/mL。结论：PE1具有较强的清除自由基活性。     

离子交换树脂吸附分离人凝血酶原复合物研究

用静态方法研究了扩张床用离子交换树脂Streamline DEAE对人凝血酶原复合物（以下简称PCC）的吸附性能，在此基础上初步考察了扩张床条件下的穿透吸附效果。结果表明：在静态条件下，离子强度和缓冲液浓度对平衡吸附量有着较大的影响；Streamline DEAE对PCC的吸附符合Langmuir模型，在15℃和25℃条件下其饱和吸附量分别为66．67mg／g和80．00mg／g。在扩张床条件下，Streamline DEAE对PCC的动态吸附量达36．36mg／mL；用2．0mol／L NaCl、0．01mol／L磷酸缓冲液浓度、pH=7．0的洗脱液对吸附了PCC的凝胶树脂进行洗脱，静态洗脱率达80％以上，动态洗脱曲线表明有较好的洗脱效果。Streamline DEAE对PCC的吸附洗脱性能表明，该树脂可用于扩张床吸附分离PCC的工艺。     

Investigation of adsorption–desorption dynamism of bovine serum albumin on crosslinked N,N′‐diethylaminoethyl dextran microbeads: Solution phase

New hydrogel microspheres based on crosslinked dextran containing N,N′‐diethylaminoethyl (DEAE) groups with different chemical structures have been used in adsorption–desorption studies. Bovine serum albumin (BSA) is frequently used in biophysical and biochemical studies. BSA has a well‐known primary structure that has been associated with binding of many different categories of small molecules. Both adsorption kinetics and equilibrium isotherms for the adsorption of BSA on crosslinked DEAE dextran have been determined experimentally. These were only slightly dependent on the initial concentration of BSA but were considerably affected by the pH of the medium. The results fitted the Freundlich–Langmuir isotherm model for pH 6.9. The adsorption capacity factor and the adsorption equilibrium constant were obtained and mathematical modeling of adsorption, adsorption rate constants, and maximum adsorption were determined. Swelling kinetics of crosslinked DEAE dextran and optimum ionic strength, pH, and mass of hydrogel were also investigated. Desorption studies were finally determined under optimum medium conditions. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006     

Multiple forms of polygalacturonase from mango (Mangifera indica L. cv Alphonso) fruit

Polygalacturonase (PG) from mango pulp revealed three isoforms (I, II, III) upon ion exchange and gel filtration chromatography, each having an abundance of 68%, 6% and 26%, and molecular weights (M_r) 40, 51 and 45 kDa, respectively. The pH optimum for the isoforms was between 3 and 4. PG-I was stable over a wide pH range (4–7.5) unlike PG II and III, which were stable at pH 4 and 5, respectively. The optimum temperature was around 40 °C for all the three isoforms. Their apparent K_m for pectic acid was in the range 0.22–0.25 mg ml⁻¹. The V_max for PG I, II and III was 5.7, 3.6 and 4.4 μmol GalA equivalent h⁻¹, respectively. Cd²⁺, Cu²⁺ and Fe²⁺ and EDTA inhibited whereas GalA, Gal, Fuc, Rha and Ara stimulated PG-I activity, in particular. The major endogenous substrates for mango PG were identified to be two rhamnogalacturonans varying in their sugar ratio. These results are discussed in the light of pectin dissolution in vivo in ripening mango.     

Streamline DEAE对蛋白酶复合物的吸附平衡研究

以人凝血酶原复合物(以下简称PCC)为目标产物,研究了扩张床用离子交换树脂Streamline DEAE对蛋白酶复合物的吸附平衡。测定了不同温度下的吸附等温线和吸附动力学曲线,并通过计算求得热力学和动力学参数。结果表明:Streamline DEAE对PCC的吸附符合Langmuir模型,吸附反应的焓热变为34.466 kJ/mol;吸附动力学曲线用拟二级速率方程拟和有着较好的吻合度。吸附过程中蛋白质分子在树脂颗粒内部的扩散为控制步骤,求得吸附反应的表观活化能为29.089 kJ/mol,表明此Streamline DEAE对PCC的吸附具有反应速率快,性能稳定等特点。     

Adsorption of BSA on DEAE-Dextran: Equilibria

Abstract

Equilibrium isotherms for adsorption of BSA on DEAE-dextran have been determined experimentally. They were little affected by the initial concentration of BSA but were considerably affected by pH. They were correlated by the Langmuir equation when pH ≥ 5.05 and by the Freundlich equation at pH 4.8 (≈ pI). The maximum amounts of inorganic anion exchanged for BSA were 1 and 0.4% of the exchange capacity of the ion exchanger at pH 6.9 and 4.8, respectively. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm but was shifted to the right. When the concentration of NaCl was 200 mol/m³, BSA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO₄ ^2? -form resin was about 3.5 times larger than that with buffer (pH 6.9). The amount of BSA adsorbed on univalent-form resin decreased when the liquid-phase equilibrium concentration of BSA was increased.     

Glucosinolates fortification of cruciferous sprouts by sulphur supplementation during cultivation to enhance anti-cancer activity

The cruciferous sprouts, including cabbage (Brassicaoleracea), broccoli (Brassicacapitata) and radish (Raphanussativus), were cultivated with supplementation of sulphur salts. With supplementation of sulphur at 60 kg/ha, a 2–5-fold increases in total glucosinolates contents in the sprouts were observed. The individual glucosinolates whose concentration increased most significantly, included progoitrin, glucoerucin, glucobrassicin, glucohirsutin and 4-methoxybrassicin. The antioxidant properties of these sulphur supplemented sprouts were also higher than that of the normal sprouts due to the increases of phenolic compounds. Consequently, the glucosinolates fortified sprouts had higher anti-proliferative activity against HepG2 human hepatocarcinoma cells than the normal sprouts, as the cell viability decreased by 22–35%. Also in CT26 mouse colorectal cancer cells, the cell viability decrease by 34–59%.