Microfluidic chip based chemiluminescence detection of L-phenylalanine in pharmaceutical and soft drinks

A sensitive chemiluminescence (CL) method on chip coupled with microfluidic system has been reported for the determination of L-phenylalanine (L-PA). A microfluidic chip device with the detection chamber capable of fast sensing light emitted from the luminol and hydrogen peroxide CL reaction catalyzed by copper sulphate was fabricated for the determination of L-PA. The microfluidic chip was fabricated by a soft-lithographic procedure using polydimethyl siloxane (PDMS). The fabricated PDMS microfluidic chip had four inlet microchannels for introducing the sample, chemiluminescent reagent, Cu(II), and oxidant, and a 500 μm wide, 250 μm deep, and 82 mm long microchannel. The detection was based on the enhancement effect of L-PA on the CL signals of luminol–H₂O₂–Cu²⁺ system in an alkaline medium. The CL intensity of the system was enhanced linearly with the concentration of L-PA in the range of 1.5 × 10⁻⁹–1.2 × 10⁻⁷ mol L⁻¹. The limit of detection was found to be 2.4 × 10⁻¹⁰ mol L⁻¹ with the relative standard deviation of 1.8%. The presented method offers a simple, rapid and easy to handle analytical technique in terms of sensitivity, dynamic range and low detection limit for the determination of L-PA in diet soft drinks and pharmaceutical injection samples.     

胡萝卜中过氧化物酶和苯丙氨酸解氨酶特性及抑制条件的研究

经硫酸铵分级沉淀、Sephadex G-100凝胶过滤层析纯化,得到部分纯化的胡萝卜过氧化物酶(POD)和苯丙氨酸解氨酶(PAL).这两种酶的纯化倍数分别为7.44倍和5.87倍,回收率分别为32.51％和5.06％.酶学性质和动力学性质研究表明,胡萝卜POD和PAL的最适pH分别为5.0和8.4;在不同pH值下POD和PAL的活力及其耐受性变化趋势相似.POD的适宜温度为30℃,当温度40～50℃时,PAL酶活力达到最高点.PAL酶液体积为1.6 mL时,反应速度最快;而POD酶液体积分别为1.0mL和1.6mL时,反应速度出现峰值.当POD和PAL底物浓度分别为44.8×10-6 mol/L和0.02 mol/L时,反应速度达到最大值.Na+、10 mmol/L Ca2+、0.1 mmol/L NL4+、Mg2+可以同时显著地(P＜o.05)抑制胡萝卜POD和PAL酶的活力.胡萝卜POD与愈创木酚的亲合力强,PAL与L-苯丙氨酸的亲合力较小.     

肉桂酸产品链消化四氯化碳的前景展望

围绕四氯化碳-肉桂酸-L-苯丙氨酸一阿斯巴甜产品链,对四氯化碳的前景进行了技术可行性和经济性的分析和展望。     

Studies of L-phenylalanine production by immobilised aminoacylase in stabilized calcium alginate beads

Aminoacylase I (EC 3.5. 1.14) was immobilised by entrapment in uncoated calcium alginate beads and calcium alginate beads coated with chitosan, polyethyleneimine and polyethyleneimine-glutaraldehyde for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine. The operational stability, thermal stability, effects of pH and temperature and kinetic constants, K_m and V_max values of free and immobilised enzymes were studied. Scanning electron micrographs revealed differences in the structures of the surfaces of coated and uncoated beads. Chitosan-coated calcium alginate beads was found to be the best among the immobilised systems studied. The activity and the optimum temperature of immobilised aminoacylase were higher than those of the free enzyme. In addition, the beads showed stable activity under operational conditions. The immobilised aminoacylase lost about 20% of its initial activity after ten cycles of reuse. Polyethyleneimine and polyethyleneimine-glutaraldehyde treatments were also found to enhance the operational stability of the enzyme but its activity was greatly reduced.     

芳香族氨基酸在732树脂上的三组分吸附平衡

研究芳香族氨基酸(L-色氨酸、L-苯丙氨酸和L-酪氨酸)在氢型732树脂上的三组分吸附平衡过程。考察了初始pH和温度对吸附等温线的影响,并用扩展的Freundlich模型对三组分吸附平衡过程进行了拟合。结果表明,L-Tyr、L-Trp和L-Phe的平衡吸附量随pH的减小和温度的升高而降低;当pH=1.0(即料液中存在过多H+)时,则会显著降低氨基酸在树脂上的平衡吸附量;三组分吸附行为表明,L-Trp是该三组分吸附系统中的强吸附质,L-Phe次之,而L-Tyr最弱;扩展的Freundlich模型能够较好地描述3种氨基酸在氢型732树脂上的三组分吸附平衡过程,所得的三组分吸附平衡方程在α=0.001水平上高度显著,其平均相对误差在0.34%～2.80%之间。     

PMVBU吸附树脂对L-苯丙氨酸的吸附行为研究

通过静态吸附试验,研究了大孔交联聚(对乙烯基苄基脲)树脂(简称PMVBU树脂)对L-苯丙氨酸的吸附热力学及动力学特性。结果表明,在研究的浓度范围内,树脂对L-苯丙氨酸吸附平衡数据符合Langmuir和Freundlich等温吸附方程,相关系数R都在0.99左右,根据热力学函数关系计算了等量吸附焓、Gibbs吸附自由能和吸附熵,表明树脂对L-苯丙氨酸的吸附均为自发、放热、熵减的物理吸附过程。吸附符合二级动力学模型,颗粒内扩散过程是影响吸附速率的主要控制步骤。     

L-苯丙氨酸对Q235钢在硫酸中的缓蚀作用

采用电化学方法,研究了L-苯丙氨酸对Q235钢在0.5mol/L H2SO4溶液中的缓蚀作用及缓蚀机理,分析了其缓蚀剂类型。结果表明:L-苯丙氨酸的缓蚀效率随浓度的增加而增大,而且随着温度的升高,Q235钢表面单个活性位点上吸附的缓蚀剂分子数量增加,缓蚀效率也增大;L-苯丙氨酸属于混合抑制型缓蚀剂,作用机理为几何覆盖效应,其在Q235钢表面的吸附为单分子吸附,且吸附规律符合El-Awady动力学模型。     

明绿豆苯丙氨酸解氨酶的分离纯化及性质初步研究

为了研究明绿豆中苯丙氨酸解氨酶(PAL)的动力学特性,采用35%和65%饱和度的硫酸铵分级沉淀、DE-52阴离子交换层析方法对明绿豆中的PAL进行了分离纯化,并对其基本性质做了初步研究。结果表明,PAL的纯化倍数为6.982 9,蛋白质得率为4.65%,酶得率为32.45%。PAL在硼砂-硼酸缓冲溶液中适宜的p H范围为7.6~9.0,最适p H有2个:8.0和8.6。PAL适宜的温度范围为35~45℃,最适反应温度为40℃。得到PAL的2个Km值,分别是:Km1为6.94×10-5mol/L,Km2为1.23×10-4mol/L。本研究结果可为进一步研究和开发明绿豆的PAL提供参考。     

电位滴定结合偏最小二乘算法用于溶液中肉桂酸和L-苯丙氨酸的同时测定

结合自动电位滴定和化学计量学算法,建立同时测定溶液中肉桂酸、L-苯丙氨酸和硫酸铵的方法。通过正交设计25组校正集溶液,通过留一验证法对校正集最佳主成分进行选择,得到偏最小二乘法较佳模型,并对模拟样品进行分析。结果显示：肉桂酸、L-苯丙氨酸、硫酸铵的回收率分别在92.9%～104.4%、94.9%～101.5%、96.6%～105.1%之间。基于PLS的方法可以方便解析重叠的滴定图谱,不需要预先分离即可预测成分浓度。     

固定化酵母细胞转化肉桂酸生产L-苯丙氨酸

建立了一套较之传统方法更为准确的测定固定化细胞生产L-苯丙氨酸(L-phe)的新方法,采用高效液相色谱法(HPLC)测定反复冲洗凝胶所得冲洗液可以更准确地测定L-phe的生成,并采用新方法研究了固定化黏红酵母细胞的活力与稳定性。复合凝胶聚乙烯醇(PVA)+琼脂糖为最适载体,最佳质量浓度为PVA 120 g/L、琼脂糖10 g/L,反复冻融4次。此法得到的固定化细胞颗粒,机械强度良好,酶活保持时间长达200 h,L-苯丙氨酸产量高。通过流化床反应器测定其半衰期为100 h,200 h后总产量为44.58 mg/10 mL,固定化细胞无崩解现象。