酶法生产L-苯丙氨酸体系的液相色谱-质谱联用分离分析

通过对高效液相色谱各主要分离条件以及质谱分析条件的优化,建立了一种有效的液质联用方法分析酶法生产L-苯丙氨酸体系中3种主要成分:L-天冬氨酸,丙酮酸和L-苯丙氨酸.该方法抗干扰能力强,简单、快速、准确、灵敏且重现性好,适用于定性及定量分析实际样品,为生产工艺优化和生产过程研究提供了依据.     

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。     

苯丙酮尿症新生儿血斑中辅酶及葡萄糖等物质的分析

苯丙酮尿症(PKU)是新生儿先天性苯丙氨酸羟化酶缺陷所引起的苯丙氨酸代谢障碍疾病.本研究采用超高效液相色谱-质谱联用技术, 测定了5例PKU新生儿出生3天和出生30天后的血斑与20例年龄相仿的正常新生儿血斑中辅酶Q10的绝对含量和辅酶Q9的相对含量,其中,健康新生儿血斑中辅酶Q10的含量为(122.1±24.9) ng/mL,PKU新生儿组的含量为(59.0±12.0) ng/mL.采用气相色谱-质谱联用技术测定了胆固醇和葡萄糖的相对含量.研究结果表明,与对照组相比,PKU新生儿血斑中辅酶Q10、Q9、胆固醇和葡萄糖的含量均显著降低,辅酶Q10的降低与血斑中苯丙氨酸含量升高呈现显著反向相关.本研究结果为PKU患儿的饮食治疗方案提供了依据.     

复合酶酶解假酸浆黄酮苷抗氧化性研究

本文研究了复合酶酶解前后假酸浆提取物的抗氧化活性能力,并采用薄层色谱法(TLC)对酶解产物中总黄酮进行了分离,利用高效液相色谱-质谱联用技术对其鉴定分析。通过对DPPH自由基、羟自由基及超氧阴离子自由基清除能力实验,酶解液抗氧化性能显著高于假酸浆提取液;酶解液的薄层及HPLC-MS法初步鉴定出假酸浆中含有桑色素、二氢槲皮素和儿茶素三种黄酮。     

质谱分析技术在纺织品检测方面的应用

介绍纺织品中有害物质标准检测方法中质谱分析技术的应用,概述了气相色谱-质谱联用、高效液相色谱-质谱联用和电感耦合等离子体-质谱联用技术在纺织品检测方面的广泛应用及其发展前景.     

固相萃取/离子阱气相色谱质谱联用对血液中多种安眠药物的同时测定

建立了气相色谱-质谱联用测定血液中多种安眠药物的分析方法,通过固相萃取提取并富集血液样品中的9种常见巴比妥类、吩噻嗪类和苯二氮杂类安眠药物,采用离子阱二级质谱检测其含量。通过优化萃取溶液pH值及气相色谱-质谱联用分析条件,对9种安眠药物进行了定量分析。采用离子阱串联质谱(MS/MS)技术消除基底干扰,给出被测物结构信息。9种安眠药物的检出限为0.04~0.10 mg/L,回收率为78%~93%。方法用于人心血中安眠药物的检测,检出安定0.6 mg/L。该方法高效、简单、灵敏度高,可用于血液中巴比妥类、吩噻嗪类和苯二氮杂类安眠药物的同时测定。     

高分子载体对米曲霉氨基酰化酶的固定化研究

合成了一系列不同结构的丙烯酸甲酯—二乙烯基苯交联共聚物,并用多乙烯多胺胺解制得了功能基化交联共聚物。研究了交联度、致孔剂用量和不同的功能基化试剂对这些载体固定化氨基酰化酶效果的影响。比较了固定化氨基酰化酶与溶液酶的酶学性质。用这种载体制成固定化酶柱,对N—乙酰—DL—蛋氨酸和N—乙酰—DL—苯丙氨酸进行连续拆分,得到了很好的效果。     

绍兴黄酒中ACE活性抑制肽的分离分析

首次将绍兴黄酒中的肽类组分进行大孔吸附树脂柱层析和高效凝胶过滤色谱以及反相色谱的多步提取纯化与抑制血管紧张素转换酶（AcE）活性试验，并首次利用基体辅助激光解吸电离-飞行时间串联质谱分析和液相色谱-电喷雾电离四极杆-飞行时间串联质谱联用分析鉴定出黄酒中4种ACE活性抑制肽的氨基酸序列为：VEDGGV、PST、NT和LY。     

高效液相色谱-电喷雾质谱联用法测定人参和西洋参的皂苷类成分

利用高效液相色谱（LC—ELSD）与电喷雾质谱（ESI—MS）联用技术对人参和西洋参中的人参皂苷类成分进行了比较研究。通过液相色谱与质谱联用技术获得了相应化合物的分子量信息;利用质谱的源内CID技术获得了相应化合物的结构信息。根据人参皂苷Rf和拟人参皂苷F11两种同分异构体在质谱中的源内CID裂解规律的不同,建立了人参和西洋参药材的LC-ESI-MS简便、准确的鉴别方法。     

基于核酸适配体的微流控芯片酶反应器用于蛋白质的分析

将核酸适配体作为胰蛋白酶固定化介质,制备了一种新型的微流控芯片酶反应器,并与高效液相色谱-串联质谱联用,搭建了在线分析平台;分别使用标准蛋白及混合蛋白样品对芯片的酶解效率及联用平台的分析能力进行了初步评价。结果表明,5 ng肌红蛋白经该平台分析后肽段覆盖率可达到37%;对500 ng混合蛋白进行3次平行分析,肽段覆盖率及相对标准偏差分别为44.3%、6.5%(牛血清白蛋白), 65.0%、2.7%(肌红蛋白)和62.0%、5.6%(细胞色素c);初步实验表明,该在线分析平台具有检测灵敏度高、重现性好、酶解效率高的特点,有望在蛋白质组学分析中发挥重要作用。     

Investigation of enzymatic hydrolysis of lipid-like substrates in monolayers

The parameters of enzymatic hydrolysis of novel lipid-like substrates assembled in monolayers at water–air interface were estimated by a simple method. The method is based on measurement of the initial velocity of the reaction registered by the decrease of the monolayer area (caused by the enzymatic hydrolysis) at surface pressure of 10 mN/m in a single–compartment trough. Hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acylated by phenylalanine, leucine and valine was characterized by catalytic constants k_cat and apparent Michael's constants K_m(app) (using lipase from Pseudomonas fluorescens as catalyst). It was found that k_cat of the synthetic pseudoglycerides (7–13 per s) are higher than k_cat of trilaurin (4 per s) that can be explained by the presence of positively charged primary aminogroups in the substrates. K_m(app) values were found to be similar for all the substrates studied (ca. 2×10⁻⁶ M). The proposed method allows estimation of the kinetic constants in traditional dimensions.     

Flow Injection Analysis of Glucose in Human Serum by Chemiluminescence

Abstract

Combining the rigidly controlable conditions of Flow Injection Analysis with detection by chemiluminescence, a method for determination of glucose in serum via enzymatic degradation by glucose in serum via enzymatic degradation by glucose oxidase is described. With a Standard deviation of less than 2% the sampling frequency is 75 samples per hour. Results obtained by the proposed procedure are compared with those acquired by a routine AutoAnalyzer method used at a local hospital. It is shown that any influence on the yield of chemiluminescence due to difference in viscosity of the injected samples effectively can be eliminated.     

Enzyme assay for chitinase catalyzed hydrolysis of tetra-N-acetylchitotetraose by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) has been used to observe the chitinase-catalyzed hydrolysis of tetra-N-acetylchitotetraose. Enzymatic hydrolysis of tetra-N-acetylchitotetraose by chitinase B from Serratia marcescens produces exclusively two molecules of di-N-acetylchitobiose allowing for the determination of a single glycosidic bond hydrolysis heat that was used to monitor the rate of the enzymatic reaction. The change in heat rate with respect to time (dQ/dt) was translated to the reaction rate, and the total heat produced was related to substrate concentration throughout the reaction. Reaction rates versus substrates concentration were fit to Michaelis-Menten plots, yielding a k_cat of 40.9 ± 0.5 s⁻¹ and a K_m of 54 ± 2 μM.     

Breath biosensing: using electrochemical enzymatic sensors for detection of biomarkers in human breath

Detection of biomarkers for disease by noninvasive methods is critical for the early diagnosis and screening of disease, enabling prompt treatment. Breath biosensors are a viable option as the exhaled breath contains several biomarkers linked to lung cancer, oxidative stress, diabetes, and other diseases. Breath analysis has been achieved by advanced analytical techniques such as gas chromatography and infrared spectroscopy. However, electrochemical enzymatic breath biosensors offer a cost-effective, sensitive platform for biomarker detection without complex analysis and interpretation by trained laboratory personnel. This review aims to summarize recent advances in the field of electrochemical enzymatic breath biosensors and offer future opportunities from other applications of nonelectrochemical enzymatic breath biosensors.     

A new biosynthetic tracer for the inline measurement of virus retention in membrane processes: part I--Synthesis protocol

In this study, a new biosynthetic tracer was developed to characterize the virus retention dynamics of membrane systems. This new tracer is a modified bacteriophage obtained by the grafting of enzymatic probes to an MS2 bacteriophage, one of the smallest non-pathogenic bacteria viruses, with an average diameter of about 30 nm. A protocol for the synthesis and purification of this new tracer was developed in this work. The production of this biosynthetic tracer was first qualitatively shown by a chromatographic characterization and an enzymatic test. The average number of probes grafted per phage was then quantified for three batches of tracers made from the same native phage suspension and the same batch of enzymatic probes. This quantification demonstrated the reproducibility of the synthesis protocol developed.     

Non‐Invasive Monitoring of Osteogenic Differentiation on Microtissue Arrays under Physiological Conditions Using Scanning Electrochemical Microscopy

In this paper, we present a non‐invasive assay using scanning electrochemical microscopy (SECM) for detecting osteogenic differentiation at physiological conditions (pH 7.5) on arrays of C2C12 microtissues. Upon exposure to bone morphogenic protein 2 (BMP‐2), C2C12 microtissues differentiate and express alkaline phosphatase (ALP), which is indirectly detected through an enzymatic assay producing an electroactive species. The latter is detected using SECM by scanning at constant height over live microtissues at physiological pH (7.5) as well as more alkaline pH (8.5). As a control, expression of ALP is confirmed using a standard colorimetric assay. Detecting differentiation on live samples at physiological conditions represents a significant improvement for continuous monitoring of tissue differentiation or further use of the microtissues for, e.g., regenerative medicine.     

葡萄糖酶电极测定环糊精聚合物中的环糊精单元

用多巴胺电氧化聚合物膜将葡萄糖氧化酶固定在铅笔电极上,制得简易的葡萄糖酶电极.用自制的葡萄糖酶电极对自制环糊精聚合物中环糊精单元进行定量检测.研究了自制的葡萄糖酶电极的检测范围和稳定性.结果表明环糊精聚合物中环糊精单元质量与葡萄糖酶电极响应电流间存在很好的线性关系,且酶电极稳定性好,可以多次反复使用,连续使用30天,响应电流只减弱7%.与用Sulfate-Phenol比色法测得的环糊精聚合物中环糊精单元结果相比,两者相对偏差在8%以内.     

Monitoring enzymatic conversions by mass spectrometry: a critical review

This review highlights recent advances in the application of electrospray ionisation and matrix-assisted laser desorption/ionisation mass spectrometry (MS) to study enzymatic reactions. Several assay schemes for different fields of application are presented. The employment of MS as a means of detection in pre-steady-state kinetic studies by rapid-mixing direct analysis and rapid-mixing quench flow techniques is discussed. Several steady-state kinetic studies of a broad range of different enzymatic systems are presented as well as enzyme inhibition studies for various target enzymes. As a promising new development multiplex assays, which monitor the conversion of several substrates simultaneously in one experiment, are described. This assay type has been used for competition studies, enzymatic activity screenings and for diagnostic purposes in clinical chemistry. Generally, it can be concluded that mass spectrometry offers an intriguing alternative as detection methodology in enzymatic bioassays. Its applicability for the monitoring the conversion of naturally occurring substrates and its overall versatility make MS an especially promising tool for the study of enzyme-catalysed processes.     

纤维素废弃物的生物转化

从纤维素酶法水解出发，叙述了纤维废弃物生物转化为葡萄糖和乙醇的研究现状和发展趋势，重点说明了加快反应速率、提高纤维素酶利用率的几种方法。     

聚羟基烷酸酯酶降解动力学研究

研究了羟基丁酸 羟基戊酸共聚物 (PHBV)在脂肪酶中的降解行为 ,用滴定法测定降解速度并进行酶促反应动力学研究 .探讨了降解速度与酶浓度和底物浓度的数学关系和Michaelis Menten常数 ,从实验上和理论上证实了PHBV的物理形态和几何尺寸对酶降解过程的影响 ,以及实验数据与非均相动力学模型的拟合