一种基于刃天青还原的荧光方法对β-葡萄糖苷酶活性的检测

建立了一种简单的荧光分析方法实现了对β-葡萄糖苷酶的定量分析检测。其原理是底物2-O-β-D-葡萄糖基-L-抗坏血酸(AA-2βG)在β-葡萄糖苷酶的水解作用下生成抗坏血酸,后者将刃天青还原生成具有强烈荧光的试卤灵,通过溶液荧光强度的变化对β-葡萄糖苷酶活性进行检测。结果发现,抗坏血酸浓度在3～400μmol/L范围内与反应体系在585 nm处的荧光强度呈线性关系,相关系数r~2=0.981。并基于此实现了对β-葡萄糖苷酶活性的定量检测:β-葡萄糖苷酶的浓度在1～30 U/L范围内与溶液在585 nm处的荧光强度呈良好的线性关系,相关系数r~2=0.995,检出限为1.0 U/L。该方法操作简单,反应灵巧,检测范围较宽,可实现对β-葡萄糖苷酶活性的定量分析检测。     

银杏叶中α-葡萄糖苷酶抑制剂的超滤质谱筛选

采用体外酶抑制活性检测方法结合超滤质谱(UF-LC/MS)筛选方法对中药提取物中的α-葡萄糖苷酶抑制剂进行了筛选.以4-硝基苯-α-D-吡喃葡萄糖苷(PNPG)为底物,阿卡波糖为阳性对照药,对5种富含黄酮类化合物的中药提取物进行了α-葡萄糖苷酶抑制活性的初步测定.结果表明,银杏叶具有最强的α-葡萄糖苷酶抑制活性,可作为进一步复筛的对象.利用超滤质谱技术对银杏叶中潜在的α-葡萄糖苷酶抑制剂进行了筛选,从中筛选出4种潜在的α-葡萄糖苷酶抑制剂,并利用液相色谱-串联质谱技术(LC-MSn)对其结构进行了鉴定.本文结果为开发新一代安全有效的降糖药物奠定了基础.     

一种凝血酶亲和色谱介质的制备方法

对凝血酶-琼脂糖亲和色谱介质的制备方法进行了研究。首先使用凝血酶和溴化氰活化的琼脂糖制备凝血酶-琼脂糖亲和色谱介质,然后用生色底物法考察亲和色谱介质上凝血酶的活性,以凝血酶活性为指标对最佳偶联条件进行了优化。结果表明最佳条件为使用pH 8.3的Na₂CO₃-NaHCO₃溶液(含0.5 mol/L NaCl)为缓冲溶液,凝血酶用量为每1 g色谱介质加入凝血酶200 U,室温反应10 h。在最佳条件下所制备的色谱介质有较好的稳定性,在4℃条件下存放40天,亲和介质上的凝血酶活性仍有70.6%保留。该亲和色谱介质可广泛用于含凝血酶抑制剂的天然药物筛选和分离纯化。     

高效液相色谱法测定人体红细胞中儿茶酚氧位甲基转移酶的活性

 采用高效液相色谱紫外检测法测定人体红细胞中儿茶酚氧位甲基转移酶 (COMT)的活性。以 3,4 二羟基苯甲酸 (DBA)作为酶反应底物 ,S 腺苷甲硫氨酸 (SAM)作为甲基供体 ,在镁离子的存在下 ,将SAM上的甲基转移到DBA 3位的氧上。色谱法测定反应产物 4 羟基 3 甲氧基苯甲酸 (4 OH 3 MBA)的生成量。人体红细胞中COMT活性的线性范围在 1U/mL～ 6 0U/mL ,最低检测限为 0 5U/mL(S/N≥ 5 ) ,方法的精密度良好 (平均RSD <10 % )。     

阴离子交换色谱-积分脉冲安培检测法分离测定烟草料液中的山梨醇和糖

采用阴离子交换色谱-积分脉冲安培检测法分离测定了烟草料液中山梨醇、葡萄糖、果糖、蔗糖和麦芽糖,研究了山梨醇和糖在阴离子交换色谱中的保留行为。采用优化的水和氢氧化钠二元梯度淋洗条件,CarboPac PA10阴离子交换色谱柱进行分离,积分脉冲安培检测器检测一次进样测定烟草料液样品中的山梨醇、葡萄糖、果糖、蔗糖和麦芽糖。各组分在测试条件下线性关系良好,线性范围为0.005～20 mg/L,检测限为0.2～1.0 μg/L,加标回收率为95.1%~102.4%,相对标准偏差为1.2%～1.9%。     

人参寡糖的固相甲基化研究及定量分析

以CH_3I为甲基化试剂、NaOH为填料的填充柱对麦芽三糖进行固相甲基化,利用液相色谱-质谱(LC-MS)检测甲基化产物并优化甲基化条件。最优条件为4.6 mm×150 mm的不锈钢填充柱,流速40μL/min和柱温25℃,在此条件下分别对麦芽糖、蔗糖、蔗果四糖及人参寡糖提取物进行固相甲基化,并以蔗果四糖为例进行方法学考察。蔗果四糖的线性范围为10.8~5400 ng/mL,r~2=0.9974,平均加标回收率为122.5%。     

测定麦芽糖转糖苷反应体系组成的高效液相色谱法

建立了分析低聚异麦芽糖组成的高效液相色谱法，采用Spherisorb-NH2色谱柱，示差折光检测器，乙腈－水（体积比70：30）为流动相，外标法定量测定；结果显示各糖质量浓度在0．1－10g/L范围内与峰面积呈良好线性关系，相关系数为0．999 0－0．999 7；应用法跟踪了pH5．0的柠檬酸－柠檬酸钠缓冲溶液中以α－葡萄糖转苷酶为催化剂在58℃温度下的麦芽糖转糖苷反应，分析了反应体系组成随时间的变化，得到了上述反应条件下麦芽糖最大限度地转化为低聚异麦芽糖的最佳反应时间为24h；该法快捷、简单、准确，可用于低聚异麦芽糖生产的质量控制。     

基于酶触发的点击化学反应测定β-葡萄糖苷酶活性

建立了一种新的β-葡萄糖苷酶活性定量检测的方法,其原理是通过β-葡萄糖苷酶对2-O-β-D-葡萄糖基-L-抗坏血酸的特异性水解,释放出抗坏血酸来还原Cu(II),原位生成的Cu(I)催化荧光较弱的香豆素和苄基叠氮进行环加成点击反应,产生高荧光强度的三氮唑,从而利用荧光光谱检测体系荧光强度的变化,反映出β-葡萄糖苷酶的活性.实验结果显示,在1～40U/L范围内,荧光强度增强程度与β-葡萄糖苷酶活性呈线性关系,可实现定量检测,其最低检测限为0.456 U/L.     

高效液相色谱法测定生物转化反应液中N,N'-乙二胺二琥珀酸

建立了高效液相色谱测定生物转化反应液中N,N'-乙二胺二琥珀酸（EDDS）含量的分析方法。采用InertSustain AQ-C₁₈色谱柱（250 mm×4.6 mm,5 μm）,以体积分数25%的甲醇水溶液（含有1.0 g/L一水乙酸铜、2.0 g/L四丁基氢氧化铵,以磷酸调节pH至2.80）为流动相,流速为1.0 mL/min,柱温为30℃,进样量为20 μL,检测波长为254 nm。该方法可在8 min内分离EDDS及其生物合成相关物质（苹果酸、柠檬酸、乙二胺四乙酸（EDTA）和富马酸）,且峰形良好。EDDS在0.06~0.6 g/L范围内线性线性关系良好（相关系数（r）为0.9995）,平均回收率为100.39%（n=9,RSD=1.15%）。EDDS生物合成反应液中EDDS含量为0.25 g/L,大部分底物被转化为苹果酸（36.56 g/L）;而EDDS的水解反应中富马酸产生较少,形成了3.05 g/L的苹果酸。该方法简便快速,灵敏可靠,适用于EDDS生物合成的研究。     

超滤亲和结合液相色谱-质谱联用和分子对接技术筛选毛菊苣种子中高亲和性α-葡萄糖苷酶抑制剂

采用超滤亲和结合液相色谱-质谱联用(UF-LC-MS) 和分子对接技术筛选毛菊苣种子中高亲和α-葡萄糖苷酶抑制剂.以4-硝基苯-α-D-吡喃葡萄糖苷(PNPG)为底物,阿卡波糖为阳性对照,评价毛菊苣种子提取物对α-葡萄糖苷酶的抑制活性,其中阿卡波糖IC50为0.003 mg/mL,毛菊苣种子IC50为0.447 mg/mL.利用UF-LC-MS技术对毛菊苣种子提取物进行筛选鉴定,获得4种化合物;通过Autodock软件筛选出2种与α-葡萄糖苷酶有较高亲和力的化合物,分别是绿原酸和异绿原酸A.结合体外酶活实验,验证了绿原酸、异绿原酸A对α-葡萄糖苷酶的抑制活性.结果表明,各化合物对α-葡萄糖苷酶的抑制活性由大到小依次是:阿卡波糖>异绿原酸A>绿原酸,其中异绿原酸A与阿卡波糖抑制率相近.     

2-Carboxy-1-naphthyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase

A new substrate, 2-carboxy-1-naphthyl phosphate (CNP), was developed for the fluorimetric determination of alkaline phosphatase (ALP) activity. The product of the enzyme reaction is 1-hydroxy-2-naphthoic acid (HNA), which is a strong fluorescent product. The amount of HNA generated is proportional to ALP activity. Optimal conditions for the determination of ALP were investigated. The linear range and detection limit for the determination of ALP are 0.01-4.8 U/L and 7.44 mU/L, respectively. This method is simple, practical and can be successfully applied to assess ALP in human serum with good accuracy and precision. The results were evaluated by comparison with a standard colorimetric assay using p-nitrophenyl phosphate as ALP substrate.     

荧光光度法测定血清中碱性磷酸酶

合成了一种新的底物水杨酸磷酸酯 (SP),用于荧光光度法测定血清中碱性磷酸酶(ALP)活力.在37.0 ℃的Tris-HCl缓冲液(pH 9.0)条件下, 碱性磷酸酶作用于荧光底物SP,水解生成强荧光产物水杨酸(SA),生成的SA的量与参与反应的ALP 的活力成正比.据此建立了荧光光度法测定血清中碱性磷酸酶活力的新方法.测定碱性磷酸酶的线性范围是0.03～6.00 U/L,检测限为7.04 mU/L.本方法适用于血清中碱性磷酸酶的测定.测定结果与临床上常用的以对硝基苯磷酸酯为底物的分光光度法相比,无显著性差异.     

Determination of lipase activity in porcine pancreas and clinical analysis of lipase in human serum with surface acoustic wave enzyme sensor system

A new kind of surface acoustic wave (SAW) enzyme sensor system has been applied to the assay of extracted lipase activity based on triacetin solution as substrate. A linear relationship between the frequency change and the enzyme activity up to 500 U/L has been obtained, and the detection limit has been evaluated to 0.3 U/L. Kinetic parameters of the extracted lipase as well as that of the standard enzyme have been estimated. The effects of temperature, pH value and surfactants have been investigated. A comparison between SAW sensor system and the conductometric method has been carried out. The technique has been applied to the determination of lipase activity in serum samples. Received: 17 January 1995 / Revised: 9 March 1995 / Accepted: 16 March 1995 Correspondence to: Shouzhuo Yao     

Determination of acetylcholinesterase and butyrylcholinesterase activity without dilution of biological samples

Two cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are known. The enzymes are important in the body and alteration of their activity has significant use in the diagnosis of poisoning, liver function, etc. Currently available methods for the determination of cholinesterases have some major drawbacks including various interferences and the inability to be used for decreasing the enzyme activity in the presence of reversible inhibitors due to sample dilution; hence, a method for dilution free assay of cholinesterases is desired. Here, microplates were modified with indoxylacetate (100 µL of 10 mmol L^?1 solution) and used for cholinesterases assay after drying at 37°C. The fact that indoxylacetate remains stable in dry state and serves simultaneously as a chromogen and substrate provide good prerequisites for the method. The limit of detection for BChE was 0.71 U while that for AChE was 2.8 U per a 100 µL sample (solution of enzyme or plasma sample). The limit of detection is low enough to allow standard examination of cholinesterasemia. The two cholinesterases can be distinguished from each other using selective inhibitors such as donepezil and iso-OMPA. The new method was also successfully validated for the standard Ellman’s assay using plasma samples with BChE activity adjusted by carbofuran. The new method based on indoxylacetate seems promising for routine tests.     

高效液相色谱法测定血管紧张素转化酶抑制剂的活性

建立了体外直接测定血管紧张素转化酶抑制剂活性的高效液相色谱分析方法。以马尿酰-组氨酰-亮氨酸为反应底物,血管紧张素转化酶为催化剂,反应所生成的马尿酸为测定指标,未加酶抑制剂的反应为空白对照。使用ZORBAX SB-C18色谱柱(4.6 mm i.d.×150 mm,填料粒径5　μm),柱温25 ℃,流动相为乙腈-超纯水(体积比为25∶75,各含0.05%（体积分数）三氟乙酸及0.1%（体积分数）三乙胺),流速0.5 mL/min,检测波长228 nm。在马尿酸浓度为0.005～1.000 mmol/L时,马尿酸浓度与其峰面积呈良好的线性关系(r＝0.9999),最小检测限为0.50 μmol/L;该方法对马尿酸的回收率为99.48%～105.64%,相对标准偏差(RSD)为2.20%(n＝6)。该方法可适用于血管紧张素转化酶抑制剂活性的体外测定,具有操作简便、精密度和准确性高的特点,为降血压药物的研制提供了方便可靠的检测手段。     

Optimization of a microwave-coupled enzymatic digestion process to prepare peanut peptides

The best enzyme to prepare peanut peptides, papain, coupled with microwave irradiation was selected from five common proteases according to the results of the yield of peanut peptides [nitrogen solution index (NSI) in trichloroacetic acid (TCA), TCA-NSI] and the degree of hydrolysis (DH). The main factors that influenced the microwave-coupled enzymatic digestion method were optimized by response surface analysis. The optimal conditions obtained were as follows: microwave extraction time, 9.5 min; power, 600 W; substrate concentration, 4%; enzymatic reaction temperature, 50 °C; enzyme quantity, 6,500 U/g; pH, 7.1 (phosphate buffer, 0.05 mol/L). Under these conditions, TCA-NSI was 62.00% and DH was 25.89%, which is higher than that obtained with either protease hydrolysis or microwave hydrolysis alone.     

Catalase biosensor for the determination of hydrogen peroxide,fluoride and cyanide

The enzyme catalase, which catalyses the decomposition of hydrogen peroxide to oxygen and water, was immobilized in a membrane by entrapping it in polyacryl amide and contacted to a Clark-type oxygen electrode. With the resulting catalase biosensor it was possible to detect the substrate hydrogen peroxide and the inhibitors fluoride and cyanide in phosphate buffer.The sensor was integrated into a flow system. In the concentration range from 5–200 mg/l a linear dependence of the peak height on the hydrogen peroxide concentration was obtained. The average decrease in activity during 30 days of storage at 6 °C was 17%. Fluoride and cyanide could be determined by measuring the inhibition of the enzymatic reaction in the same flow system. The analysis was executed in three steps; namely determination of the original activity by pumping substrate solution, inhibition of the enzyme by pumping inhibitor solution, and determination of the activity after the inhibition.The decrease in activity correlated with the inhibitor concentration of the sample, but a linear dependence was not found. The inhibition of fluoride and cyanide was both reversible, the enzyme membrane could be reactivated completely by pumping substrate solution. The detection limit was 1 mg/l for fluoride and 1.5 mg/l for cyanide.     

Development of nitrilase-mediated process for phenylacetic acid production from phenylacetonitrile

This study aimed at developing an efficient biotransformation process for phenylacetic acid production from phenylacetonitrile by using recombinant Escherichia coli M15 harboring a double mutant MG nitrilase (I113M/Y199G) from Burkholderia cenocepacia J2315. A yield of 2310 U/mL nitrilase was obtained by fermentation after the optimization of cultivation conditions, with a specific activity of 64 U/mg dcw. The MG nitrilase showed high substrate tolerance and completely hydrolyzed 100 mM phenylacetonitrile in 30 min under optimal conditions. To alleviate substrate inhibition, periodic or continuous batch-feeding of substrate was used during the biotransformation. Up to 164 g/L substrate was completely hydrolyzed in 9 h with continuous batch-feeding using resting cells, corresponding to 400 U/mL of nitrilase activity, and leading to production of 163.4 g/L phenylacetic acid. The hydrolysis process has potential application for phenylacetic acid production on a large scale.     

Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant K_m was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn²⁺ and K⁺ could enhance the activity of PPO; that Ba²⁺ and Ca²⁺ could inhibit the activity of PPO; and that Cu²⁺ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.