固定化细胞酶法拆分N-乙酰-D,L-3-甲氧基丙氨酸

利用氨基酰化酶固定化细胞酶法拆分了N-乙酰-D,L-3-甲氧基丙氨酸. 考察了温度、pH值、底物浓度、金属离子和拆分时间对酶促反应的影响. 确定了氨基酰化酶固定化细胞手性拆分N-乙酰-D,L-3-甲氧基丙氨酸的最佳工艺条件为pH=7.0, 反应温度50 ℃及底物浓度500 mmol/L. 10^-4 mol/L的Co²⁺和Mg²⁺对氨基酰化酶有显著激活作用, Cu²⁺和Zn²⁺对酶促反应有明显抑制作用. 在最佳条件下, 氨基酰化酶固定化细胞对N-乙酰-L-3-甲氧基丙氨酸的摩尔转化率达96%.     

Interactions between carbonic anhydrase and some decarboxylating enzymes as studied by a new bioelectrochemical approach.

This work presents the results of a study, carried out by recently developed amperometric bioelectrodes, on the interactions between carbonic anhydrase (CA) and the decarboxylating enzymes arginine decarboxylase (ADC), L-lysine decarboxylase (LDC), and L-ornithine decarboxylase (ODC). These are all pyridoxal-phosphate dependent enzymes and catalyze the decarboxylation reaction of the respective amino acids, to give carbon dioxide and the corresponding diamine (agmatine, cadaverine, and putrescine, respectively). The rate of each decarboxylase catalyzed reaction was measured by monitoring the production of the respective diamine by a plant tissue diamino oxidase (DAO) based bioelectrode. DAO is the enzyme which catalyzes the oxidation of agmatine, cadaverine, and putrescine with the production of NH and H2O2. DAO-based bioelectrodes consist of an amperometric H2O2 electrode, coupled to the biocatalytic membrane formed by a whole plant tissue (lentil cotyledon) containing the enzyme DAO, immobilized on a dialysis membrane by polyazetidine prepolymer (PAP). The bioelectrodes were calibrated and characterized in standard solutions of agmatine, cadaverine, and putrescine. Kinetic studies to measure decarboxylase activity were performed in the presence of different concentrations of ADC, LDC, and ODC, resulting in a lowest detection limit of 10, 25, and 10 U l(-1), respectively. The effect of bovine CA II (bCAII) was evaluated in the presence of 500 U l(-1) of each decarboxylase, showing a marked increase of the rate of the decarboxylation reaction. These results suggest that (i) CA can be used to enhance the performance of decarboxylase-based biosensors, and (ii) it possibly plays further physiological roles, acting synergistically, at specific cellular and subcellular sites, with low-activity decarboxylating enzymes.     

壳聚糖固定化乳酸脱氢酶的制备及酶学性质研究

以磁性壳聚糖作为载体,戊二醛作为交联剂,对乳酸脱氢酶(LDH)进行固定化.固定化的最适条件为:戊二醛浓度6%,pH值7.5,酶的偶联时间2 h.对游离及固定化LDH酶学性质的研究表明,酶促反应的最适pH值为9.2,最适温度分别为37℃和50℃,对乳酸的表观米氏常数分别为1.6 mmol/L和0.9 mmol/L.游离酶和固定化酶在40℃放置150 min后,其活力分别为最初的56.5%和76.1%.固定化酶在4℃贮存4周后,活力仍保留50%以上.固定化酶在室温下与底物重复反应6次后,活力仍保留60%以上,说明固定化酶具有较好的热稳定性、贮存稳定性和复用性.     

Effect of transmembrane Ca2+ gradient on conformation and enzyme activity of reconstituted adenylate cyclase.

Adenylate cyclase from bovine brain cortex was reconstituted into asolectin liposomes with (500-fold) or without transmembrane Ca2+ gradient. The enzyme activity of four types of proteoliposomes (the active center of enzyme exposing outside) was compared. The highest adenylate cyclase activity was observed in the vesicles with outside lower Ca2+ concentration (approximately 10(-6) mol/L, similar to the physiological condition). If the transmembrane Ca2+ gradient was in the inverse direction (i.e. outside higher Ca2+ concentration, 0.5 mmol/L), a lowest enzymatic activity would appear. The difference in enzymatic activity between the two types of proteoliposomes could be diminished following the addition of Ca2+ ionophore A23187. Proteoliposomes without transmembrane Ca2+ gradient exhibited intermediate activities. The conformation difference of adenylate cyclases in the above-mentioned proteoliposomes was also detected by measuring intrinsic fluorescence and fluorescence quenching with KI.     

Determination of agmatine, arginine, citrulline and ornithine by reversed-phase liquid chromatography using automated pre-column derivatization with o-phthalaldehyde

A method is presented for the pre-column derivatization of agmatine, arginine, citrulline or ornithine with o-phthalaldehyde-2-mercaptoethanol, and subsequent separation of the derivatives by reversed-phase liquid chromatography. Fluorescent response is linear from 10 to 150 pmol of injected analyte and detection limits range from 28 to 100 fmol. Response factors relative to the internal standard, homocysteic acid, were 1.16 (agmatine and arginine), 1.03 (citrulline) and 0.34 (ornithine). The applicability of the method to the measurement of arginase, arginine deaminase, arginine decarboxylase and other enzyme activities in bacterial extracts was examined.     

中空纤维膜固定化甲酸脱氢酶催化CO_2合成甲酸

以紫外光表面接枝改性的聚乙烯(PE)中空纤维膜为载体,采用共价结合的方式固定化甲酸脱氢酶(FDH),考察了CO2通入方式、溶液pH值、缓冲液种类和还原型烟酰胺腺嘌呤二核苷酸(NADH)的浓度对酶催化CO2合成甲酸反应的影响.结果表明,与加压法相比,CO2鼓泡法更有利于甲酸的生成;磷酸盐缓冲液优于Tris-HCl和盐酸三乙醇胺缓冲液;体系pH值对反应的影响较大,固定化FDH的最佳pH值仍为6.0,但pH耐受性增强;随着辅酶NADH浓度的增加,反应初速度加快,收率下降;游离酶和固定化酶的最大酶活分别为0.246和0.138mmol/(L.h);固定化FDH在4℃贮存两周后活性仅下降4%,而游离酶活性下降50%.FDH催化膜重复利用10次后,活性没有明显降低.     

乙酰鸟氨酸脱乙酰酶固定化细胞拆分D,L-缬氨酸

报道了一种利用具有乙酰鸟氨酸脱乙酰酶活性的固定化细胞拆分D,L-缬氨酸的新方法. 该酶促反应最适条件: pH=6, 反应温度50 ℃, 底物N-乙酰-D,L-缬氨酸浓度200 mmol/L, 固定化细胞用量0.2 g/mL(或100 U/mL). 0.1 mmol/L CoCl₂条件对该酶促反应有显著的激活作用. 在以上条件下反应2～3 h, 测得产物L-缬氨酸浓度95 mmol/L. 该固定化细胞连续10次使用, 平均转化率为90.8%(以N-乙酰-L-缬氨酸计), 显示出了良好的工业化应用前景.     

聚碳酸乙烯撑酯与双端氨基聚乙二醇交联体系固定化酶载体的合成与表征

将聚碳酸乙烯撑酯(PVCA)与α,ω-双端氨基聚乙二醇(H₂N-PEG-NH₂)溶于DMF,于液蜡中进行交联反应制得亲水性固定化酶载体,将其与胰蛋白酶进行偶联反应制备了固定化胰蛋白酶.酶蛋白的比活力及其于载体上的结合量与反应条件有关,当w(PVCA)/w(H₂N-PEG-NH₂)为0.5时,二者均处于最高值.此固定化酶酶促反应的最适pH值和Km值均较之溶液酶有显著提高,但二者的最适酶促反应温度却相当一致.     

近平滑假丝酵母细胞催化乙酰基三甲基硅烷不对称还原反应

 采用近平滑假丝酵母细胞用于催化乙酰基三甲基硅烷不对称还原反应, 可高选择性地生成 (R)-1-三甲基硅乙醇. 结果表明, 固定化于海藻酸钙的细胞催化该反应的产物收率比游离细胞的高. 不同辅底物对该反应的影响显著, 以葡萄糖为辅底物时, 反应的初速率较快, 产物收率较高. 该反应的最适条件为: 辅底物 (葡萄糖) 浓度 110 mmol/L, 振荡速度 180 r/min, 缓冲液 pH 值 6.0, 反应温度 30 oC, 底物浓度 20 mmol/L. 在此反应条件下反应的初速率、产物收率和产物的 ee 值分别为 11.4 μmol/h, 96.5% 和 99.9%.     

Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase

Whole cells ofTrigonopsis variabilis were immobilized by entrapment in Ca²⁺-alginate and used for the production of α-keto acids from the corresponding D-amino acids. The D-amino acid oxidase within the immobilized cells has a broad substrate specificity. Hydrogen peroxide formed in the enzymatic reaction was efficiently hydrolyzed by manganese oxide co-immobilized with the cells. The amino acid oxidase activity was assayed with a new method based on reversed-phase HPLC. Oxygen requirements, bead size, concentration of cells in the beads, flow rate, and other factors were investigated in a “ trickle-bed ” reactor.     

醇脱氢酶在反胶束溶液中的催化动力学性质

以十六烷基三甲基溴化铵(CATB)-辛烷-己醇反胶束体系对醇脱氢酶(ADH)进行固定化,试验了含水量、酶液pH值、CTAB和己醇浓度对ADH固定化的影响。对游离酶和固定化酶的催化动力学性质研究表明：酶促反应的最适pH值分别为8.2和8.8,最适温度分别是31℃和20℃,对乙醇的米氏常数Km分别为12mmol/L和7.4mmol/L。在30℃时,游离酶存放150min后失活90%,固定化酶失活50%,表明反胶束固定化ADH有较好的热稳定性。     

有机溶剂/缓冲液双相体系中固定化Acetobacter sp.CCTCC M209061细胞催化1-(4-甲氧基)-苯基乙醇不对称氧化反应

在有机溶剂/缓冲液双相体系中, 利用固定化醋酸杆菌Acetobacter sp.CCTCC M209061细胞高对映体选择性地催化1-(4-甲氧基)-苯基乙醇(MOPE)的不对称氧化反应, 成功地拆分外消旋MOPE得到对映体纯(S)-MOPE. 与游离细胞相比, 固定化细胞催化反应速度有所降低, 但其稳定性(包括操作稳定性、 热稳定性和储藏稳定性)明显提高. 固定化细胞连续使用10批次(每批次12 h)后, 仍能保留其初始催化活性的58%以上, 而游离细胞仅保留约20%的相对活性. 在所考察的不同有机溶剂中, 正己烷不仅能较好地溶解底物, 而且对细胞的生物相容性相对较好, 因而提高了反应底物浓度、 反应初速度、 对映体回收率及残留底物e.e.值, 是反应体系中最适宜的有机相. 该反应的最适宜正己烷体积分数为60%, 辅底物为50 mmol/L丙酮, 底物浓度为40 mmol/L, 缓冲液pH=6.5, 反应温度为30 ℃; 在此条件下, 反应初速度为80.4 μmol/min, 反应12 h后, 对映体回收率和残留底物e.e.值分别为51.0%和99.9%, 明显好于水单相反应体系.     

Biohydrogen Production from Cheese Whey Wastewater in a Two-Step Anaerobic Process

Cheese whey-based biohydrogen production was seen in batch experiments via dark fermentation by free and immobilized Enterobacter aerogenes MTCC 2822 followed by photofermentation of VFAs (mainly acetic and butyric acid) in the spent medium by Rhodopseudomonas BHU 01 strain. E. aerogenes free cells grown on cheese whey diluted to 10 g lactose/L, had maximum lactose consumption (～79%), high production of acetic acid (1,900 mg/L), butyric acid (537.2 mg/L) and H(2) yield (2.04 mol/mol lactose; rate,1.09 mmol/L/h). The immobilized cells improved lactose consumption (84%), production of acetic acid (2,100 mg/L), butyric acid (718 mg/L) and also H(2) yield (3.50 mol/mol lactose; rate, 1.91 mmol/L/h). E. aerogenes spent medium (10 g lactose/L) when subjected to photofermentation by free Rhodopseudomonas BHU 01 cells, the H(2) yield reached 1.63 mol/mol acetic acid (rate, 0.49 mmol/L/h). By contrast, immobilized Rhodopseudomonas cells improved H(2) yield to 2.69 mol/mol acetic acid (rate, 1.87 mmol/L/h). The cumulative H(2) yield for free and immobilized bacterial cells was 3.40 and 5.88 mol/mol lactose, respectively. Bacterial cells entrapped in alginate, had a sluggish start of H(2) production but outperformed the free cells subsequently. Also, the concomitant COD reduction for free cells (29.5%) could be raised to 36.08% by immobilized cells. The data suggest that two-step fermentative H(2) production from cheese whey involving immobilized bacterial cells, offers greater substrate to- hydrogen conversion efficiency, and the effective removal of organic load from the wastewater in the long-term.     

Incorporation of glucose oxidase into Langmuir-Blodgett films based on Prussian blue applied to amperometric glucose biosensor

Glucose oxidase (GOx) was immobilized in the organic-inorganic Langmuir-Bldogett (LB) films consisting of octadecyltrimethylammonium (ODTA) and nanosized Prussian blue (PB) clusters. The amperometric glucose biosensors based on the LB films were fabricated and tested. It was found that the sensors exhibited a clear response current under an applied voltage of 0.0 V (vs Ag/AgCl). The linearity of current density versus glucose concentration was confirmed below 15 mmol/L concentration. This is the first observation of biosensor function of the hybrid organic-inorganic LB films. The successful preparation of glucose sensors operating at the very low potential indicates that the adsorbed PB clusters in the LB films act as an electrocatalyst for the electrochemical reduction of hydrogen peroxide, which is the final product of the enzymatic reaction sequence. The observed low potential applicability is estimated to inhibit the responses of interferants such as ascorbic acid, uric acid, and acetominophen. It was also found that an electrostatic interaction between positively charged ODTA+ and the adsorbed species of both GOx and PB provided a stabilized adsorption state in the LB films. Such stable immobilization contributes to the steady amperometric response current observed in the present ODTA/PB/GOx LB films.     

单分散磁性纳米粒子固定化猪胰脂肪酶的研究

借助溶热法制备了一种亲水及生物相容良好的Fe3O4磁性纳米粒子,用γ-氨丙基三乙氧基硅烷直接对所得磁性粒子表面改性,然后用戊二醛偶联法制得了固定化猪胰脂肪酶.表征研究显示,所得磁性粒子粒径约200 nm,具有良好的单分散性和磁响应性.考察了戊二醛浓度、给酶量和反应时间对脂肪酶固定化过程的影响,并通过游离酶与固定化酶的比...     

基于次血红素六肽的过氧化氢和葡萄糖灵敏比色方法

采用次血红素六肽(DhHP-6)催化H₂O₂氧化4-氨基安替吡啉-氯代苯酚显色体系,建立了测定H₂O₂和葡萄糖的方法。 研究了pH值、底物浓度和DhHP-6浓度对实验的影响,检测了比色方法的反应线性、稳定性、相关性和回收率。 在最佳反应条件下,DhHP-6在不同时间及温度条件下,活性要优于过氧化物酶(POD);DhHP-6催化H₂O₂ 的米氏常数(K_m)和最大反应速率(v_max)分别为0.171 mmo/L和4.22×10^-6 mol/s;H₂O₂响应的线性范围为0.39~25.0 mmol/L;高、中、低 3水平测定的变异系数(CV)和加标回收率分别在1.29%~2.16%和94.5%~101.1%之间;与葡萄糖商品试剂盒比较相关系数R²=0.9946;36例血液样品中的葡萄糖浓度在4.26~17.48 mmol/L之间。 与葡萄糖检测商品试剂盒之间的两组数据经统计差异不显著(P>0.05)。 该方法是一种简单、廉价、方便、灵敏的比色测定方法。     

羊肝源穿山龙薯蓣皂苷-α-L-鼠李糖苷酶的分离及其动力学特性

对羊肝中含有的水解穿山龙薯蓣皂苷鼠李糖糖基的穿山龙薯蓣皂苷-α-L-鼠李糖苷酶进行了分离、纯化, 并对其动力学特性进行了研究. 结果表明, 粗酶液经DEAE-Cellulose离子交换层析柱纯化后, 其比活提高了19.9倍. 在pH＝6.8、反应温度为42 ℃、反应时间为8 h和底物浓度为23 mmol/L的条件下, 该酶达到其最高活力. 在10—200 mmol/L范围内, Fe3+和Cu2+对酶活力有明显的抑制作用, Mg2+和Zn2+对酶活力有微弱的激活作用, 而Ca2+对酶有较强的激活作用. 采用SDS-PAGE方法测得酶蛋白分子量为71000. 选择穿山龙薯蓣皂苷、人参皂苷Re和芦丁为酶反应底物, 进行酶的底物专一性研究发现, 穿山龙薯蓣皂苷-α-L-鼠李糖苷酶对其底物具有高度专一性.     

固定化过氧化氢酶的制备及其抗氧化作用

以烟用醋酸纤维的生物化学改性为目标,研究了以壳聚糖为载体时,吸附交联固定化过氧化氢酶的条件,并考察了固定化酶的性质。结果表明,固定化的最佳条件为:加酶量(酶活2×104C IU/m l)6m l,3%壳聚糖20m l,乙二醛浓度6%(w/v),交联剂用量100m l,吸附时间0.5 h,交联时间2.5h,酶活收率可达42.9%。过氧化氢酶固定化后,动学参数Km值为61.7mmol/L;对活性氧具有较好清除作用。     

毛细管胶束电动色谱法测定血管紧张素转化酶的活性

 建立了应用毛细管胶束电动色谱 (MECC)灵敏、快速的测定血管紧张素转化酶 (ACE)活性的方法。通过对电压、上样时间、电极缓冲液体系等影响因素的优化 ,探讨了方法的可行性 ,确立了最佳测定条件 (电压 :8 1kV ;上样时间 :1s;电极缓冲液 :2 0mmol/L硼酸盐缓冲液 (pH 9 0 ,含 5 0mmol/LSDS) ;检测波长 :2 2 8nm)。方法的最低ACE活性检测限为 5pmol/min(以 2倍的信噪比计 )。     

硫酸铜诱导烟草多酚氧化酶抗变性的研究

In order to investigate the interaction between exogenous Cu2+ and Polyphenoloxidase (PPO) from Nictiana Tobacum, the Cu2+-induced resistant-denaturation against guanidine hydrochloride(Gdn-HCl) was studied by the following enzymatic activity assay, fluorescence spectroscopy and circular dichroism (CD). The results show that the GdnHCl-induced unfolding is a two-state process with no detectable inter mediate state in the absence of exogenous Cu2+, while the GdnHCl-induced unfolding in the presence of 10.0 mmol/L exogenous Cu2+ follows a three-state transition with an intermediate state, which results from the fact that Cu2+ increases the structural stability of native PPO and its intermediate. In terms of enzymatic activities, 6 mol/L GdnHCl makes PPO lose 81.4% of its original activity after 5 min, inactivate completely after 30 min, while in the presence of 10.0 mmol/L exogenous Cu2+, only 39.4% and 75.1%, after 5 and 30 min respectively. According to the CD measurements, the relative average fractions of α-helix, anti-parallel2-sheet, 2-turn/parallel 2-sheet, aromatic residues and disulfide bond, and random coil/γ-turn are 1.1%, 3.8%, 3.3%, 7.5% and 84.3%, respectively, in the 6.0mol/L GdnHCl containing no CuSO4, but34.2%, 13.7%, 21.0%, 9.5% and 21.6%, respectively, in the same concentration of Gdn-HCl containing 10mmol/L CuSO4.