巨大芽孢杆菌青霉素G酰化酶在新型环氧载体ZH-HA上的固定化

巨大芽孢杆菌青霉素G酰化酶共价结合在新型环氧-氨基型载体ZH-HA 上,通过对酶浓度、固定化时间、pH以及缓冲液浓度等条件的考察,确定了最优固定化条件:50 mg比活力6000 U/g的巨大芽孢杆菌青霉素G酰化酶蛋白和1g ZH-HA悬浮于pH 9.01 mol/L磷酸缓冲液,室温搅拌6 h,制得固定化巨大芽孢杆菌青霉素G酰化酶,活力2126 U/g湿载体,活力回收率7.67%.比较研究了固定化酶与原酶性质,原酶最适温度45℃,最适pH为8.0.固定化酶则分别是50℃和9.0,分别比溶液酶偏移5℃、1.0个pH单位.经过40批连续水解青霉素G钾盐,固定化巨大芽孢杆菌青霉素酰化酶仍保持80%的活力,显示出良好的工作稳定性.     

菜籽蛋白酶解液制备N-酰化肽的研究(Ⅰ) ——酰化肽制备工艺的优化

对N-酰化肽的合成条件进行了研究,得出较佳的工艺条件为：温度10-25℃,酶解液与脂肪酰氯的摩尔比为0.7∶1,pH值为9.0。温度对氨基氨的转化率影响不大,在实际生产中可选择室温条件。pH值对反应的影响较大,pH9时比pH8时转化率提高近50%。氨基氮与酰氯的配比为0.7时,能使90%的氨基氮转化为酰化肽。     

菜籽蛋白酶解液制备N酰化肽的研究(Ⅰ)——酰化肽制备工艺的优化

对N 酰化肽的合成条件进行了研究 ,得出较佳的工艺条件为 :温度 1 0 2 5℃ ,酶解液与脂肪酰氯的摩尔比为0 .7:1 ,pH值为 9.0。温度对氨基氨的转化率影响不大 ,在实际生产中可选择室温条件。pH值对反应的影响较大 ,pH9时比 pH8时转化率提高近 50 %。氨基氮与酰氯的配比为 0 .7时 ,能使 90 %的氨基氮转化为酰化肽。     

固定化青霉素酰化酶的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键连接到醋酸纤维素载体上,制成的固定化青霉素酰化酶的表观活力达2000 u／g左右(PDAB法)。水解lO％(w／v)的青霉素G钾盐落液,使用30批,保留活力70％以上。6-氨基青毒烷酸(6-APA)总收率平均达88．37％。固定化青霉素酰化酶水解青霉素G的最适pH为9．95,最适温度为55℃,表观米氏常数为1.093×10-2mol／L,在pH 5．8-10．7,温度45℃以下酶的活力稳定。     

Identification and optimal degradation conditions for cellulase-degrading enzyme of a methanol-utilizing and cellulase-producing bacterium

采用刚果红染色法,从废弃矿山周边土壤中筛选出一株产纤维素酶的甲醇利用细菌,命名为xt - 04.形态特征、生理试验及16S rDNA序列和gyrB序列分析表明,该菌株属于Bacillus methylotrophicus.为提高该菌所产纤维素酶的降解能力,首先通过单因子实验考察了底物CMC -Na浓度、反应温度及缓冲液pH值对纤维素酶活力的影响;然后采用响应面分析法对影响纤维素酶活力的3个单因子进行了优化.结果表明,单因素实验得出的适宜反应温度、缓冲液pH和底物浓度分别为70℃、5.0和2％ (20 mg/mL);响应面法得出的最高酶活力条件:反应温度、pH和底物浓度分别为66.1℃、4.81和19.01mg/mL.在最优条件下,酶活力达到17.85 U/mL,比优化前的酶活力12.84 U/mL提高了39.01％.因此,鉴于这种纤维素酶能耐受较高温度和酸性条件,该菌株所产纤维素酶可能在工业中具有良好的应用前景.     

一株产纤维素酶的甲醇利用细菌的鉴定及其纤维素降解条件优化

采用刚果红染色法,从废弃矿山周边土壤中筛选出一株产纤维素酶的甲醇利用细菌,命名为xt-04。形态特征、生理试验及16SrDNA序列和gyrB序列分析表明,该菌株属于Bacillusmethylotrophicus。为提高该菌所产纤维素酶的降解能力,首先通过单因子实验考察了底物CMC—Na浓度、反应温度及缓冲液pH值对纤维素酶活力的影响;然后采用响应面分析法对影响纤维素酶活力的3个单因子进行了优化。结果表明,单因素实验得出的适宜反应温度、缓冲液pH和底物浓度分别为70℃、5．0和2％（20mg／mL）;响应面法得出的最高酶活力条件：反应温度、pH和底物浓度分别为66．1℃、4．81和19．01mg／mL。在最优条件下,酶活力达到17．85U／mL,比优化前的酶活力12．84U／mL提高了39．01％。因此,鉴于这种纤维素酶能耐受较高温度和酸性条件,该菌株所产纤维素酶可能在工业中具有良好的应用前景。     

功能基化聚丙烯酸甲脂固定化氨基酰化酶的研究

以功能基化聚丙烯酸甲酯为载体制备固定化氨基酰化酶时,载体制备时的交联度,致孔剂用量和混合致孔剂比例均会影峋酶的固定化效率。结果表明25％交联度和loo％致孔剂用量制备的载体最佳。用此载体固定化氨基酰化酶可提高酶对高底物浓度、离子强度、温度、pH、有机溶剂和蛋白质变性剂的耐受能力。用固定化酶柱式反应器连续拆分N－乙酰基－DL－苯丙氨酸,使用一个月仍可保留90％的酶活力。     

活性炭吸附固定化粪产碱杆菌青霉素G酰化酶

目的：以活性炭为载体固定化粪产碱杆菌来源的青霉素G酰化酶,考察固定化酶的性质。方法：对影响酶固定化的因素优化筛选,确定有显著影响的因素：pH、离子强度、酶量、固定化时间进行L934的正交实验,获得最佳固定化条件,并对固定化酶的最适反应温度、pH及批次稳定性进行研究。结果：最佳固定化条件为：载体0.3g,酶量5mL,总反应体系为12mL,离子强度1mol/L,温度4℃,pH 7.0,固定化40h;最高固定化酶活性为135.9U/g湿载体。固定化酶性最适反应温度为55℃,最适pH为10,重复使用12次后没有活性损失。结论：活性炭吸附固定化青霉素G酰化酶的活性高,批次反应稳定,具有工业应用潜力。     

刺孢小克银汉霉菌体氨基酰化酶的性质研究

首次选育出有较高氨基酰化酶活性的菌株刺孢小克银汉霉（Cunninghamella echinulata）9980,并进行液体培养,比较了3种不同培养基中菌体细胞氨基酰化酶活性,考察了几种因素对菌体细胞酶活的影响。结果表明：蛋白胨培养基中菌体细胞酶活最高,达680u／g。菌体细胞酶活最适温度55％,最适pH7．0,最佳底物浓度为0．2mol／L,缓冲液中的无机离子对酶活有抑制作用,10^-3-10^-4mol／L的Co^2＋对酶活有激活作用。     

青霉素酰化酶在新型复合载体上的固定化研究

通过γ-氯丙基三甲氧基硅烷的媒介,将聚乙烯亚胺(PEI)化学偶联在硅胶微粒表面,制备了新型复合载体PEI/silica gel,然后通过双官能团试剂戊二醛的作用,将青霉素酰化酶固定在复合载体上;考察了戊二醛用量、pH值、固定化温度、固定化时间及给酶量等条件对固定化青霉素酰化酶表观活力、活性回收率等性能的影响;并通过测定复合载体在固定化前的ζ电位,探索了复合载体PEI/silica gel固定化酶的作用机理。研究结果表明,由于PEI分子链中含有大量胺基,共价键联与物理吸附相结合,使青霉素酰化酶被快速稳定地固定化,并具有高的催化活性与活力回收率。复合载体PEI/silica gel(0.5 g)固定青霉素酰化酶的适宜固定化条件为:固定化温度为30℃;固定化时间为14~15 h;戊二醛用量为1.2 mmol/g;pH=7.92;给酶量为0.1 mL/g。     

ISOELECTRIC POINTS FOR THE MYCELIUM OF FUNGI

1. Mycelium of Rhizopus nigricans when stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.0. 2. When grown on potato dextrose agar the reaction of which was varied with phosphoric acid the extent of colony growth of Rhizopus nigricans plotted against the initial Sörensen value of the agar produced a double maximum curve with the minimum between the two maxima at initial pH 5.2. 3. When grown in potato dextrose broth the reaction of which was varied with phosphoric acid the dry matter produced by Rhizopus nigricans plotted against the Sörensen value of the broth produced a double maximum curve with the minimum between the two maxima at initial pH 5.2 or average pH 4.9. 4. Mycelium of Rhizopus nigricans placed in buffer mixtures of 0.01 M phosphoric acid and sodium hydroxide of pH 4.1 to 6.3, changed the reaction in most cases toward greater alkalinity. 5. Mycelium of Fusarium lycopersici stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.5.     

New Buffers to Improve the Quantitative Real-Time Polymerase Chain Reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.     

New buffers to improve the quantitative real-time polymerase chain reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.     

Acid phosphatase activity of human gingival fibroblasts measured using a simultaneous coupling semi-permeable membrane technique

Summary Acid phosphatase activity has been measured in cultured human gingival fibroblasts using a validated histochemical simultaneous coupling semi-permeable membrane technique. The histochemical reaction was linear over a three hour incubation period and had a pH optimum of 5.0. The activity was not increased by prior exposure to hypotonic acetate buffer and was inhibited by fluoride and molybdate but not by formaldehyde. These results indicate that the semi-permeable membrane technique described may be used for observing and measuring acid phosphatase activity in cultured fibroblasts. From results obtained using inhibitors, it appears that in these cells most of the acid phosphatase observed is lysosomal. The absence of any activation of activity following pre-incubation with hypotonic buffer indicates that the method is not suitable for monitoring lysosomal membrane function.     

Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.     

Acid-catalyzed hydration of reduced nicotinamide adenine dinucleotide and its analogues.

The rate of the primary acid modification reaction of 1,4-dihydronicotinamide adenine dinucleotide (NADH) and 1,4-dihydro-3-acetylpyridine adenine dinucleotide (APADH) and their analogues has been studied over a wide pH range (pH 1-7) with a variety of general acid catalysts. The rate depends on [H+] at moderate pH and becomes independent of [H+] at low pH. This behavior is attributed to substrate protonation at the carbonyl group (pK of NADH = 0.6). The reaction is general acid catalyzed; large solvent deuterium isotope effects are observed for the general acid and lyonium ion terms. Most buffers cause a linear rate increase with increasing buffer concentration, but certain buffers cause a hyperbolic rate increase. The nonlinear buffer effects are due to complexation of the buffer with the substrate, rather than to a change in rate-limiting step. The rate-limiting step is a proton transfer from the general acid species to the C5 position of the substrate. Anomerization is not a necessary first step in the case of the primary acid modification reaction of beta-NADH, in which beta to alpha anomerization takes place.     

Liquid chromatographic method for simultaneous determination of mycophenolic acid and its phenol- and acylglucuronide metabolites in plasma

A simple, sensitive and reproducible HPLC method is presented for the simultaneous determination of mycophenolic acid (MPA) and its metabolites phenolic MPA-glucuronide (MPAG) and acyl glucuronide (AcMPAG) in human plasma. Sample purification requires protein precipitation with 0.1 M phosphoric acid/acetonitrile in the presence of Epilan D as an internal standard (IS). Separation was performed by reversed-phase HPLC, using a Zorbax SB-C18 column, 32% acetonitrile and a 40 mM phosphoric acid buffer at pH 3.0 as mobile phase; column temperature was 50 degrees C, flow rate 1.4 ml/min, and measurement by UV detection was at 215 nm (run time 12 min). The method requires only 50 microl plasma. Detection limits were 0.1 microg/ml for MPA and AcMPAG, and 2.0 microg/ml for MPAG, respectively. Mean absolute recovery of all three analytes was >95%. This analytical method for the determination of MPA and its metabolites is a reliable and convenient procedure that meets the criteria for application in routine clinical drug monitoring and pharmacokinetic studies.     

Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1.

1. The EPR spectrum at 15 degrees K of soybean lipoxygenase-1 in borate buffer pH 9.0 has been studied in relation to the presence of substrate (linoleic acid), product (13-L-hydroperoxylinoleic acid) and oxygen. 2. The addition of 13-L-hydroperoxylinoleic acid to lipoxygenase-1 at pH 9.0 gives rise to the appearance of EPR lines at g equals 7.5, 6.2, 5.9 and 2.0, and an increased signal at g equals 4.3. 3. In view of the effect of the end product on both the kinetic lag period of the aerobic reaction and the fluorescence of the enzyme, it is concluded that 13-L-hydroperoxylinoleic acid is required for the activation of soybean lipoxygenase-1. Thus it is proposed that the enzyme with iron in the ferric state is the active species. 4. A reaction scheme is presented in which the enzyme alternatingly exists in the ferric and ferrous states for both the aerobic and anaerobic reaction.     

Determination of fenofibric acid in human plasma using automated solid-phase extraction coupled to liquid chromatography

The pharmacokinetic studies of fenofibrate require a rapid, selective and robust method to allow the determination of fenofibric acid, its active metabolite, in different biological matrixes (such as plasma, serum or urine). A new fully automated method for the determination of fenofibric acid in plasma has been developed, which involves the solid-phase extraction (SPE) of the analyte from plasma on disposable extraction cartridges (DECs) and reversed-phase HPLC with UV detection. The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with octadecyl silica was first conditioned with methanol and pH 7.4 phosphate buffer. A 0.8-ml volume of diluted plasma sample containing the internal standard (sulindac) was then applied on the DEC. The washing step was performed with the same buffer (pH 7.4). Finally, the analytes were successively eluted with methanol (1.0 ml) and 0.04 M phosphoric acid (1.0 ml). After a mixing step, 100 μl of the resultant extract was directly introduced into the HPLC system. The liquid chromatographic (LC) separation of the analytes was achieved on a Nucleosil RP-8 stationary phase (5 μm). The mobile phase consisted of a mixture of methanol and 0.04 M phosphoric acid (60:40, v/v). The analyte was monitored photometrically at 288 nm. The method developed was validated. In these conditions, the absolute recovery of fenofibric acid was close to 100% and a linear calibration curve was obtained in the concentration range from 0.25 to 20 μg/ml. The mean RSD values for repeatability and intermediate precision were 1.7 and 3.9% for fenofibric acid. The method developed was successfully used to investigate the bioequivalence between a micronized fenofibrate capsule formulation and a fenofibrate Lidose™ formulation.     

高效液相色谱法测定必特螺旋霉素发酵液中有机酸

必特螺旋霉素是运用基因工程技术获得的工程茵产生的一组以异戊酰螺旋霉素为主要成分的多组分新型抗生素,其前体为乙酸、丙酸、丁酸和异戊酸等有机酸。本研究利用高效液相色谱法以0．01mol／L磷酸缓冲液（pH2．3）和甲醇为流动相,分别在反相C8（α-酮戊二酸、乙酸、柠檬酸、琥珀酸、丙酸）和C18（丁酸、异戊酸）柱上定量测定了必特螺旋霉素前体酸和三羧酸循环相关有机酸。所建立的测定方法的相对标准偏差为0．10％～0．42％,回收率为93．19％～102．08％。