从酵母菌中分离纯化超氧化物岐化酶

超氧化物岐化酶(SOD)由于具有消除氧自由基的功能,在医药、保健品中具有重要应用价值。我国目前SOD产品主要是从牲畜血液中提取,这个方法所用原料有限,SOD质量不稳定。80年代后,美国和日本先后开发了用发酵法生产SOD,大大地降低了生产成本。但由于SOD为胞内蛋白,因而发酵法产生SOD后提取工艺极为复杂,至少需要六步,一般经过细胞破碎、离心、盐析、透析、离子交换层析(2～3次)和凝胶层析等才能达到比活>3000u/mg。由于细胞破碎主要为机械法(如球磨和超声波法),胞内所有蛋白都释放出来,SOD比活只有10～30u/mg,因而后续SOD纯化     

从酵母菌中分离纯化超氧化物岐化酶

超氧化物岐化酶(SOD)由于具有消除氧自由基的功能,在医药、保健品中具有重要应用价值。我国目前SOD产品主要是从牲畜血液中提取,这个方法所用原料有限,SOD质量不稳定。80年代后,美国和日本先后开发了用发酵法生产SOD,大大地降低了生产成本。但由于SOD为胞内蛋白,因而发酵法产生SOD后提取工艺极为复杂,至少需要六步,一般经过细胞破碎、离心、盐析、透析、离子交换层析(2～3次)和凝胶层析等才能达到比活>3000u/mg。由于细胞破碎主要为机械法(如球磨和超声波法),胞内所有蛋白都释放出来,SOD比活只有10～30u/mg。     

固体发酵法制取植酸酶

对植酸酶产生菌进行了诱变育种,分离纯化;采用固体培养法生产植酸酶及对酶的热稳定性进行了研究,研究结果表明以麸皮为主要培养基成分,添加2%葡萄糖,1%（NH4）2SO4,0.02%MgCl2,40ml水,29℃恒温培养8d-10d后,其酶活可达5615u/g.ssc。用含CaCl22%的pH5.0的醋酸缓冲液抽提效果好,相对酶活达94%。经提取。粗酶粉活力可达80130u/g。温度升高70℃,处理1h,相对酶活为90%。     

改良的连苯三酚自氧化测定超氧化物歧化酶活性的方法

超氧化物歧化酶(superoxide dismutase简称SOD)的测活方法很多。目前,国内外采用的连苯三酚自氧化法,其测定缓冲液均用HCl配制,而Cl~-对SOD有一定的抑制作用。为了提高SOD对超氧阴离子(O_2~-)的竞争能力,本法改用K_2HPO_4KH_2PO_4缓冲液。实验表明,改良法不仅比在TrisHCl缓冲液中测定灵敏度提高了50％,而且因减少了SOD的加入量,使酶被O_2~-所饱和。实验还表明,SOD在自氧化速率30—65％范围内,酶的加入量与抑制率成线性关系,见图1。     

药用中性蛋白酶的研究

枯草杆菌(Baceillus subtilis) AS 1.398中性蛋白酶的粗酶(5万u／g),用自来水(1：10)抽提12小时,在这抽提液中加入20％(v／v)CaCl2溶液(在100ml水中加60g无水CaCl2)：再加25％固体硫酸铵使酶沉淀,过滤后得酶饼,用0．005MpH7．2磷酸缓冲液溶解,经Sephadex G-25柱脱盐,冷冻干燥得片状粉末,含酶活60一100万u/g,纯度提高4倍,本制剂具有较强的抗炎消肿及溶解纤维蛋白的作用。     

从蚯蚓中联合提取抗氧化酶SOD、CAT的方法研究

本研究采用闪式提取技术,固液比为1:4(m/V)的2.5 mmol/L pH 7.0磷酸缓冲液,提取转速5500 rpm,提取时间2 min,从蚯蚓体内提取出SOD、CAT,并通过羧甲基纤维素CM-22离子交换层析实现SOD和CAT的联合提取分离,SOD、CAT的活性回收率分别达到88.23%和69.5%。在纯化工艺中经过丙酮沉淀和柱层析技术得到蚯蚓SOD纯品,比活达到9352 U/mg,产物在SDS-PAGE上为单一条带,其亚基分子量约为17 kD;通过柱层析纯化了蚯蚓CAT,比活达到22606 U/mg。     

猪血Cu，Zn-SOD的分离纯化

用加热变性、盐析和凝胶过滤层析的方法从猪血中提取分离出有多种生理活性的Cu ,Zn SOD ,经过分析得知 ,其比活为 1 0 0 3 5u/mg、分子量为 3 1 70 0u的电泳纯单组分。     

少孢根霉RT—3胞外产超氧化物歧化酶的研究

利用少抱根霉RT－3行固态发酵,从培养物中可以直接拍提得到SOD。固体发酵培养物发酵前后的酶活分析表明少抱根霉RT－3可向胞外分泌SOD。酶抽提液中含有Cu、Zn型和Mh型SOD。对其初步纯化所得粗酶制剂比活力为444．8u／mg。酶的紫外吸收峰在258nnl。酶在60℃以下较稳定,活性稳定pH为5．5～9．5。     

少孢根霉RT-3胞外产超氧化物歧化酶的研究

利用少抱根霉RT－3行固态发酵,从培养物中可以直接拍提得到SOD。固体发酵培养物发酵前后的酶活分析表明少抱根霉RT－3可向胞外分泌SOD。酶抽提液中含有Cu、Zn型和Mh型SOD。对其初步纯化所得粗酶制剂比活力为444．8u／mg。酶的紫外吸收峰在258nnl。酶在60℃以下较稳定,活性稳定pH为5．5～9．5。     

普通脱硫弧菌产氢化酶条件及提取贮存因素研究

普通脱硫弧菌D-2氧化酶在培养基N上产量较高。金属螯合剂EDTA能增加菌体酶含量,其最适浓度为0.02g/L。碱性缓冲液(含有EDTA)在30℃、15分钟内能抽提约90%的周质氢化酶。该酶在低温、充氮条件下能保持很高的起始酶活,且牛血清白蛋白(BSA)能明显增加酶在空气中的稳定性,但还原的氢化酶纯样品氧失活比较明显。     

Selective release of superoxide dismutase from Saccharomyces cerevisiae with isopropanol

Summary A new and inexpensive approach by which the release and purification of intracellular protein can be carried out simultaneously has been developed. Alcoholic solvents were used to selectively release superoxide dismutase (SOD) from Saccharomyces cerevisiae. 90%(v/v) isopropanol was used to treat the cells for 120 minutes and then 50 mM phosphate buffer(pH7.0) was added after removal of the solvent by filtration. The recovery of SOD was up to 90% , while the specific activity of SOD increased 25 times compared to traditional methods such as ultrasonic method.     

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-monooxygenase

7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.     

pH-dependent interconversion of two forms of tyrosinase in human skin.

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.     

Spherical alginate granules formulated for quick-release active subtilisin

Novel attrition-resistant and spherical enzyme granules encapsulating active subtilisin were formed by emulsification of 2% alginate sol loaded with active enzyme, instantaneous gelation triggered through in situ release of Ca(2+) (internal gelation), particle separation, and finally acetone extractive drying. Granular subtilisin was highly active, readily dispersible, and mechanically robust. This technique serves as a new and attractive alternative to established enzyme granulation processes, such as fluid bed coating, extrusion followed by marumerization, drum granulation, or prilling, for use in industrial enzyme applications such as detergents, textile manufacturing, and food processing. The formulation and encapsulation conditions were optimized to maximize the resistance of the granule to compression and impact forces, consistent with enzyme release and particle dispersion in detergent solutions. Well characterized alginates, with specified guluronic/mannuronic acid (G/M) content and molecular weight, were used in the formulation. The characteristics of the resulting microspheres, including their size and distribution, morphology, shrinkage, compression resistance, impact strength, solubility and encapsulation yield, were examined. Spherical dry granules were formulated with a mean diameter of 500 microm with particle sizes ranging from 300 to 800 microm. Dry alginate granules were discrete, spherical, and glossy white and exhibited impact strength, compression resistance, and solubility difference dependent on composition. Reduced starch levels, high alginate concentration, low alginate molecular weight, and use of high guluronate alginates resulted in the lowest dust level and highest compression resistance. Subtilisin mass yields were approximately 50%, and specific activity yields ranged from 60% to 100%. A formulation consisting of 3% SG150 alginate, 10% starch, 10% TiO(2), and 1% CaCO(3) provided granules appropriate for use in detergent application.     

Purification and characterization of 7beta-hydroxysteroid dehydrogenase from rabbit liver microsomes

7beta-Hydroxysteroid dehydrogenase (7beta-HSD), a specific enzyme active in the metabolization of 7beta-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2'5'ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity. The specific activity of the purified enzyme was 276 nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was beta-NADP+. The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine-NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7beta-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7beta-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7beta-HSD had sequence similarity to rabbit type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD), indicating that 7beta-HSD may belong to the rabbit type I 11beta-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols.     

高产菊粉酶酵母筛选、发酵和酶学性质研究

筛选到1株菊粉酶高产克鲁维酵母菌株,采用酵母高密度细胞发酵方法,最高菊粉酶产量达到288.78u/mL,比80～90年代国际上报道的克鲁维酵母菊粉酶最高产量高6.8倍。该酶的菊粉酶/转化酶活性比为1/24.72;菊糖m=13.3mmol/L,蔗糖Km=62.6mmol/L;最适反应pH值为4.4,但在pH3.8～5.6的范围内均保持了较高的活性,相当于最适pH值下活性的90%;最适反应温度为55℃,在50～575℃范围内能够保持较高活性,50℃下酶的半衰期约为16h;外加Mg2+提高酶活性11.28%。     

Optimizing Immobilization and Stabilization of Feruloyl Esterase from Humicola Insolens and its Application for the Feruloylation of Oligosaccharides

Feruloyl esterase (FAE)-catalyzed esterification reaction is as a potential route for the biosynthesis of feruloylated oligosaccharides as functional ingredients. Immobilization of FAE from Humicola insolens on metal chelate-epoxy supports was investigated. The study of effects of immobilization parameters using response surface methodology revealed the significance of enzyme/support ratio (3.25-29.25 mg/g support), immobilization time (14-38 h), buffer molarity (0.27-1.25 M) and pH (4.0-8.0). The interactions between enzyme-to-support ratio/buffer molarity and enzyme-to-support ratio/pH were found to be critical for the modulation of the immobilization activity yield and the retention of specific activity, respectively. Optimum conditions for FAE-immobilization on metal chelate Sepabeads® EC-EP R were identified to be 22.75 mg FAE/g support, pH of 5.0, 27.7 h and buffer molarity of 0.86 M. At these conditions, an activity yield of 82.4%, a specific activity retention of 143.4%, and an enzyme activity of 395.4 μmol/min. g support were achieved. Further incubation of the immobilized FAE at pH 10.0 improved its thermostability. Increasing the pore size of the epoxy support improved the retention of FAE hydrolytic activity and the esterifying efficiency of the immobilized biocatalyst. Optimally immobilized and stabilized FAE on metal chelate-epoxy support retained up to 92.9% of the free enzyme feruloylation efficiency to xylooligosaccharides..     

Computational biotechnology: Prediction of competitive substrate inhibition of enzymes by buffer compounds with protein-ligand docking

In vitro enzymatic activity highly depends on the reaction medium. One of the most important parameters is the buffer used to keep the pH stable. The buffering compound prevents a severe pH-change and therefore a possible denaturation of the enzyme. However buffer agents can also have negative effects on the enzymatic activity, such as competitive substrate inhibition. We assess this effect with a computational approach based on a protein-ligand docking method and the HYDE scoring function. Our method predicts competitive binding of the buffer compound to the active site of the enzyme. Using data from literature and new experimental data, the procedure is evaluated on nine different enzymatic reactions. The method predicts buffer-enzyme interactions and is able to score these interactions with the correct trend of enzymatic activities. Using the new method, possible buffers can be selected or discarded prior to laboratory experiments.     

粪产碱杆菌青霉素G酰化酶在大肠杆菌中组成型表达及分离纯化

将粪产碱杆菌青霉素G酰化酶基因构建重组表达质粒pKKFPGA ,pKKFPGA再转化宿主菌DH5α,所得重组菌不需诱导便能高效表达青霉素G酰化酶 ,表达量达 2590u L ,比野生型粪产碱杆菌表达量高432倍 ,其菌体比活力达300 (u L) A₆₀₀。菌体破碎后的上清液经DEAE-SepharoseCL 6B离子交换层析和Butyl-SepharoseCL 4B疏水层析 ,即可得纯度提高 20倍、比活为 686u mg的青霉素G酰化酶 ,两步纯化的总收率达 91%。Western印迹分析表明5%的原前体青霉素酰化酶在胞内形成了包涵体 ,说明其成熟的限速步骤在胞内的运输阶段.