有机溶剂对脂肪酶ipozyme^IM催化活性的影响

脂肪酶Ｌｉｐｏｚｙｍｅ＾ＩＭ经有机溶剂浸泡处理后活性显著提高。该文系统地探讨了有机溶剂特性,有机溶剂水含量以及有机溶剂包温度和浸泡时间对Ｌｉｐｏｚｙｍｅ＾ＩＭ催化活性的影响。发现Ｌｉｐｏｚｙｍｅ＾ＩＭ经疏水性较强的有机沉沦 泡处理后活性成倍增长;浸泡的最佳温度为６０℃左右,浸泡时间对酶活影响不大;随着有机溶剂水含量的增加,酶活性急剧下降。     

介导巨噬细胞摄取氧化修饰极低密度脂蛋白的受体

用未标记氧化修饰极低密度脂蛋白（ｏｘ－ＶＬＤＬ）、ｎ－ＶＬＤＬ、乙酰ＬＤＬ竞争１２５Ｉ－ｏｘ－ＶＬＤＬ与巨噬细胞的结合。在浓度为２００μｇ蛋白／ｍｌ时,分别抑制标记ｏｘ－ＶＬＤＬ结合量的７０～７８％、６０～７０％和２５～３５％。用未标记ｏｘ－ＶＬＤＬ竟争１２５Ｉ－ｎ－ＶＬＤＬ与巨噬细胞的结合,能抑制７７％。结果说明ｏｘ－ＶＬＤＬ主要通过ｎ－ＶＬＤＬ受体进入巨噬细胞。以ｏｘ－ＶＬＤＬ与ｏｘ－ＬＤＬ进行交叉竞争时,ｏｘ－ＶＬＤＬ与ｏｘ－ＬＤＬ自身可抑制标记ｏｘ－ＶＬＤＬ或ｏｘ－ＬＤＬ的７５～８２％,而ｏｘ－ＶＬＤＬ或ｏｘ－ＬＤＬ的交叉竞争仅３８～４０％。表明ｏｘ－ＶＬＤＬ与ｏｘ－ＬＤＬ有部分共同的构象与巨噬细胞的脂蛋白受体结合,但ｏｘ－ＶＬＤＬ不是经ｏｘ－ＬＤＬ受体被巨噬细胞摄取。     

介导巨噬细胞摄取氧化修饰极低密试脂蛋白的受体

用未标记氧化修饰极低密度脂蛋白（ｏｘ－ＶＬＤＬ）、ｎ－ＶＬＤＬ、乙酰ＬＤＬ竞争＾１２５Ｉ－ｏｘ－ＶＬＤＬ与巨噬细胞的结合。在浓度为２００μｇ蛋白／ｍｌ时,分别抑制标记ｏｘ－ＶＬＤＬ结合量的７０￣７８％、６０￣７０％和２５￣３５％。用未标记ｏｘ－ＶＬＤＬ竞争＾１２５Ｉ－ｎ－ＶＬＤＬ与巨噬细胞的结合,能抑制７７％。结果说明ｏｘ－ＶＬＤＬ主要通过ｎ－ＶＬＤＬ受体进入巨噬细胞。以ｏｘ－ＶＬＤＬ与ｏｘ－ＬＤ     

体内足迹法研究乙肝病毒DNA与蛋白质的相互作用

以基因组中整合了乙型肝炎病毒基因的ＨｅｐＧ２．２．１５细胞为材料,应用连接介导的ＰＣＲ体内足迹分析法,研究了乙肝病毒增强子Ⅱ和核心启动子区在体内条件下与核蛋白因子的相互作用。结果发现,已在体外条件确定的增强子Ⅱ区的一些转录因子的结合位点,例如,ＳＰ１位点（ｎｔ１６１６－１６２８,ｎｔ１７３３－１７４２,ｎｔ１７４５－１７５４）,Ｃ／ＥＢＰ位点（ｎｔ１６３９－１６４７,ｎｔ１６５１－１６５９,ｎｔ１     

反应介质对脂肪酶稳定性的影响

脂肪酶Ｌｉｐｏｚｙｍｅ＾ＩＭ在有机溶剂中的热稳定性显著提高,在水溶液中,热处理温度高于５０℃后,其催化甘油三酯水解的活力迅速下降,温度升到６０℃时,水解活力仅残存１３．６％,温度高于７０℃该酶完全失活,而在有机溶剂正庚烷中,温度高达８５℃仍表现出较高的活性,反应介质的疏水性越强,酶抗超声辐射变性作用的能力越强,经同样的超声辐射处理后,正已烷中Ｌｉｐｏｚｙｍｅ＾ＩＭ的酯水解活力仅损失１１．８％,而在     

氧化修饰极低密度脂蛋白增强其致动脉粥样硬化作用

研究了氧化修饰极低密度脂蛋白（ｏｘ－ＶＬＤＬ）对小白鼠腹腔巨噬细胞内脂质堆积作用及其机制。经Ｃｕ~（２＋）修饰后ＶＬＤＬ的电泳迁移率及脂质过氧化物含量均显著增加。ｏｘ－ＶＬＤＬ更易导致小鼠腹腔巨噬细胞内脂质堆积。以相同浓度（３００μｇＴＧ／ｍＬ）或不同浓度（２００─５００μｇＴＧ／ｍＬ）的ｏｘ－ＶＬＤＬ及正常ＶＬＤＬ（ｎ－ＶＬＤＬ）与巨噬细胞温育２４ｈ,前者使巨噬细胞内ＴＧ堆积均比后者显著（Ｐ＜０．０１）。同时,随ｏｘ－ＶＬＤＬ的脂质过氧化物含量（ＴＢＡＲＳ水平）增加,巨噬细胞内ＴＧ含量的百分率相应增加。以５０μｇ蛋白／ｍＬ的ｎ－ＬＤＬ,ｏｘ－ＬＤＬ,ｎ－ＶＬＤＬ及ｏｘ－ＶＬＤＬ与巨噬细胞温育６０ｈ。细胞内ＣＥ堆积中氧化组均比正常组高（Ｐ＜０．０１）。巨噬细胞对~（１２５）Ｉ－ｎ－ＶＬＤＬ与~（１２５）Ｉ－ｏｘ－ＶＬＤＬ的结合、降曲线均有饱和趋势。两结合曲线无明显差异,但细胞对后者降解的量比前者多。结合的竞争实验表明,ｎ－ＶＬＤＬ能抑制大部分~（１２５）Ｉ－ｏｘ－ＶＬＤＬ与细胞结合,而Ａｃ－ＬＤＬ只能抑制小部分。结果表明ｏｘ－ＶＬＤＬ主要通过受体途径：大部分经过ｎ－ＶＬＤＬ受体,小部分经过清道夫受体被巨噬细胞摄     

硝基左旋精氨酸对睡眠抑制作用的机制研究

本文观察了硝基左旋精氨酸（Ｌ－ＮＮＡ,５０ｍｇ／ｋｇ,ｉｐ）和Ｌ－精氨酸（Ｌ－ａｒｇ,１１０ｍｇ／ｋｇ,ｉｐ）对慢性植入电极的大鼠睡眠－觉醒周期的影响及中缝核５－羟色胺（５－ＨＴ）神经元免疫阳性反应的变化。结果表明：Ｌ－ＮＮＡ显著抑制慢波睡眠和快眼动睡眠,使平均动脉压（ＭＡＰ）升高。Ｌ－ａｒｇ则使ＭＡＰ显著降低,对睡眠无明显影响。预先给予Ｌ－ａｒｇ可逆转Ｌ－ＮＮＡ的效应。腹腔给予Ｌ－ＮＮＡ后２ｈ,     

巨噬细胞新型氧化低密度脂蛋白结合蛋白的研究

小鼠腹腔巨噬细胞（ＭＰＭ）膜上存在能结合氧化低密度脂蛋白（ｏｘ－ＬＤＬ）的Ａ类清道夫受体（ＳＲ－Ａ）,但用配体印迹技术研究制备ＭＰＭ膜蛋白,发现还存在一种新型ｏｘ－ＬＤＬ膜结合蛋白,其分子量低于ＳＲ－Ａ,为９２ｋＤ它不结合乙酰化低密度脂蛋白ａｃ－ＬＤＬ）,与配体的结合也不受还原剂的影响,但唾液酸酶处理则明显减弱其与ｏｘ－ＬＤＬ的结合,未标记ｏｘ－ＬＤＬ能竞争性抑制（＾１２５Ｉ）ｏｘ－ＬＤＬ与９２ｋ     

中国林蛙乳酸脱氢酶多基因系统及基因间连锁关系的研究

（１）用聚丙烯酰胺凝胶电泳对山西产中国林蛙４个地理群的３３３只林蛙进行了分析,结果表明：中国林蛙的ＬＤＨ由ＬＤＨ－Ａ,ＬＤＨ－Ｂ和ＬＤＨ－Ｃ３个基因决定。ＬＤＨ－Ａ是单态座位,ＬＤＨ－Ｂ和ＬＤＨ－Ｃ均为多态座位,每个多态座位均有两个等位基因。ＬＤＨ－Ｂ与ＬＤＨ－Ｃ呈紧密连锁关系。认为ＬＤＨ－Ｃ是ＬＤＨ－Ｂ的重复产物。（２）热稳定性、尿素处理稳定性及组织特异性研究表明：ＬＤＨ对温度和尿素处理稳定性顺序为Ａ４＞Ｂ’４＞Ｂ４＞Ｃ４。Ａ４在骨骼肌和肝脏等组织中活力最大,Ｂ４在心肌和卵巢中活力最强,ＬＤＨ－Ｃ主要在眼球和卵巢中表达。（３）Ｌｄｈ－ｂ和Ｌｄｈ－ｂ＇在不同地理群间呈差异分布,随着纬度的增高,Ｌｄｈ－ｂ在种群中的频率增大。     

新鲜蜂王浆和6-BA对水稻种子萌发和离体白菜叶片叶绿素含量的影响

经用１０－３ｇ／Ｌ、１０－２ｇ／Ｌ、１０－１ｇ／Ｌ６ＢＡ和１０－２,１０－１、１ｇ／Ｌ新鲜蜂王浆处理水稻种子和离体白菜叶片后,水稻种子发芽率均高于对照值,其中１０－２ｇ／Ｌ的６ＢＡ和１ｇ／Ｌ的蜂王浆效果最佳。而离体白菜叶片叶绿素含量也比对照高,其中１０－３ｇ／Ｌ的６ＢＡ和１ｇ／Ｌ的蜂王浆效果最佳。１０－２ｇ／Ｌ的６ＢＡ和１０－２ｇ／Ｌ的蜂王浆混合处理白菜叶片,其效果要比单独用６ＢＡ和蜂王浆处理好。     

有机相中酶促有机硅烷醇的转酯

在有机介质中,脂肪酶L-1754可以催化非天然有机硅烷醇与脂肪酸酯的转酯反应.反应介质、反应体系水活度和底物的结构对酶促转酯反应有显著的影响.适宜的反应介质为正辛烷,反应体系最适水活度为0.55.     

Two-Step biocatalytic conversion of an ester to an aldehyde in reverse micelles

Lipases from Candida cyclindracea (L-1754) and wheat germ (L-3001) have been used to hydrolyze esters to their corresponding alcohols and acids in reverse micelles. Alcohol dehydrogenase from baker's yeast (YADH) was subsequently used to reduce the alcohol products to aldehydes. Cofactor recycling in the redox reaction was achieved using a sacrificial cosubstrate, as described previously. Four surfactants (sodium dioctylsulfosuccinate, Nonidet P-40 with Triton X-35, polyoxyethylene, 10-cetyl-ether, polyoxyethylene sorbitan trioleate) were employed to determine the effect of amphiphile on ester hydrolysis and redox reaction rates separately. The effect of type of organic solvent, W(0) [(water]/[surfactant)], and substrate concentration on separte enzyme activity were also investigated. A brief investigation of a single phase, two-step reaction catalyzed by the combination of lipase and YADH in reverse micelles is also reported. The activities of the enzymes are significantly different when used together instead of independently. (c) 1994 John Wiley & Sons, Inc.     

Isolation of myo-Inositol from Solutions by Enzymatic Biotransformation

The transferase reaction between phospholipids and inositol catalyzed by phospholipase D was studied at interfaces in water–organic solvent systems. Optimum conditions were determined for phosphatidylinositol synthesis in heterogeneous water–organic solvent systems. Hydrophobic components (phospholipids) were readily separated from water-soluble products (alcohols) in systems with organic solvents. In the hexane–water system, addition of methanol (an alcohol substrate) to the reaction medium displaced myo-inositol from the molecule of phosphatidylinositol. myo-Inositol was isolated from the mixture of its isomers using a two-step transferase reaction catalyzed by phospholipase D.     

有机相脂肪酶催化合成乙酸肉桂酯

对有机相中酶法催化合成乙酸肉桂酯的转酯化反应进行研究。结果发现:Candida anatarctic脂肪酶(Novozyme435)、根霉脂肪酶(Rhizopus niveus lipase)和荧光假单胞菌脂肪酶(Pseudomonas fluore lipase)均有较好的催化活性。同时考察各反应参数(温度、反应溶剂、体系水活度、酰化剂类型、肉桂醇与酰化剂摩尔比、肉桂醇浓度等)对脂肪酶Novozyme435合成乙酸肉桂酯反应的影响,确定了反应体系最优工艺条件:在10 mL甲基叔丁基醚中,肉桂醇200 mmol/L,n(肉桂醇)∶n(乙酸乙烯酯)=1∶1.5,初始水活度αw=0.84,温度35℃,酶加量0.02 g,反应3 h后肉桂醇转化率可达到99%,产物经质谱(MS)鉴定。固定化酶经过10个批次反应,反应转化率都保持在90%以上。     

Native and modified subtilisin 72 as a catalyst of peptide synthesis in media with low water content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.     

Reaction Kinetics of Immobilized α-Chymotrypsin in Organic Media 1. Influence at Solvent Polarity

Esterification of N-acetyl phenylalanine with ethanol catalyzed by immobilized α-chymotrypsin (EC 3.4.21.1) was studied in various reaction media. The effect of reaction medium polarity on enzymatic activity as well as equilibrium yield was measured. The reaction rate increased with increasing amounts of added water, reaching an optimum corresponding to water saturation of the reaction medium. Further additions of water resulted in decreased activity. Bell-shaped activity profiles were obtained for all reaction media tested. Reaction media consisting of pure solvents and of mixtures of solvents were used. The enzymatic activity and the equilibrium yield increased with decreased polarity of the medium. Maximum activity was found in a reaction medium consisting of 80% diisopropyl ether and 20% heptane. The enzymatic activity obtained at optimal water additions in the different solvents and solvents mixtures could be correlated to the solubility of water and the log P of the medium. The equilibrium yield of the reaction was much more closely correlated to the solubility of water than the log P. Much lower enzymatic activity was obtained when solvent mixtures producing water-miscible media were created, than in mixtures producing water-immiscible media, such as mixtures of acetonitrile and diisopropyl ether. The equilibrium yield could be increased by decreasing the water content in the reaction medium, which reduced the water activity.     

Thermodynamic parameters monitoring the equilibrium shift of enzyme-catalyzed hydrolysis/synthesis reactions in favor of synthesis in mixtures of water and organic solvent

The main strategy developed to shift the equilibrium state of a hydrolase-catalyzed hydrolysis/synthesis reaction consists in reducing water activity by addition of organic solvents in the reaction medium. We have used several mixtures of water and 1,4-butanediol, ranging from pure water to pure 1,4-butanediol, to study the hydrolysis/synthesis reaction of the N-Cbz-L-tryptophanyl-glycineamide dipeptide, catalyzed by alpha-chymotrypsin. In the presence of 1,4-butanediol, alpha-chymotrypsin also catalyzed the esterification reaction between this diol and N-Cbz-L-tryptophan; this ester hydrolysis/synthesis reaction has thus also been examined. The dipeptide and ester equilibrium concentrations increase when the water content of the reaction medium is decreased. Using our experimental data, we have determined the equilibrium constants of the hydrolysis/synthesis equilibria involving the nonionized forms of the protected amino acids, the estimated values of which are Ksp = 8 10(5) for the dipeptide and Kse = 78 for the ester respectively. They are true thermodynamic equilibrium constants, each related to a single, well-defined reaction equilibrium and with water activity being taken into account. If an organic solvent is added to the reaction medium these equilibria can be shifted towards synthesis by decreasing the water activity but also by modifying the ionization/neutralization equilibrium constant of the ionizable groups. These two effects depend both on the water content and on the nature of the organic solvent used, and, in particular, on its dielectric constant. Because of the importance of this parameter in our study, we discuss using it as an indicator to select an appropriate organic solvent to perform an enzyme-catalyzed synthesis.     

Ring-opening polymerization of ɛ-caprolactone catalyzed by a novel lipase Candida sp. 99-125

The ring-opening polymerization of ?-caprolactone catalyzed by a novel lipase Candida sp. 99-125 was achieved in organic solvents. ?-Caprolactone was found to be polymerized in quantitative yield in toluene at 80?°C for 5 days. The poly (?-caprolactone) obtained had a number-average molecular weight of 14,120 (polydispersity index?=?1.161). The effect of enzyme concentration, temperature, reaction time, reaction medium, and water content on monomer conversion and product molecular weight were investigated. Kinetic analysis showed that the enzyme displayed Michaelis–Menten kinetics and showed stronger affinity for ?-caprolactone (with a K_m value of 6.1?mmol/L) than other reported lipases.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

Lipase-catalyzed transesterification of soybean oil for biodiesel production in <Emphasis Type="Italic">tert</Emphasis>-amyl alcohol

Lipase-catalyzed transesterification of soybean oil and methanol for biodiesel production in tert-amyl alcohol was investigated. The effects of different organic medium, molar ratio of substrate, reaction temperature, agitation speed, lipase dosage and water content on the total conversion were systematically analyzed. Under the optimal conditions identified (6 mL tert-amyl alcohol, three molar ratio of methanol to oil, 2% Novozym 435 lipase based on the soybean oil weight, temperature 40°C, 2% water content based on soybean oil weight, 150 rpm and 15 h), the highest biodiesel conversion yield of 97% was obtained. With tert-amyl alcohol as the reaction medium, the negative effects caused by excessive molar ratio of methanol to oil and the by-product glycerol could be reduced. Furthermore, there was no evident loss in the lipase activity even after being repeatedly used for more than 150 runs.