宫粉郁金的组织培养和快速繁殖

１植物名称宫粉郁金（Ｃｕｒｃｕｍａ　ｋｗａｎｇｓｉｅｎｓｉｓ）。２材料类别　块茎萌动芽。３培养条件　萌动芽生长培养基：（１）ＭＳ＋６－ＢＡ１ｍｇ·Ｌ－１（单位下同）＋ＮＡＡ０．２。不定芽增殖与愈伤组织诱导培养基：（２）ＭＳ＋６－ＢＡ１０＋ＫＴ５;（３）ＭＳ＋６．ＢＡ１０;（４）ＭＳ＋６－ＢＡ５＋ＫＴ２．５;（５）ＭＳ＋６－ＢＡ５;（６）ＭＳ＋６－ＢＡ２＋ＫＴ１。生根培养基：（７）ＭＳ＋ＮＡＡ０．５;（８）ＭＳ＋６－ＢＡ０．５＋ＮＡＡ０．５;（９）ＭＳ。以上培养基均加０．７％琼脂,３％蔗糖,ｐＨ５…     

矮生鸡冠花的离体快繁及试管苗开花

１植物名称矮生鸡冠花（Ｃｅｌｏｓｉａｃｒｉｓｔａｔａ）。２材料名称无菌种子苗顶芽、腋芽。３培养条件基本培养基为ＭＳ。培养基组合为：（１）ＭＳ＋ＢＡ１－２ｍｇ·Ｌ~（－１）（单位下同）＋ＮＡＡ０．１～０．２;（２）ＭＳ＋ＢＡ２＋ＩＡＡ０．２;（３）ＭＳ＋ＫＴ２;（４）ＭＳ＋ＢＡ０．１～０．５。以上培养基均含３％蔗糖,０．６％琼脂,ｐＨ为５．８,培养温室为（２６±２）℃,光照１２ｈ．ｄ~（－１）（２０００ｌｘ）。４生长与分化情况４．１无菌材料的获得成熟的种子置７０％酒精中摇动５０ｓ后,转入饱和漂白粉溶…     

微水——有机溶剂两相体系中消旋萘普生的酶法拆分

研究了微水－有机溶剂两相体系中固定化脂肪酶催化的萘甲酯的立体选择性水解反应,固定化酶活性受载体极性、水含量、有机溶剂的ｌｏｇＰ值,产物抑制的影响,据此构建了一种可以连续拆分产生（Ｓ）－（＋）－萘普生的微水－有机溶剂两相体系。反应在一个具有回路的连续流搅拌反应器中进行,反应器中添加有采用吸附法固定化的脂肪酶,截体为一种弱极性的合成载体,水相连同固定化酶颗粒一起永久保持在反应器中,有机流动相带入底物,     

香石竹的叶片培养及植株再生

１植物名称香石竹（Ｄｉａｎｔｈｕｓｃａｒｙｏｐｈｙ－ｌｌｕｓ）,别名康乃馨。２材料类别无菌苗叶片。３培养条件以ＭＳ为基本培养基。分化培养基附加：（１）６－ＢＡ１．０ｍｐ·Ｌ－１（单位下同）＋ＮＡＡ０．３;（２）６－ＢＡ１．０＋ＮＡＡ０．１;（３）６－ＢＡ１．０＋ＮＡＡ０．０５。增殖培养基附加６－ＢＡ０．５＋ＮＡＡ０．１。分化和增殖培养基均加蔗糖３％、琼脂０．７％,ｐＨ５．８。生根培养基为１／２ＭＳ附加ＮＡＡ０．１,蔗糖２％,琼脂０．６％,ｐＨ５．８。培养温度为（２５±１）℃,光照１２ｈ·ｄ－１,…     

5—羟色胺对肺动脉平滑肌细胞在缺氧条件下增殖的作用

本研究应用细胞培养、＾３Ｈ－ＴｄＲ掺入,核酸分子杂交、免疫组织化学染色技术,探讨无氧（０％Ｏ２＋９５％Ｎ２＋５％ＣＯ２）和／或低氧（２．５￣３％Ｏ２＋９２％Ｎ２＋５％ＣＯ２）对新生小牛肺动脉平滑肌细胞增殖和５－羟色胺转载体基因表达的影响。结果表明：无氧２４ｈ可刺激ＰＡＳＭ的ＤＮＡ合成,＾３Ｈ－ＴｄＲ的掺入增加（Ｐ〈０．０５）,加入５－羟色胺能非常显著地促进无氧ＰＡＳＭ增殖（Ｐ〈０．００１）,而对常     

链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２ 株脂肪酶产生菌中,链霉菌Ｚ９４２ 产脂肪酶活力为５９６ｕ／ ｍＬ,其最适培养基（ｇ／Ｌ） 为：糊精１０ 、黄豆饼粉３０ 、尿素１０ 、Ｋ２ＨＰＯ４ ０－５ 、ＭｇＳＯ４ ０－５ 、ＮａＣｌ １ 和ＡＥＯ９ ０ ．５ ,产酶的最适条件为：初始ｐＨ９ ．５ ～１０－０ ,在２６ ℃培养４８ｈ 。用ＰＶＡ 橄榄油乳化系统测定该酶的最适ｐＨ９ ．８ ,最适温度３７ ℃,在ｐＨ８－６ ～１０－２ 于５ ℃存放２４ ｈ ,酶活力不变。０－１４ｍｏｌ／Ｌ 的氯化钙有较大的激活作用。     

蝴蝶兰根段的组织培养

１　植物名称　蝴蝶兰（ＰｈａｌａｅｎｏｐｓｉｓＭｅｌｌｅｒＧｏｌｄ“ＮＦＳ”）。２　材料类别　根段。３　培养条件　（１）愈伤组织的诱导及分化培养基：Ｂ５＋ＮＡＡ１．５ｍｇ·Ｌ－１（单位下同）＋ＫＴ０．２＋ＣＭ１５０ｍｌ·Ｌ－１＋３％蔗糖;（２）原球茎增殖培养基：Ｂ５＋ＧＡ０．０５＋ＣＨ１２０＋３％蔗糖;（３）小苗生长培养基：１／２ＭＳ＋２０％香蕉泥＋２％蔗糖;（４）诱导生根培养基：１／２ＭＳ＋ＩＢＡ０．３＋２％蔗糖。上述培养基均加０．２％活性炭,０．５８％琼脂粉,ｐＨ为５．５;培养基在１２１℃高…     

苎麻疫霉雄器侧生性状的遗传研究

在来自江苏、江西棉花、苎麻和构树的１２个苎麻疫霉菌株中均观察到侧生雄器,其比率为４．０－１６．５％,在以菌ＪＳ－５和ＰＭ－８（雄器侧生比率分别为１６．５％和９．５％）为亲本所建立的连续２－３代单游动孢子无性一代中,雄器侧生性状可以遗传,但单孢株间雄器侧手比率有一定差异,其分布范围分别为９．０－３４．０％和２．５－１５．５％,进一步诱导菌株ＪＳ－５的单游动孢子株的卵孢子萌发,分别对具侧生雄器和具围生     

紫菀花序芽培养及植株再生

１植物名称紫菀（Ａｓｔｅｒｔａｔａｒｉｃｕｓ）。２材料类别花序芽。３培养条件愈伤组织、不定芽诱导及增殖培养基：（１）ＭＳ＋６－ＢＡ０．５～２．０ｍｇ·Ｌ－１（单位下同）;生根培养基：（２）ＭＳ＋ＮＡＡ０．５～２．０＋６－ＢＡ０．２,（３）１／２ＭＳ（大量元素减半）＋ＮＡＡ０．５～２．０＋６－ＢＡ０．２。上述培养基均加０．８％琼脂和３％蔗糖,高温高压灭菌前ｐＨ值调至５．８。培养温度为（２３１２）℃,每天光照１２ｈ,光照度为１５００～２０００ｌｘ４生长与分化情况４．１愈伤组织及不定芽的诱导剪取长约０…     

刺槐宽叶和四倍体无性系的组织培养

１植物名称刺槐（Ｒｏｂｉｎｉａｐｓｅｕｄｏａｃａｃｉａ）优良无性系：Ｔｅｔｒａｐｌｏｉｄｌｏｃｕｓｔ、Ｇｌｇａｓｔｙｐｅｌｏｃｕｓｔ。２材料类别带腋芽的茎段。３培养条件（１）启动培养基：ＭＳ＋６－ＢＡ０．２５ｍｇ·Ｌ－１（单位下同）＋ＮＡＡ０．０５。（２）分化培养基和继代培养基：ＭＳ＋６－ＢＡ０．５＋ＮＡＡ０．１＋ＡｇＮＯ３１０,ＭＳ＋６ＢＡ０．５＋ＮＡＡ０．１。上述培养基均添加３％蔗糖、０．６％琼脂。（３）生根培养基：１／２ＭＳ＋ＩＢＡ０．２＋ＮＡＡ０．２,添加２％蔗糖０．６％琼脂。培养基ｐＨ…     

Lipase‐catalyzed dynamic resolution of naproxen 2,2,2‐trifluoroethyl thioester by hydrolysis in isooctane

A lipase‐catalyzed enantioselective hydrolysis process under continuous in situ racemization of substrate by using trioctylamine as an organic base was developed for the production of (S)‐naproxen from racemic naproxen thioesters in isooctane. Naproxen 2,2,2‐trifluoroethyl thioester and 45°C were selected as the best substrate and temperature, respectively, by comparing the time‐course variations for the racemization of (S)‐naproxen thioesters containing an electron‐withdrawing group. A detailed investigation of the effect of trioctylamine concentration on the kinetic behaviors of the thioester in racemization and enzymatic reaction was conducted, in which more than 70% conversion of the racemate (or 67.2% yield of (S)‐naproxen) with ee_p value higher than 92% was obtained. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 120–126, 1999.     

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity.     

Enantioselective synthesis of (S)‐naproxen using immobilized lipase on chitosan beads

S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane and Tween‐80 were 3:7 and 0.1% (v /v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.     

Enantioselective enzymatic hydrolysis of racemic drugs by encapsulation in sol–gel magnetic sporopollenin

Candida rugosa lipase was encapsulated within a sol–gel procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of octyltriethoxysilane and tetraethoxysilane in the presence of magnetic sporopollenin. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e., the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of racemic naproxen methyl ester, mandelic acid methyl ester or 2-phenoxypropionic acid methyl ester that were studied in aqueous buffer solution/isooctane reaction system. The encapsulated magnetic sporopollenin (Spo-M-E) was found to give 319 U/g of support with 342% activity yield. It has been observed that the percent activity yields and enantioselectivity of the magnetic sporopollenin encapsulated lipase were higher than that of the encapsulated lipase without support. The substrate specificity of the encapsulated lipase revealed more efficient hydrolysis of the racemic naproxen methyl ester and 2-phenoxypropionic acid methyl ester than racemic mandelic acid methyl ester. It was observed that excellent enantioselectivity (E > 400) was obtained for encapsulated lipase with magnetic sporopollenin with an ee value of S-Naproxen and R-2 phenoxypropionic acid about 98%.     

A magnetically separable biocatalyst for resolution of racemic naproxen methyl ester

Candida rugosa lipase (CRL) was encapsulated via the sol–gel method, using 5, 11, 17, 23-tetra-tert-butyl-25,27-bis(2-aminopyridine)carbonylmethoxy-26, 28-dihydroxy-calix[4]arene-grafted magnetic Fe₃O₄ nanoparticles (Calix-M-E). The catalytic activity of encapsulated lipase (Calix-M-E) was tested both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic naproxen methyl ester. The present study demonstrated that the calixarene-based compound has the potential to enhance both reaction rate and enantioselectivity of the lipase-catalyzed hydrolysis of racemic naproxen methyl ester. The encapsulated lipase (Calix-M-E) had great catalytic activity and enantioselectivity (E > 400), as well as remarkable reusability as compared to the encapsulated lipase without supports (E = 137) for S-Naproxen.     

Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes

The regulation of avian lipoprotein lipase by dibutyryl cyclic AMP in cultured adipocytes was studied with quantitative and specific methods for the measurements of enzyme catalytic activity, enzyme protein mass, and immunoadsorption of labeled enzyme. Incubation of adipocytes in 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline results in a time-dependent decrease in cell lipoprotein lipase catalytic activity. The activity is decreased by 70% in 4 h and over 90% by 12 h. The decrease in cellular catalytic activity is due to a decrease in both enzyme content and enzyme catalytic efficiency. 4 h after exposure of adipocytes to cAMP, enzyme protein was decreased from 3.58 +/- 0.5 to 1.92 +/- 0.1 ng/dish and specific activity from 15.1 +/- 2.1 to 8.4 +/- 1.1 nmol/ng. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in lipoprotein lipase activity was half-maximal at less than 25 microM dibutyryl cyclic AMP. The rate of lipoprotein lipase synthesis was estimated by measuring the incorporation of L-[35S]methionine into enzyme protein during 30 min. A method for the quantitative immunoadsorption of lipoprotein lipase from cell lysates was developed. Utilizing this immunoadsorption technique, the rate of incorporation of L-[35S]methionine into lipoprotein lipase was 0.0026 +/- 0.002%, when expressed as a percentage of that incorporated into total trichloroacetic acid-precipitable counts. By 2 h after exposure of adipocytes to 0.5 mM dibutyryl cAMP, the relative synthesis rate had already decreased to 64 +/- 4% of the control rate. After 16 h the synthesis rate was 43.2 +/- 13.8% of the control rate. The observed decreased synthesis rate could account for most of the decreased cellular enzyme content and diminished enzyme secretion rate.     

Integration of reactive membrane extraction with lipase-hydrolysis dynamic kinetic resolution of naproxen 2,2,2-trifluoroethyl thioester in isooctane

Lipases immobilized on polypropylene powders have been used as the biocatalyst in the enantioselective hydrolysis of (S)-naproxen from racemic naproxen thioesters in isooctane, in which trioctylamine was added to perform in situ racemization of the remaining (R)-thioester substrate. A detailed study of the kinetics for hydrolysis and racemization indicates that increasing the trioctylamine concentration can activate and stabilize the lipase as well as enhance the racemization and non-stereoselective hydrolysis of the thioester. Effects of the aqueous pH value and trioctylamine concentration on (S)-naproxen dissociation and partitioning in the aqueous phase as well as the transportation in a hollow fiber membrane were further investigated. Good agreements between the experimental data and theoretical results were obtained when the dynamic kinetic resolution process was integrated with a hollow fiber membrane to reactively extract the desired (S)-naproxen out of the reaction medium.     

The kinetics of heparin inhibition of the esterase and basal lipase activities of lipoprotein lipase

The kinetics of inhibition of the esterase and lipase activities of bovine milk lipoprotein lipase (LPL) were compared. The esterase LPL activity against emulsified tributyrylglycerol was not affected by the enzyme activator apolipoprotein C-II (C-II) and amounted to about 15% of the "plus activator" lipase enzyme activity. Heparin at concentrations of 20 micrograms/ml inhibited 25% of the esterase activity. The reaction followed Henri-Michaelis-Menten kinetics and the inhibition by heparin followed a linear, intersecting, noncompetitive kinetic model. On the other hand, the basal lipase activity of LPL against emulsified trioleoylglycerol (TG) was very sensitive to inhibition by heparin: 1 microgram/ml inhibited about 80% of the reaction and 3 micrograms/ml drove the reaction to zero. The velocity curve for the uninhibited basal LPL activity was sigmoidal with an apparent nH(TG) of 2.94. Heparin inhibited the lipase activity competitively: heparin decreased nH(TG) and increased[TG]0.5 6.4-fold, while TG decreased the nH(Heparin) from 2.14 to 0.95 and caused a 3-fold increase in [Heparin]0.5. C-II, at concentrations lower than 2.5 X 10(-8) M (i.e., lower than KA), countered the inhibitory effects of heparin: at constant inhibitor concentrations, C-II increased nH(TG) from 1.78 to 2.52 and decreased [TG]0.5 about 10-fold; it also increased the apparent Vmax. At the lower C-II concentrations, nH(C-II) was approximately equal to 1.0 and increasing the TG concentrations decreased [C-II]0.5 from 3.8 X 10(-8) to 8.5 X 10(-9) M, with no effect on the nH(C-II). At the higher C-II concentrations, nH(C-II) was 2.5 and TG decreased [C-II]0.5 about 2-fold with no effect on the nH(C-II). In the absence of heparin, C-II had no effect on nH(TG) nor on [TG]0.5, but it increased the apparent Vmax. On the other hand, TG had no effect on nH(C-II) nor on [C-II]0.5, but at any given C-II concentration, the reaction velocity increased with increasing TG concentrations. It is concluded that TG and heparin as well as C-II and heparin are mutually exclusive and that lipoprotein lipase is a multisite enzyme, possibly a tetramer, with three high-affinity catalytic sites, and an equal number of sites for C-II and heparin per oligomer. However, LPL differs from classical allosteric enzymes in that its activator has no effect on substrate cooperativity nor on [S]0.5; its only effect is to increase Vmax by increasing the catalytic rate constant kp by inducing conformational changes in the enzyme.     

Candida rugosa lipase mediated multigram synthesis of acid part of S(+)-atliprofen,a new NSAID and molecular modeling studies aimed at predicting selectivity of the enzyme

An efficient procedure to prepare S-4-(3-thienyl)phenyl-α-methylacetic acid, an intermediate of a recently approved non-steroidal anti-inflammatory cyclooxygenase inhibitor atliprofen by enantioselective hydrolysis of the corresponding esters in presence of candida rugosa lipase is reported. The methyl and butyl esters of the racemic acid 2 were synthesized and subjected to enantioselective hydrolysis by the lipase to give S-4-(3-thienyl)phenyl-α-methylacetic acid upto 97.86% ee. The observed enantioselectivity during the hydrolysis of the substrate by the lipase was rationalized by molecular modeling studies. The methyl esters of both R and S-enantiomers of 4-(3-thienyl)phenyl-α-methylacetic acid, naproxen and ketoprofen were taken for the modeling studies. The results of the modeling studies are in conformity with the experimental observations.