triton和flask的区别_tornado和flask区别

       大家好，今天我来给大家讲解一下关于triton和flask的区别的问题。为了让大家更好地理解这个问题，我将相关资料进行了整理，现在就让我们一起来看看吧。

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       matter 物质

       energy 能

       vacuum 真空

       liquid 液体

       fluid 流体

       solid 固体

       body 物体

       mass 质量

       weight 重量

       density 密度

       specific gravity 比重

       gravity 重力

       velocity 速度

       kinetic energy 动能

       intensity 强度

       friction 磨擦力

       pressure 压力,压强

       to exert a force 施作用力

       vector 矢量,向量

       work 功

       temperature 温度

       heat 热

       conduction 传导

       conductor 导体

       radiation 辐射

       expansion 膨胀

       quantum theory 量子论

       dynamics, kinetics 动力学

       kinematics 运动学

       statics 静力学

       torque 转矩

       axis of rotation 转动轴

       moment of inertia 转动惯量

       electric current 电流

       electron 电子

       proton 质子

       positron 阳电子

       charge 电荷

       positive 正

       negative 负

       electromotive force 电动势

       electrode 电极

       anode 阳极,正极

       cathode 阴极,负极

       electropositive 电阳性的,电正性的

       electronegative 电阴性的,电负性的

       magnetism 磁学,磁性

       magnetic field 磁场

       flux 磁通量

       magnetic induction 磁感应

       magnet 磁体,磁铁

       electromagnet 电磁体

       electromagnetic 电磁的

       pole 磁极

       coil 线圈

       light 光

       ray 线,射线

       source 源

       beam 束,柱,道

       diffraction 衍射

       reflection 反射

       refraction 折射

       incident ray 入射线

       angle of incidence 入射角

       refractive index 折射率

       lens 透镜

       image 像

       focus, focal point 焦点

       focal length 焦距

       convergent 全聚的

       divergent 发散的

       concave 凹

       convex 凸

       biconcave, concavo-concave 凹凹,双凹

       biconvex, convexo-convex 凸凸,双凸

       mirror 镜

       以上是物理的。

       Bunsen burner 本生灯

       product 化学反应产物

       flask 烧瓶

       apparatus 设备

       PH indicator PH值指示剂,氢离子(浓度的)负指数指示剂

       matrass 卵形瓶

       litmus 石蕊

       litmus paper 石蕊试纸

       graduate, graduated flask 量筒,量杯

       reagent 试剂

       test tube 试管

       burette 滴定管

       retort 曲颈甑

       still 蒸馏釜

       cupel 烤钵

       crucible pot, melting pot 坩埚

       pipette 吸液管

       filter 滤管

       stirring rod 搅拌棒

       element 元素

       body 物体

       compound 化合物

       atom 原子

       gram atom 克原子

       atomic weight 原子量

       atomic number 原子数

       atomic mass 原子质量

       molecule 分子

       electrolyte 电解质

       ion 离子

       anion 阴离子

       cation 阳离子

       electron 电子

       isotope 同位素

       isomer 同分异物现象

       polymer 聚合物

       symbol 复合

       radical 基

       structural formula 分子式

       valence, valency 价

       monovalent 单价

       bivalent 二价

       halogen 成盐元素

       bond 原子的聚合

       mixture 混合

       combination 合成作用

       compound 合成物

       alloy 合金

       metal 金属

       metalloid 非金属

       Actinium(Ac) 锕

       Aluminium(Al) 铝

       Americium(Am) 镅

       Antimony(Sb) 锑

       Argon(Ar) 氩

       Arsenic(As) 砷

       Astatine(At) 砹

       Barium(Ba) 钡

       Berkelium(Bk) 锫

       Beryllium(Be) 铍

       Bismuth(Bi) 铋

       Boron(B) 硼

       Bromine(Br) 溴

       Cadmium(Cd) 镉

       Caesium(Cs) 铯

       Calcium(Ca) 钙

       Californium(Cf) 锎

       Carbon(C) 碳

       Cerium(Ce) 铈

       Chlorine(Cl) 氯

       Chromium(Cr) 铬

       Cobalt(Co) 钴

       Copper(Cu) 铜

       Curium(Cm) 锔

       Dysprosium(Dy) 镝

       Einsteinium(Es) 锿

       Erbium(Er) 铒

       Europium(Eu) 铕

       Fermium(Fm) 镄

       Fluorine(F) 氟

       Francium(Fr) 钫

       Gadolinium(Gd) 钆

       Gallium(Ga) 镓

       Germanium(Ge) 锗

       Gold(Au) 金

       Hafnium(Hf) 铪

       Helium(He) 氦

       Holmium(Ho) 钬

       Hydrogen(H) 氢

       Indium(In) 铟

       Iodine(I) 碘

       Iridium(Ir) 铱

       Iron(Fe) 铁

       Krypton(Kr) 氪

       Lanthanum(La) 镧

       Lawrencium(Lr) 铹

       Lead(Pb) 铅

       Lithium(Li) 锂

       Lutetium(Lu) 镥

       Magnesium(Mg) 镁

       Manganese(Mn) 锰

       Mendelevium(Md) 钔

       Mercury(Hg) 汞

       Molybdenum(Mo) 钼

       Neodymium(Nd) 钕

       Neon(Ne) 氖

       Neptunium(Np) 镎

       Nickel(Ni) 镍

       Niobium(Nb) 铌

       Nitrogen(N) 氮

       Nobelium(No) 锘

       Osmium(Os) 锇

       Oxygen(O) 氧

       Palladium(Pd) 钯

       Phosphorus(P) 磷

       Platinum(Pt) 铂

       Plutonium(Pu) 钚

       Polonium(Po) 钋

       Potassium(K) 钾

       Praseodymium(Pr) 镨

       Promethium(Pm) 钷

       Protactinium(Pa) 镤

       Radium(Ra) 镭

       Radon(Rn) 氡

       Rhenium(Re) 铼

       Rhodium(Rh) 铑

       Rubidium(Rb) 铷

       Ruthenium(Ru) 钌

       Samarium(Sm) 钐

       Scandium(Sc) 钪

       Selenium(Se) 硒

       Silicon(Si) 硅

       Silver(Ag) 银

       Sodium(Na) 钠

       Strontium(Sr) 锶

       Sulphur(S) 锍

       Tantalum(Ta) 钽

       Technetium(Tc) 锝

       Tellurium(Te) 碲

       Terbium(Tb) 铽

       Thallium(Tl) 铊

       Thorium(Th) 钍

       Tin(Sn) 锡

       Thulium(Tm) 铥

       Titanium(Ti) 钛

       Tungsten(W) 钨

       Uranium(U) 铀

       Vanadium(V) 钒

       Xenon(Xe) 氙

       Ytterbium(Yb) 镱

       Yttrium(Y) 钇

       Zinc(Zn) 锌

       Zirconium(Zr) 锆

       organic chemistry 有机化学

       inorganic chemistry 无机化学

       derivative 衍生物

       series 系列

       acid 酸

       hydrochloric acid 盐酸

       sulphuric acid 硫酸

       nitric acid 硝酸

       aqua fortis 王水

       fatty acid 脂肪酸

       organic acid 有机酸

       hydrosulphuric acid 氢硫酸

       hydrogen sulfide 氢化硫

       alkali 碱,强碱

       ammonia 氨

       base 碱

       hydrate 水合物

       hydroxide 氢氧化物,羟化物

       hydracid 氢酸

       hydrocarbon 碳氢化合物,羟

       anhydride 酐

       alkaloid 生物碱

       aldehyde 醛

       oxide 氧化物

       phosphate 磷酸盐

       acetate 醋酸盐

       methane 甲烷,沼气

       butane 丁烷

       salt 盐

       potassium carbonate 碳酸钾

       soda 苏打

       sodium carbonate 碳酸钠

       caustic potash 苛性钾

       caustic soda 苛性钠

       ester 酯

       gel 凝胶体

       analysis 分解

       fractionation 分馏

       endothermic reaction 吸热反应

       exothermic reaction 放热反应

       precipitation 沉淀

       to precipitate 沉淀

       to distil, to distill 蒸馏

       distillation 蒸馏

       to calcine 煅烧

       to oxidize 氧化

       alkalinization 碱化

       to oxygenate, to oxidize 脱氧,氧化

       to neutralize 中和

       to hydrogenate 氢化

       to hydrate 水合,水化

       to dehydrate 脱水

       fermentation 发酵

       solution 溶解

       combustion 燃烧

       fusion, melting 熔解

       alkalinity 碱性

       isomerism, isomery 同分异物现象

       hydrolysis 水解

       electrolysis 电解

       electrode 电极

       anode 阳极,正极

       cathode 阴极,负极

       catalyst 催化剂

       catalysis 催化作用

       oxidization, oxidation 氧化

       reducer 还原剂

       dissolution 分解

       synthesis 合成

       reversible 可逆的

       以上是化学。

       head 头

       throat 喉咙, 咽喉

       armpit hair 腋毛

       nipple 乳头

       chest 胸部

       pit 胸口

       navel 肚脐

       abdomen 腹部

       private parts

       thigh 大腿

       neck 脖子

       shoulder 肩

       back 背

       waist 腰

       hip 臀部

       buttock 屁股

       skull 颅骨, 头盖骨

       collarbone 锁骨

       rib 肋骨

       backbone 脊骨, 脊柱

       shoulder joint 肩关节

       shoulder blade 肩胛骨

       breastbone 胸骨

       elbow joint 肘关节

       pelvis 骨盆

       kneecap 膝盖骨

       bone 骨

       skeleton 骨骼

       sinew 腱

       muscle 肌肉

       joint 关节

       blood vessel 血管

       vein 静脉

       artery 动脉

       capillary 毛细血管

       nerve 神经

       spinal marrow 脊髓

       brain 脑

       respiration 呼吸

       windpipe 气管

       lung 肺

       heart 心脏

       diaphragm 隔膜

       exhale 呼出

       inhale 呼入

       internal organs 内脏

       gullet 食管

       stomach 胃

       liver 肝脏

       gall bladder 胆囊

       pancreas 胰腺

       spleen 脾

       duodenum 12指肠

       small intestine 小肠

       large intestine 大肠

       blind gut 盲肠

       vermiform appendix 阑尾

       rectum 直肠

       anus 肛门

       bite 咬

       chew 咀嚼

       knead 揉捏

       swallow 咽下

       digest 消化

       absord 吸收

       discharge 排泄

       excrement 粪便

       kidney 肾脏

       bladder 膀胱

       penis

       testicles 睾丸

       scroticles 阴囊

       urine 尿道

       ovary 卵巢

       womb 子宫

       vagina 阴道

       pine 松

       cerdar 雪松类

       larch 落叶松

       juniper 杜松

       cone 松果

       cypress 柏树

       bamboo 竹

       box 黄杨

       poplar 白杨

       cottonwood 三角叶杨

       osier 紫皮柳树

       willow 垂柳

       birch 白桦

       maple 枫树

       sequoia 红杉

       fir 冷杉

       hemlock spruce 铁杉

       spruce 云杉

       yew 紫杉

       eucalytus 桉树

       locust 洋槐

       wattle 金合欢树

       camphor tree 樟树

       rosewood 紫檀

       ebony 乌檀

       sandalwood 檀香木

       satinwood 椴木

       linden 椴树

       rowan 欧洲山梨

       teak 柚木树

       elm 榆木树

       oak 橡树

       acorn 橡树果

       sycamore 美国梧桐

       ginkgo 银杏树

       holly 冬青

       coco 椰树

       date 枣椰树

       hickory 山核桃树

       plane tree 悬铃树

       beech 山毛榉

       horse chestnut 七叶树

       blackthorn 黑刺李

       baobab 猴面包树

       elder 接骨木

       myrtle 桃金娘科植物

       cycad 苏铁

       oil palm 油棕榈树

       treetop 树梢

       branch 树枝

       twig 小树枝

       bough 大树枝

       knot 树节

       trunk 树干

       leaf 树叶

       sprout 新芽

       sapling 树苗

       stump 树桩

       root 树根

       root hair 根毛

       taproot 主根

       bark 树皮

       resin 树脂

       pith 木髓

       cambium 形成层

       ring 年轮

       wood 木材

       rose 玫瑰花

       tulip 郁金香

       balsam 凤仙花

       canna 美人蕉

       lily 百合花

       jasmine 茉莉

       sweet pea 香豌豆花

       sunflower 向日葵

       geranium 大竺葵

       morning-glory 牵牛花

       cosmos 大波斯菊

       pansy 三色堇

       poppy **花

       marigold 金盏花

       carnation 麝香石竹

       amaryllis 孤挺花

       dahlia 大丽花

       pink 石竹花

       crocus 番红花

       iris 蝴蝶花

       hyacinth 风信花

       daffodil 黄水仙

       chrysanthemum 菊

       marguerite, daisy 雏菊

       gladiolus 剑兰

       cantury plant 龙舌兰

       magnolia 木兰

       yucca 丝兰

       orchid 兰花

       freesia 小苍兰

       cyclamen 仙客来

       begonia 秋海棠

       anemone 银莲花

       wisteria 柴藤

       redbud 紫荆

       dogwood 山茱萸

       hawthorn 山楂

       camellia 山茶

       hydrangea 八仙花

       hibiscus 木槿

       peony 芍药

       azalea 杜鹃

       rhododendron 杜鹃花

       daphne 瑞香

       gardenia 栀子

       lilac 紫丁香

       night-blooming cereus 仙人掌

       apple 苹果

       pear 梨

       orange 桔子

       quince 柑橘

       apricot 杏

       plum 洋李

       pistil 雌蕊

       ovary 子房

       petal 花瓣

       anther 花药

       stamen 雄蕊

       nectar gland 蜜腺

       sepal 萼片

       stalk 花柄

       pollen 花粉

       snake 蛇

       adder, viper 蝰蛇

       boa 王蛇

       cobra 眼镜蛇

       copperhead 美洲腹蛇

       coral snake 银环蛇

       grass snake 草蛇

       moccasin 嗜鱼蛇

       python 蟒蛇

       rattlesnake 响尾蛇

       lizard 蜥蜴

       tuatara 古蜥蜴

       chameleon 变色龙,避役

       iguana 鬣蜥

       wall lizard 壁虎

       salamander, triton, newt 蝾螈

       giant salamander 娃娃鱼, 鲵

       crocodile 鳄鱼, 非洲鳄

       alligator 短吻鳄, 美洲鳄

       caiman, cayman 凯门鳄

       gavial 印度鳄

       turtle 龟

       tortoise 玳瑁

       sea turtle 海龟

       frog 青蛙

       bullfrog 牛蛙

       toad 蟾蜍

       carp 鲤鱼

       crucian 鲫鱼

       chub 鲢鱼

       eel 鳗鱼

       herring 青鱼, 鲱

       mullet 乌鱼, 黑鱼

       perch 鲈鱼

       pike 梭子鱼

       salmon 鲑鱼

       trout 鳟鱼

       anchovy 凤尾鱼

       anglerfish 安康鱼

       cod 鳕鱼

       hake 无须鳕

       mackerel 鲭,鲐

       plaice 鲽

       red mullet, surmullet 羊鱼

       ray 鳐鱼

       sardine 沙丁鱼

       sailfish 旗鱼

       sea bream 海鲷

       sea horse 海马

       shark 鲨鱼

       skipjack 鲣鱼

       sole 舌鳎

       swordfish 剑鱼

       sturgeon 鲟鱼

       sunfish 翻车鱼

       tarpon 大海鲢

       tunny, tuna 金枪鱼

       turbot 大菱鲆

       whiting 小无须鳕

       ant 蚂蚁

       queen ant 蚁后

       male ant 雄蚁

       termite 蚁

       white ant 白蚁

       worker ant 工蚁

       bee, honeybees 蜜蜂

       bumble bee 大黄蜂

       drone 雄蜂

       queen bee 蜂王

       wasp 黄蜂, 胡蜂

       beetle 甲虫, 金龟子

       Japanese beetle 日本金龟子

       fly 苍蝇

       horsefly, gadfly 厩蝇,牛虻

       flea 跳蚤

       silverfish 蠹虫

       louse, lice 虱子, 白虱

       spider 蜘蛛

       mosquito 蚊

       anopheles 按蚊,疟蚊

       wiggler 孑孓

       ladybird 瓢虫

       glowworm, firefly 萤火虫

       cicada 蝉

       dragonfly 蜻蜓

       cricket 蟋蟀

       locust 蝗虫

       grasshopper 蚱蜢

       praying mantis 螳螂

       caterpillar 毛虫

       centipede 蜈蚣

       butterfly 蝴蝶

       sulphur butterfly 白蝴蝶

       cabbage butterfly 纹白蝶

       pale clouded yellow 纹黄蝶

       swallowtail 凤尾蝶

       moth 蛾

       silkworm moth 蚕蛾

       bedbug, bug 臭虫

       stink bug 椿象

       cockroach 蟑螂

       tarantula 多毛毒蜘蛛

       scorpion 蝎子

       snail 蜗牛

       cuttloefish 乌贼

       squid 枪乌贼,鱿鱼

       octopus 章鱼

       clam 蚌

       cockle 鸟蛤

       mussel 淡菜,贻贝

       oyster 牡蛎

       scallop 扇贝

       sea urchin 海胆

       prawn 虾

       crayfish 小龙虾, 喇蛄

       lobster 龙虾

       shrimp 对虾

       large prawn 大对虾

       Norway lobster 蝉虾

       spiny lobster, rock lobster 大螯虾

       crab 蟹

       hermit crab 寄居蟹

       spider crab 蜘蛛蟹

       earthworm 蚯蚓, 地龙

       leech 蚂蝗, 水蛭

       tapeworm 绦虫

       trichina 旋毛虫

       以上是生物。

       Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate

       by Escherichia coli transformant cells coexpressing

       the carbonyl reductase and glucose dehydrogenase genes

       由共表达碳酰还原酶和葡萄糖脱氢酶的大肠杆菌转化细胞合成

       纯光学（S）-4-氯-3-乙酯

       Abstract The asymmetric reduction of ethyl 4-chloro-3-

       oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate

       ((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the

       catalyst. In an organic-solvent-water two-phase system,(S)-CHBE formed in the organic phase amounted to 2.58 M (430 g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulated 1.25 M (208 g/l) (S)-CHBE in an aqueous monophase system by continuously feeding on COBE, which is unstable in an aqueous solution. In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems. The aqueous system used for the reduction reaction involving E. coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple. Furthermore, the system does not require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.

       本本篇文献研究了利用COBE不对称合成（S）-4-氯-3-乙酯（CHBE）。大肠杆菌细胞作为催化剂同时表达了来自念珠菌属magnoliae的碳酰还原酶和来自巨大芽孢杆菌的葡萄糖脱氢酶基因。在水/有机溶剂两相体系中，(S)-CHBE在有机相中的浓度可以达到2.58M（430g/l），摩尔产率达到85%。大肠杆菌的副产物S1和GDH也达到了1.25M（208g/l），COBE在水相中不稳定，所以(S)-CHBE可以在水单相中不停的生成。在这种情况下，适当的从NADP+到CHBE的转变达到了21,600 mol/mol。所形成的CHBE的旋光度在这种体系中100%对映体过量。在水相中用携带含有S1和GDH基因质粒的E. coli HB101作为催化剂不对称还原是比较简单的。并且，这种体系并不额外需要商业GDH或者有机溶剂。因此，这种体系对于实际合成纯光学活性的(S)-CHBE是非常方便的。

       Optically active 4-chloro-3-hydroxybutanoic acid esters are useful chiral building blocks for the synthesis of pharmaceuticals. The (R)-enantiomer is a precursor of L-carnitine (Zhou et al. 1983), and (S)-enantiomer is an important starting material for hydroxymethylglutaryl- CoA (HMG-CoA) reductase inhibitors (Karanewsky et

       al. 1990). Many studies have described the microbial or enzymatic asymmetric reduction of 4-chloro-3-oxobutanoic acid esters (Aragozzini and Valenti 1992; Bare et al.1991; Hallinan et al. 1995; Patel et al. 1992; Shimizu et al. 1990; Wong et al. 1985) based on the reduction by baker’s yeast (Zhou et al. 1983).We have previously showed that Candida magnoliae AKU4643 cells reduced ethyl 4-chloro-3-oxobutanoate (COBE) to (S)-CHBE with an optical purity of 96% enantiomeric excess (e.e.) (Yasohara et al. 1999). As this yeast has at least three different stereoselective reductases (Wada et al. 1998, 1999a, b), the (S)-CHBE produced by this yeast was not optically pure. From among these three enzymes, an NADPH-dependent carbonyl reductase, designated as S1, was purified and characterized in some detail (Wada et al. 1998). We cloned and sequenced the gene encoding S1 and overexpressed it in Escherichia coli cells. This E. coli transformant reduced COBE to optically pure (S)-CHBE in the presence of glucose, NADP+, and commercially available glucose dehydrogenase (GDH) as a cofactor generator (Yasohara

       et al. 2000).

       Here, we describe the construction of three E. coli transformants coexpressing the S1 from C. magnoliae and GDH from Bacillus megaterium genes and analyze the reduction of COBE catalyzed by these strains. Previous reports on the enzymatic reduction of COBE to (R)-CHBE with an optical purity of 92% e.e. (Kataoka et al. 1999; Shimizu et al. 1990) recommended an organic- solvent two-phase system reaction for an enzymatic or microbial reduction, because the substrate (COBE) is unstable in an aqueous solvent and inactivates enzymes. We examined the reduction of COBE to optically pure (S)-CHBE by E. coli transformants in a water monophase system reaction and discuss the possible use of this type of reaction system in industrial applications。

       具有旋光性的（S）-4-氯-3-乙酯在药物制剂的合成中是重要的手性化合物。其右旋体是L-卡尼汀的前体，其左旋体是羟甲基戊二酰辅酶A还原酶抑制剂的起始材料。许多研究描述了以面包酵母为基础微生物或者酶的COBE的不对称还原。我们先前已经知道利用来自念珠菌属magnoliae AKU4643 细胞催化COBE生成光学纯度96%的CHBE。这种酵母至少有三种立体选择性的还原酶，这种酵母产生的CHBE并非纯光学的，在这三种酶之中，NADPH-依赖碳酰还原酶，我们克隆并测序编码S1的基因，并在大肠杆菌中过表达。大肠杆菌转化细胞在葡萄糖，NADP+和商业化的葡萄糖脱氢酶作为辅酶因子的启动子催化COBE生成纯光学的CHBE。

       我们构建这三种大肠杆菌转化细胞共表达来自的S1和来自巨大芽孢杆菌的GDH，并分析COBE被这几种菌株催化还原的反应机理。先前的报道表明，利用酶催化还原COBE生成CHBE光学纯度可达92%，也提到了因为底物（COBE）在水相中不稳定，并且酶容易钝化，所以利用酶或者微生物在有机溶剂/水两相体系中催化反应。我们研究了在水单相体系中由COBE还原生成纯光学的CHBE，还讨论了这种反应体系在工业应用中可能的用途。

       Materials and methods

       Bacterial strain and plasmids

       The E. coli strains used in this study were JM109 and HB101.Plasmid pGDA2, in which the GDH gene from B. megaterium is inserted into pKK223-3, was kindly provided by Professor I. Urabe, Osaka University (Makino et al. 1989). Plasmids pSL301 and pTrc99A were purchased from Invitrogen (USA), and Amersham Pharmacia Biotech (UK), respectively. Plasmids pUC19 and pSTV28 (Homma et al. 1995; Takahashi et al. 1995) were purchased from Takara Shuzo (Japan).

       材料和方法

       菌株和质粒

       本次实验中使用的大肠杆菌是JM109 and HB101。来自B. megaterium的GDH基因插入到Pkk233-3质粒中，而带有GDH基因片段的pGDA2质粒由到由大阪大学的urabe教授提供。质粒pSL301和 pTrc99A是由美国的Invitrogen公司和英国的公司分别购买的。质粒pUC19和pST28是由日本takara公司购买的。

       The recombinant plasmid used in this study was constructed as follows (Fig. 1): Plasmid pGDA2 was double-digested with EcoRI and PstI to isolate a DNA fragment of about 0.9 kilobase pairs (kb) including the GDH gene. This fragment was inserted into the EcoRI-PstI site of plasmid pSL301 to construct plasmid pSLG. Plasmid pSLG was double-digested with EcoRI and XhoI to isolate a DNA fragment of about 0.9 kb including the GDH gene.

       这次实验使用的重组质粒构建如下：质粒pGDA2 被EcoRI 和 PstI双酶切从而分离出一个大小约为0.9kb的包含有GDH基因的DNA片段。这个片段被插入到质粒Psl301的EcoRI-PstI酶切位点从而构建出质粒pSLG。质粒pSLG被EcoRI和XhoI

        To construct plasmid pNTS1G, this 0.9-kb fragment was inserted into the EcoRI-SalI site of pNTS1, which was constructed to overproduce S1 as described previously (Yasohara et al. 2000). To construct plasmid pNTGS1, plasmid pNTG was first generated. Two synthetic primers (primer 1, TAGTCCATATGTATAAAGATTTAG,and primer 2 TCTGAGAATTCTTATCCGCGTCCT) were prepared for polymerase chain reaction (PCR) using pGDA2 as the template. The PCR-generated fragment was double- digested with NdeI and EcoRI and then inserted into the NdeI EcoRI site of plasmid pUCNT, which was constructed from pUC19 and pTrc99A, as reported (Nanba et al. 1999), to obtain pNTG. To construct plasmid pNTGS1, two synthetic primers (primer 3, GCCGAATTCTAAGGAGGTTAATAATGGCTAAGAACTTCTCCAACG, and primer 4, GCGGTCGACTTAGGGAAGCGTGTAGCCACCGTC) were prepared using pUCHE, which contains the S1 gene as the template. The PCR-generated fragment was double-digested with EcoRI and SalI and then inserted into the EcoRI-SalI site of pNTG to obtain pNTGS1. Plasmid pNTS1G, pNTGS1 or pNTG was transformed into E. coli HB101.

       构建pNTS1是为了过表达前文所提到的S1，这个0.9kb大小的片段被插入到pNTS1的EcoRI-SalI酶切位点从而构建pNTS1G。为了构建质粒pNTGS1，首先需要构建pNTG。两个合成引物（引物1，TAGTCCATATGTATAAAGATTTAG和引物2，TCTGAGAATTCTTATCCGCGTCCT）和作为模板的pGDA2是PCR反应需要的。PCR得到的片段是由NdeI 和EcoRI双酶切和并插入到质粒pUCNT的NdeI EcoRI酶切位点来得到pNTG。根据报道，pUCNT是由pUC19和 pTrc99A构建而来。为了构建质粒pNTGS1，两个合成引物(引物 3, GCCGAATTCTAAGGAGGTTAATAATGGCTAAGAACTTCTCCAACG, and 引物 4, GCGGTCGACTTAGGGAAGCGTGTAGCCACCGTC)，包括了S1基因作为模板。Pcr产物片段被EcoRI和SalI双酶切然后被插入到pntg的EcoRI-SalI酶切位点得到pntg1.质粒pNTS1G, pNTGS1或者 pNTG都是导入大肠杆菌HB101.

       Plasmid pGDA2 was double-digested with EcoRI and PstI to isolate a DNA fragment of about 0.9 kb including the GDH gene. To construct plasmid pSTVG, this fragment was inserted into the EcoRI-PstI site of plasmid pSTV28. Plasmid pSTVG was transformed into E. coli HB101.

        质粒pGDA2被EcoRI 和 PstI双酶切得到包含GDH基因的0.9kb大小的DNA片段。为了构建pSTVG质粒，这个片段被插入到pSTV28质粒的EcoRI-PstI的酶切位点。pSTVG质粒被导入到E. coli HB101。

       Medium and cultivation

       The 2×YT medium comprised 1.6% Bacto-tryptone, 1.0% yeast

       extract, and 0.5% NaCl, pH 7.0. E. coli HB 101 carrying pNTS1,

       pNTG, pNTS1G, or pNTGS1 was inoculated into a test tube containing

       2 ml 2×YT medium supplemented with 0.1 mg/ml ampicillin,

       followed by incubation at 37 °C for 15 h with reciprocal shaking.

       This preculture (0.5 ml) was transferred to a 500-ml shaking

       flask containing 100 ml 2×YT medium. The cells were cultivated

       at 37 °C for 13 h with reciprocal shaking. E. coli HB101 carrying

       pNTS1 and pSTVG was similarly cultivated in 2×YT medium

       supplemented with 0.1 mg/ml ampicillin and 0.1 mg/ml chloramphenicol.

       培养基和培菌

       2*YT培养基 包含有1.6%细菌用胰蛋白胨，1.0%酵母提取物，0.5% NaCl，pH7.0.

       携带有pNTS1,pNTG, pNTS1G, 或 pNTGS1的大肠杆菌HB101被接种到有0.1mg/ml氨苄青霉素的2ml的2*YT培养基，37°C摇床15小时。将0.5ml菌液接种到100ml2*YT培养基的500ml烧瓶中。在37°C摇床培养13小时。携带有pNTS1 和 pSTVG质粒的大肠杆菌HB101在2*YT培养基中培养方法相似，只是培养基中要加入0.1 mg/ml的氨苄青霉素和 0.1 mg/ml的氯霉素。

       Preparation of cell-free extracts and the enzyme assay

        Cells were harvested from 100 ml of culture broth by centrifugation, suspended in 50 ml of 100 mM potassium phosphate buffer (pH 6.5), and then disrupted by ultrasonication. The cell debris was removed by centrifugation; the supernatant was recovered as the cell-free extract. Carbonyl reductase S1 activity (COBE-reducing activity) was determined spectrophotometically as follows: The assay mixture consisted of 100 mM potassium phosphate buffer (pH 6.5), 0.1 mM NADPH, and 1 mM COBE. The reactions were incubated at 30 °C and monitored for the decrease in absorbance at 340 nm. The assay mixture for GDH activity consisted of 1 M Tris-HCl buffer (pH 8.0), 100 mM glucose, and 2 mM NADP+. The reactions were incubated at 25 °C and monitored for the increase in absorbance at 340 nm. One unit of S1 or GDH was defined as the amount catalyzing the reduction of 1 μmol NADP+ or oxidation of 1 μmol NADPH per minute, respectively. Protein concentrations were measured with a protein

       assay kit containing Coomassie brilliant blue (Nacalai Tesque, Japan),

       using bovine serum albumin as the standard (Bradford 1976).

       无细胞抽提液和酶鉴定

       将100ml培养液离心收获菌体，用50ml0.1mol/LpH为6.5的磷酸缓冲液悬浮，然后超声粉碎。细胞碎片通过离心可以去除，收集上层清液就是无细胞抽提物。碳酰还原酶S1的活性由分光光度计测量如下：测定的混合物包括：0.1mol/LpH6.5的磷酸二氢钾缓冲液，0.1mMNADPH和1mMCOBE。反应在30°C条件下反应，并且随时监测其在340nm处的吸光值。测GDH混合物包括：1M pH 8.0的Tris-HCl的缓冲液，100mM的葡萄糖，2mM的NADP+。反应在25°C下进行，监测其在340nm处的吸光值。一个单位S1或GDH被定义为每分钟催化还原1μmol NADP+或氧化1 μmol NADPH的量。蛋白质的测定通过含有考马斯亮蓝的蛋白质测定试剂利用牛血清白蛋白作为标准进行测定。

       Study of enzyme stability

       One milliliter of 100 mM potassium phosphate buffer (pH 6.5) containing the cell-free extracts of E. coli HB101 carrying pNTS1 (S1: 20 U/ml) was mixed with an equal volume of each test organic solvent in a closed vessel. After the mixture was shaken at 30 °C for 48 h, the remaining enzyme activities in an aqueous phase were assayed as described above. The mixture, containing 100 mM potassium phosphate buffer (pH 6.5), S1 (20 U/ml), and various concentrations of CHBE, was incubated at 30 °C for 24 h in order to study the enzyme’s stability in the presence of CHBE.The remaining enzyme activities were assayed as described above.

       酶稳定性的研究

       一毫升含有含有pNTS1质粒的E. coli HB101的无细胞抽提液的100mM磷酸氢二钾缓冲液（pH6.5）与等体积的有机溶剂混合。混合物在30 °C震摇48小时后，水相中残留的酶活力即是上述的酶活力。

       COBE reduction with E. coli cells expressing the S1 gene and E. coli cells expressing GDH genes in a two-phase system reaction

       The reaction mixture comprised 15 ml culture broth of E. coli HB101 carrying pNTG, 17 ml culture broth of E. coli HB101 carrying pNTS1, 1.6 mg NADP+, 4 g glucose, 2.5 g COBE, 25 ml n-butyl acetate, and about 25 mg Triton X-100. The pH of the reaction mixture was controlled at 6.5 with 5 M sodium hydroxide. At 2 h, 1.25 g COBE and 2.5 g glucose were added to the reaction mixture. To compare the reaction by E. coli transformant coexpressing the GDH and S1 genes, 30 ml culture broth of E. coli

       HB101 carrying pNTS1G was used instead of culture broth of E. coli HB101 carrying pNTG and E. coli HB101 carrying pNTS1. Other components and the procedure were the same as described above.

       表达S1基因和GDH基因的大肠杆菌细胞在两相反应体系中的还原反应

       混合物包含有带有pNTG质粒的大肠杆菌HB101的菌液15ml，pNTS1质粒的大肠杆菌HB101的菌液17ml，1.6 mg NADP+，4 g葡萄糖，2.5g的COBE，25ml的n-butyl acetate丁酰醋酸盐和大约25mg的聚乙二醇辛基苯基醚Triton X-100。用5M的NaOH溶液将pH控制在6.5。在反应两小时后，加入1.25gCOBE和2.5g葡萄糖到该混合物中。比较大肠杆菌转化细胞共表达GDH和S1基因，携带有pNTS1G质粒的大肠杆菌HB10130ml菌液取代了携带有pNTG和pNTS1质粒的大肠杆菌HB101菌液。其他的成分和步骤和上述的方法相似。

        COBE reduction to (S)-CHBE in a two-phase system reaction

        The reaction mixture contained 50 ml of culture broth of an E. coli HB101 transformant, 3.2 mg NADP+, 11 g glucose, 10 g COBE, 50 ml n-butyl acetate, and about 50 mg Triton X-100. The reaction mixture was stirred at 30 °C, and the pH was controlled at 6.5 with 5 M sodium hydroxide. Five grams of COBE/5.5 g glucose and 10 g COBE/11 g glucose were added to the reaction mixture at 3 h and 7 h, respectively; 3.2 mg NADP+ was added at 26 h.

       COBE在两相系统中还原生成（S）-CHBE

       反应混合物包含50ml E. coli HB101转化细胞的培养液，3.2mgNADP+,11g

       葡萄糖，10gCOBE，50ml丁酰醋酸，和大概50mg聚乙二醇辛基苯基醚Triton X-100.

       在30°C温度下将其混合均匀，并用5M的NaOH溶液将pH控制在6.5。在第3小时加入5gCOBE和5.5g葡萄糖或者在第7小时加入10gCOBE和11g葡萄糖，分别在第26小时加入3.2gNADP+。

        COBE reduction to (S)-CHBE in an aqueous system reaction

       The reaction mixture was made up of 50 ml of culture broth of an E. coli HB101 transformant, 3.1 mg NADP+, 11 g glucose, and about 50 mg Triton X-100. The reaction mixture was stirred at 30 °C. Fifteen grams of COBE was fed continuously by means of a micro-feeding machine at a rate of about 0.02 g/min for about 12 h. The pH of the reaction mixture was controlled at 6.5 with 5 M sodium hydroxide. The reaction mixture was extracted with 100 ml ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and then evaporated in vacuo.

       COBE在水相中还原成(S)-CHBE的反应

       反应的体系是由50ml大肠杆菌HB101转化细胞的菌液，3.1mgNADP+，11g葡萄糖和大约50mg聚乙二醇辛基苯基醚Triton X-100。反应混合物在30°C15mg的COBE通过微量添加机器以0.02 g/min的速率连续12小时恒定的加入到体系中。用5M的NaOH溶液将pH控制在6.5。反应混合物用100ml乙酸乙酯萃取。有机层用无水硫酸钠吸干，并在真空中脱水。

       Analysis

       The organic layer was obtained on centrifugation of the reaction mixture and was assayed for CHBE and COBE by gas chromatography. Optical purity of CHBE was analyzed by high-performance liquid chromatography (HPLC), as described previously (Yasohara et al. 1999).

       Enzymes and chemicals

       Restriction enzymes and DNA polymerase were purchased from

       Takara Shuzo (Japan). COBE (molecular weight: 164.59) was purchased

       from Tokyo Kasei Kogyo (Japan). Racemic CHBE (molecular

       weight: 166.60) was synthesized by reduction of COBE with

       NaBH4. All other chemicals used were of analytical grade and

       commercially available.

       分析

       离心反应混合物得到的有机层通过气相色谱法测定其CHBE和COBE。COBE的光学纯度如前所述通过高效液相色谱法进行分析。

       酶和化学试剂

       限制性内切酶和DNA聚合酶由takara公司购得，COBE（分子量：164.59）由东京Tokyo Kasei Kogyo公司购得，消旋体CHBE（分子量166.6）通过COBE及NaBH4合成。所有其他化学试剂都是分析等级和商业化的试剂。

       Construction of E. coli transformants overproducing S1 and GDH

       To express the carbonyl reductase S1 and GDH genes in the same E. coli cells, four expression vectors were constructed (Fig. 1). Plasmids pNTS1G and pNTGS1 contain the S1 gene from C. magnoliae, the GDH gene from B. megaterium, the lac promoter derived from pUC19, and the terminator derived from pTrc99A. Plasmid pNTS1 contains the S1 gene, the lac promoter derived from pUC19, and the terminator derived from pTrc99A. The enzyme activities in cell-free extracts of the E. coli transformants are shown in Table 1. E. coli HB101 cells carrying the vector plasmid pUCNT had no detectable S1 or GDH activity. E. coli HB101 carrying either pNTS1G or pNTGS1 showed S1 and GDH activity without isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The S1 activities of these two transformants were lower than the GDH activities. To obtain a transformant whose S1 activity was equal to or greater than the level of GDH activity, we used a lower copy vector, pSTV28 (Homma et al. 1995; Takahashi et al. 1995), to express the GDH gene. It may be possible to raise the S1 activity by lowering the GDH activity. Plasmid pSTVG contains the GDH gene, the lac promoter, the chloramphenicol resistance gene, and the replicative origin derived from pACYC184 for compatibility with the plasmid pNTS1. In E. coli HB101 carrying pNTS1 and pSTVG, the S1 activity was higher than the GDH activity, but this GDH

       level may be too low to regenerate in a COBE reduction reaction as described below.

       过产生S1和GDH的大肠杆菌转化细胞的构建

       为了在同一大肠杆菌细胞中表达碳酰还原酶S1和GDH基因，要构建四个表达型载体。质粒pNTS1G 和 pNTGS1包含有来自C. magnoliae的S1基因，来自B. megaterium的GDH基因，来自pUC19的LAC启动子，从pTrc99A的来的终止子，质粒pNTS1包含有S1基因，来自pUC19的LAC启动子，从pTrc99A的来的终止子。在大肠杆菌转化细胞的无细胞抽提物的酶活力如表一所示。携带有运输质粒pUCNT的大肠杆菌细胞无法检测到其S1和GDH活性。携带有pNTS1G 或 pNTGS1质粒在没有IPTG的诱导下有S1和GDH的活性。在这两个转化菌种中，S1的活力小于GDH的活力。为了得到S1活性等于或者大于GDH的大肠杆菌转化菌株，我们使用低拷贝的载体pSTV28，来表达GDH基因。它可能可以通过降低GDH的活性从而提高S1的活性。质粒pSTVG包含有GDH基因，lac启动子，和氯霉素抗性基因，以及与pNTS1具有相容性的从pACYC184得来的复制起始位点。在携带有pNTS1和pSTVG的大肠杆菌转化细胞中，S1的活性要高于GDH的活性，但是GDH的活性可能会太低而在COBE还原反应中不能再生。

       太长了，字数有限制，所以不能发完。分数我无所谓啦，我很少登录的。这应该算是基因工程的吧，是我以前自己翻的，不是很好。如果你要的话可以联系我的邮箱。iamecho23@163.com

       好了，今天关于“triton和flask的区别”的话题就讲到这里了。希望大家能够通过我的介绍对“triton和flask的区别”有更全面的认识，并且能够在今后的实践中更好地运用所学知识。如果您有任何问题或需要进一步的信息，请随时告诉我。