火山岩滤料固定化恶臭假单胞菌条件的优化及其吸附铜离子的研究

以多孔介质火山岩滤料为载体,探讨了温度、转速、反应器的底面积等因素对滤料固定化恶臭假单胞菌的影响,比较了固定化恶臭假单胞菌野生菌和重组菌吸附Cu2+的效果.结果表明,火山岩滤料固定化恶臭假单胞菌的最优条件为选择底面积较大的反应器、30℃、静置条件下吸附3.5h.固定化野生菌、重组菌滤料及空白滤料对Cu2+的吸附率依次为:74.76％、89.36％和55.09％.为多孔载体固定化微生物在废水处理中的应用提供了实验依据.     

生物催化剂在手性药物合成中的应用

基因表达及功能分析需要高通量的表达系统,因而简单、经济且高效地将PCR产物或其他来源的目的基因片段构建到质粒载体中就成为了亟需解决的问题.传统克隆方法使用限制性内切酶和连接酶,其缺点是受到基因序列中原有的酶切位点的限制以及依赖连接酶导致的较低的连接转化率.为了克服这些缺点,一些不需要限制酶和连接酶的克隆方法被开发出来并运用到基因克隆中,获得了更佳的实验结果.该文就LIC、UDG克隆和Gateway重组克隆这三种不依赖连接反应的高通量克隆方法的原理及特性进行介绍.     

恶臭假单胞菌HspB的单晶培养及结晶条件优化

为了获得可用于X射线衍射的恶臭假单胞菌尼古丁代谢途径中关键单加氧酶Hsp B的单晶。定点突变PCR构建重组质粒,大肠杆菌中诱导表达,镍柱亲和层析、烟草蚀纹病毒(Tobacco etch virus,TEV)蛋白酶酶切和凝胶过滤层析纯化,悬滴扩散法进行结晶。成功构建重组质粒并获得高表达;比较了TEV蛋白酶柱上及透析酶切的效率,TEV蛋白酶透析酶切效率更高;确定了该纯化路线,获得电泳纯级的Hsp B蛋白。结晶条件初筛和正交优化后获得可培养Hsp B蛋白单晶的条件为22%PEG3350、0.1 mol/L Bis-Tris p H6.5、0.21 mol/L Mg Cl2、18℃、1?50比例加晶种。去除标签后的Hsp B蛋白获得了分辨率1.8?的单晶。     

假单胞菌L-半胱氨酸合成酶的纯化和性质研究

假单胞菌TS-1138的细胞浆液通过硫铵沉淀、SephadexG-75凝胶过滤、DEAE-Cellulose52离子交换、SephadexG 100凝胶过滤等分离纯化手段分别将从L-ATC合成L 半胱氨酸的两个酶——L-ATC水解酶和L-SCC水解酶纯化了 83.9和 90.3倍。SDS-PAGE鉴定均为单一条带 ,两种酶的相对分子质量分别为 37.5和 42.8kD ;酶反应的最适温度均为 35℃ ,最适pH分别为 7.0和 8.0 ;酶的米氏常数分别为 0.67mmol L和 0.15mmol L ,     

产芳香腈水解酶的恶臭假单胞菌Pseudomonas putida CGMCC3830的筛选、鉴定及发酵优化

近年来微生物腈水解酶水解腈类化合物制备有机酸已逐步受到关注。本研究分离到一株表现出较高腈水解酶活力的细菌菌株,通过形态学、生理生化实验以及16S rRNA基因序列分析将其鉴定为恶臭假单胞菌Pseudomonas putida CGMCC3830。结合单因素及响应面法对该菌株产腈水解酶的发酵条件进行了优化,获得最适培养条件为:甘油13.54 g/L,胰蛋白胨11.59 g/L,酵母粉5.21 g/L,KH2PO4 1 g/L,NaCl 1 g/L,脲1 g/L,初始pH 6.0及培养温度30℃。通过优化,酶活由2.02 U/mL提升至36.12 U/mL。对该菌株底物特异性的考察结果表明,恶臭假单胞菌腈水解酶对芳香族腈类化合物具有较高的水解活力。将其应用于烟酸的生物合成中,2 mg/mL游离细胞能90 min内将20.8 g/L 3-氰基吡啶彻底转化,制备得到相应烟酸。这些结果表明恶臭假单胞菌P.putida CGMCC3830在烟酸的规模化生产中具有一定的应用潜力。     

假单胞菌L-11产生生物表面活性剂发酵条件的优化

确定了假单胞菌L-11用葡萄糖为底物产生生物表面活性剂的最适发酵培养基组成和发酵条件。在1L的发酵罐中,L-11的发酵液（10％）与原油的界面张力可以达到5.3×10^-3mN/m。该产品可以用于微生物提高原油采收率的实验研究。对其放大工艺也进行了初步的研究。     

杂交鲇恶臭假单胞菌的分离鉴定及其病理损伤研究

为确定一起杂交鲇皮肤溃疡症的病原,实验从病鱼体内分离到几株优势菌（DYJ140914-DYJ140917）,根据4株分离菌的形态、生理生化特性,结合16S rRNA和gyrB基因序列测定（GenBank登录号分别为KP693689和KP693690）与系统发育分析,将其鉴定为恶臭假单胞菌（Pseudomonas putida）。在此基础上以腹腔注射的方式进行人工感染试验,证实其为杂交鲇溃疡症的病原菌。病鱼组织器官具有典型的病理变化,其主要靶器官为肝脏、皮肤肌肉以及肾间质,分别引起多灶性坏死性肝炎、坏死性肌炎及坏死性间质性肾炎。此外,还可引起心外膜、心内膜炎及坏死性脾炎。药敏结果显示该菌对强力霉素、诺氟沙星和左氧氟沙星等药物敏感;对青霉素、氟苯尼考、磺胺甲基异恶唑、头孢西丁、阿奇霉素等药物耐药。     

恶臭假单胞菌S1产胞内海藻糖合成酶发酵培养基的优化

采用摇瓶发酵法研究了不同碳源、氮源对恶臭假单胞菌S1干重和胞内海藻糖合成酶酶活力的影响。通过正交试验得出其最佳培养基配方为(g.L-1):葡萄糖25,玉米浆15,麦芽糖20,豆粕水解液10 mL.L-1,Na3C6H5O7.2 H2O 0.5,Na2HPO4.12 H2O 0.5。优化后菌体密度、单位细胞产酶活力分别提高了1.44倍和72.16%。     

一种新型环氧化物水解酶的发现及其催化机理的阐明（英文）

【目的】环氧化物水解酶（epoxide hydrolases,EHs）在手性药物的合成中起着重要作用。为了补充和发现更多的高性能环氧化物水解酶,通过基因调取技术探索新的环氧化物水解酶。【方法】通过基因调取技术鉴定了一种来自卡尔斯巴德曲霉的新型环氧化物水解酶（Aspergillus carlsbadensis epoxide hydrolase,Ac EH）。采用Auto Dock2预测Ac EH的关键水解位点,通过计算设计阐明重要位点对Ac EH的结构和催化机制的影响。【结果】对新型Ac EH一级结构的分析揭示了3个特征性α/β EH基序的存在：HGWP、GYTFS和GGDIGS。Ac EH酶表现出高活性,可在15 min内完全水解氧化苯乙烯（styrene oxide,SO）,比活性为13 951 U/g。K_m、V_max和k_cat/K_m值分别为（107.07±57.98） mmol/L、（37.22±17.85）μmol/（min·mg）和1.17 mmol/（L·s）。Ac EH的关键水解位点是催化三联体的Asp192-His372-Glu346和2个保守酪氨酸Tyr251/314。某些突变（R49L、R49Y）导致酶失活,而其他突变（Y45L）导致无活性包涵体的形成。相互作用网络分析显示,第49个氨基酸残基的变化破坏了重要活性位点残基的相互作用,导致酶失活。另一方面,第45个氨基酸残基的改变使酶的结构不稳定,导致包涵体的形成。【结论】本研究发现了一种新的环氧化物水解酶,并分析了其水解机制,为进一步研究这种酶及其工业化应用提供了有价值的见解。     

假单胞菌L-11产生生物表面活性剂发酵条件的优化

确定了假单胞菌L-11用葡萄糖为底物产生生物表面活性剂的最适发酵培养基组成和发酵条件。在1L的发酵罐中,L-11的发酵液（10％）与原油的界面张力可以达到5.3×10-3mN/m。该产品可以用于微生物提高原油采收率的实验研究。对其放大工艺也进行了初步的研究。     

Biocatalysis in natural products chemistry

The properties of enzymes and microbial cells as biocatalysts useful in natural products chemistry are discussed from the perspective of the chemical transformations they catalyse. Attention is focused on numerous reactions of value to natural products chemists, including the acyloin condensation, Baeyer-Villiger oxidation, regio- and enantioselective ester hydrolyses, oxidations of aromatic and non-aromatic substrates, oxidoreduction and O- and N-dealkylations. Compounds considered in this review include amino acids, alkaloids, antibiotics, coumarins, naphthoquinones, quassinoids, rotenoids and mono-, sesqui-, di- and triterpenoid substrates. The value of biocatalysis compared with traditional chemical catalysis is considered within the broad framework of natural products chemistry, and the potential for using immobilized enzyme and cell technology is presented.     

Development of chiral drugs in Japan: An update on regulatory and industrial opinion

The purpose of this commentary is to provide information on the present status of the racemate/enantiomer debate in Japan and current industrial and regulatory attitudes to chiral drugs in Japan. It provides an update of our previous paper (Shindo and Caldwell, Chirality 3:91–93, 1991), and the interested reader is referred to this for background information. © 1995 Wiley-Liss, Inc.     

Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.     

Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.     

Biocatalysis of nitro substituted styrene oxides by non-conventional yeasts

Yeast strains (410) from more than 45 different genera were screened for the enantioselective hydrolysis of nitro substituted styrene oxides. These strains included 262 yeasts with known epoxides hydrolase activity for various other epoxides. Epoxide hydrolase activity for p-nitrostyrene oxide (pNSO) (177 strains) and m-nitrostyrene oxide (mNSO) (148 strains) was widespread in the yeasts, while activity for o-nitrostyrene oxide (oNSO) was less ubiquitous (22 strains). The strains that displayed enantioselectivity in the hydrolysis of one or more of the nitro substituted styrene oxides (35 strains) were also screened against styrene oxide (SO). Rhodosporidium toruloides UOFS Y-0471 displayed the highest enantioselectivity for pNSO (ee 55%, yield 35%) while Rhodotorula glutinis UOFS Y-0653 displayed the highest enantioselectivity for mNSO (ee >98%, yield 29%), oNSO (ee 39%, yield 19%) and SO (ee >98%, yield 19%). (R)-Styrene oxide was preferentially hydrolysed to the corresponding (R)-diol with retention of configuration at the stereogenic centre. In the case of the nitro substituted styrene oxides the absolute configurations of the remaining epoxides and the formed diols were not established.     

Biocatalysis of theophylline oxidation by microbial theophylline oxidase in the presence of non-physiological electron acceptors

Microbial theophylline oxidase (ThOx) is a redox enzyme catalysing 8-hydroxylation of theophylline to form 1,3-dimethyluric acid. In this work, ThOx has been characterized as a fragile haem-containing protein complex composed of several non-covalently bound dynamic domains with molecular weights of around 60 and 210 kDa, and capable of formation of 1.5 MDa assemblies as well. The rate of theophylline oxidation by ThOx with the non-physiological electron acceptor ferricyanide was 0.17 s^?1, approaching that with cytochrome c, 0.33 s^?1. The apparent catalytic constant depended on the electron acceptor concentration. At concentrations lower than 0.2 mM the reaction did not fit the Michaelis–Menten scheme, and some non-catalytic processes dominated in the overall reaction. The kinetics of ThOx catalysis were also studied at electrodes modified with self-assembled monolayers (SAM) of hydroxyl- and amine-terminated alkanethiols. Different compositions of the SAM provide different orientations of ThOx on these layers. Depending on the orientation of ThOx onto the SAM-modified electrodes, the heterogeneous electron transfer (ET) constant, k_s, which characterizes the ET reaction between the electrodes and the haem of ThOx (E^o/ of 87 mV (NHE)) was 0.4 s^?1 and 3.2 s^?1. Only the low-ET-rate orientation appeared to be productive for the electrocatalytic function of ThOx, giving a reaction similar to that with ferricyanide and cytochrome c. The apparent efficiency of ThOx bioelectrocatalysis in the absence of mediators was substantially lower than that mediated by ferricyanide or cytochrome c. This lower efficiency is consistent with a correspondingly lower amount of ThOx being in direct ET contact with the electrodes and thus involved in electrocatalysis.     

RNA药物的发展及其在水产上的应用

基于RNA的RNA干扰和基因组编辑技术被应用于许多领域.由于其作用的特异性,使RNA成为靶点药物的候选分子.基于RNA的疾病治疗药物的研发近年来有了迅速的发展.随着养殖业的发展,病害引起的损失日益严重.运用小分子RNA药物有效保护水产动物抵抗病毒、寄生虫等的危害的研究也取得了一些成果.综述了RNA干扰和CRISPR的作...     

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

A Specific Effect of Antipsychotic Drugs on Protein Synthesis in Human Lymphomononuclear Cells

We have studied the effects of psychotropic drugs on patterns of protein synthesis in human lymphomononuclear cells by two-dimensional gel electrophoretic analysis. Drugs effective in treatment of schizophrenia specifically increased the relative synthesis of a 30-kDa polypeptide in cultured human lymphomononuclear cells whereas dopamine (DA) or psychoactive drugs lacking antipsychotic properties did not. The effect was stereospecific with respect to the clinically active and inactive isomers of flupenthixol. Synthesis of the 30-kDa polypeptide appears therefore to be correlated with antipsychotic properties but not with DA receptor binding. It is possible that such effects may be associated with the clinically beneficial effect of antipsychotic drugs in the brain.     

Regioselective Enzymatic Synthesis of Non-Steroidal Anti-Inflammatory Drugs Containing Glucose in Organic Media

Enzymatic transesterification of glucose with the vinyl ester of non-steroidal anti-inflammatory drugs (NSAIDs) was in organic media performed for synthesis of novel NSAIDs-glucose conjugates. Glucose was regioselectively acylated at the 6-hydroxyl group. The indomethacin-glucose conjugate and ketoprofen-glucose conjugate were produced by the catalysis of alkaline protease from Bacillus subtilis in the respective yields of 42% (over 48 h) and 63% (over 40 h). The etodolac-glucose conjugate was obtained in 26% yield (over 144 h) by lipase from Candida antarctica.