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 ,     

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%。     

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。     

假单胞菌胞外多糖发酵条件的研究

研究了假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓｓｐ．６）利用木糖产胞外多糖的发酵条件。实验表明ＫＨ２ＰＯ４,Ｏ２和高碳氮比对多糖合成有促进作用,在发酵后期补加木糖有助于多糖产量的提高     

甲基对硫磷降解菌假单胞菌WBC-3的筛选及其降解性能的研究

从农药污染土样中分离出的一株细菌具有彻底降解甲基对硫磷的能力。该菌经生理生化特性分析和16S rDNA序列同源性分析,鉴定为假单胞菌属,命名为Pseudomonas sp.WBC\|3。该菌在pH7～8、温度23℃～30℃范围内均生长良好,对甲基对硫磷的耐受浓度在单纯无机盐培养基中可达到800mg/L,在含有01%葡萄糖的培养基中可达到2000mg/L。该菌能够以甲基对硫磷作为唯一碳源、氮源,将其彻底降解作为生长基质,对于300mg/L甲基对硫磷的降解速度达15mg/L\5h,于22h后达到其稳定生长期。该菌对于多种有机磷农药及部分芳烃类化合物具有生化代谢能力。从该菌的细胞周质组分中纯化出的有机磷水解酶在SDSPAGE胶上显示为分子量约为33.5×103的条带。     

生物信息学在工业生物催化研究中的应用

随着石油等不可再生资源的日益减少以及环境污染问题的日益严重,应用工业生物催化技术改造或取代传统化工工艺已经成为新世纪化学工业可持续发展的研究热点。工业生物催化技术的研究对象是生物催化剂及其催化过程。近来,利用生物信息学技术进行工业生物催化研究已经越来越受到人们的重视。随着工业生物催化的发展,生物信息学将直接指导并加快新型高效生物催化剂的发现及功能改造进程。     

环氧化物的生物不对称合成

手性环氧化物是有机化学合成中重要的中间体;与以往化学合成方法相比,手性环氧化物的生物合成有其独特之优点;本文从直接加氧、间接环氧和酶解拆分三个途径全面地介绍该领域的研究成果和最新进展,并对其未来的发展和方向进行了展望 。     

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.     

手性药物合成中的生物转化

目前手性药物的发展非常迅速,本文介绍了利用微生物及其酶系作为生物催化剂,进行外消旋底物的拆分或前手性底物的不对称化,以合成手性药物的生物转化方法;并评述了生物转化在合成手性药物这一领域的应用现状及今后的发展趋势。     

Application of lipases in kinetic resolution of racemates

Lipases have been well established as valuable catalysts in organic synthesis. This review article focuses on some of the recent developments in the rapidly growing field of lipase-catalyzed kinetic resolution of racemates as a versatile method for the separation of enantiomers. The literature search dates back to the last five years and covers some comprehensive examples. The main emphasis is on the use of lipases in organic solvents.     

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.