转dct结构基因固氮斯氏假单胞菌工程菌株构建及其特性研究

固氮斯氏假单胞菌(Pseudomonas stutzeri)A1501是一种定殖于水稻根部的联合固氮菌。将含有A1501四碳Z-羧酸转运系统(Dct-transport system)结构基因dctPQM的亚克隆大片段克隆到具有广泛宿主范围转移特性的质粒载体pSZ21上。通过三亲接合试验，重组质粒pSZY6中所携带的dctPQM基因随机转座插入到菌株A1501的染色体基因组中，构建了含有额外拷贝dct结构基因的遗传212程菌株。在A15卡那霉素(50μg／mol)平板上，共筛选到约60株转接合子。其中工程菌株A-136、A-115和A-142在以不同的四碳二羧酸如琥珀酸、延胡索酸和苹果酸，以及乳酸为唯一碳源生长时，表现了较高的固氮活性，其固氮水平明显高于野生型菌株A1501。     

固氮斯氏假单胞菌固氮调节基因ntrB的功能研究

利用三亲接合的方法将含有ntrB基因部分序列的自杀载体导入固氮斯氏假单胞菌（Pseudomonas stutzeri）A1501中,获得了mrB基因的非极性突变株（AdnB）。RT-PCR实验证明,AdnB菌株中与ntrB连锁的下游基因ntrC能正常转录;ntrB基因的非极性突变导致细胞完全丧失固氯能力,但其突变株能在含NH4^＋的基本培养基K中正常生长;在以硝酸盐为唯一氯源时,AdnB比野生型A1501的细胞生长延迟约14～18h,认为ntrB基因与硝酸盐的氮代谢有关。功能互补实验结果表明,AdnB的功能互补菌株能完全恢复细胞的固氮作用和对硝酸盐的利用。     

敲除Ras1基因对裂殖酵母生长及产孢影响

目的:探究Ras1基因对裂殖酵母生长及产孢的影响。方法:培养将野生型与基因敲除的突变株,分析其生长情况;野生型与突变菌株交配,探讨其产孢情况。结果:在25℃及37℃培养条件下,敲除基因后的突变菌株其生长慢于野生型菌株。产孢实验结果表明,野生型100%产生4个孢子,而突变菌株75%产生1个孢子、25%产生2个孢子,且突变菌株的孢子略小于野生型。结论:敲除基因Ras1会影响裂殖酵母的生长和产孢。     

氯代芳香族污染物生物降解菌株的分离及其降解质粒的初步研究

从工业废水污染的水体和污泥中,分离并筛选到６株对氯苯和苯酚类有机污染物高浓度耐受力的细菌菌株。６株降解细菌能在氯代苯和氯代酚作为唯一碳源的培养介质中生长,具有不同程度降解这类有机污染物的代谢能力,除２号和５号菌外,所筛选的其它４株降解细菌均含有一个或多个质粒。采用２,４－Ｄ单加氧酶基因ｔｆｄＡ的保守引物和ＰＣＲ扩增技术,证明在２号和６力的染色体和３号菌的大质粒上含有ｔｆｄＡ基因的同源序列。     

导入四碳二羧酸转移酶基因对费氏中华根瘤菌共和固氮效率的影响

将来自苜蓿中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｍｅｌｉｌｏｔｉ）的四碳二羧酸转移酶基因（ｄｃｔＡＢＤ）经ｐＩＪ２９２５克隆到广宿主、稳定性质粒ｐＴＲ１０２上,获得诱导型表达的重组质粒ｐＨＮ２０２。在此基础上再引入来自ｐＤＢ３０所含的发光酶基因（ｌｕｘＡＢ）作分子标记,以ｐＴＲ１０２为基础建成带有ｄｃｔＡＢＤ和ｌｕｘＡＢ的重组质粒ｐＨＮ２０５。＃? ｄｇ ｕｓ ｓｇ ｒｕｖ ｗｇｋ ｌｆｔｑ,ｕ     

导入四碳二羧酸转移酶基因对费氏中华根瘤菌共生固氮效率的影响

将来自苜蓿中华根瘤菌(Sinorhizobium meliloti)的四碳二羧酸转移酶基因(dctABD)经pIJ2925克隆到广宿主、稳定性质粒pTR102上,获得诱导型表达的重组质粒pHN202.在此基础上再引入来自pDB30所含的发光酶基因(luxAB)作分子标记,以pTR102为基础构建成带有dctABD和luxAB的重组质粒pHN205.经三亲本接合转移,将重组质粒pHN205导入费氏中华根瘤菌(Sinorhizobium fredii)HNO1,GR3和YC4.与出发菌相比较的盆栽试验结果表明:HN01(pHN205)和GR3(pHN205)分别在宁镇一号和川早一号大豆上能显著提高植株地上部分干重(生物量)和总氮量, YC4(pHN205)在黑龙33大豆上能同时显著提高植株地上部分干重(生物量),总氮量和根瘤鲜重.本研究结果表明:导入dctABD基因对共生固氮效率的增效性与受体根瘤菌和大豆品种等因素有关.以luxAB为报告基因进行的转移接合子培养条件下分离单菌落和共生条件下形成根瘤的发光活性检测结果表明:pHN205可在供试费氏中华根瘤菌中稳定遗传.     

生物絮凝剂产生菌的N+注入诱变选育及其培养条件研究

为获得高絮凝活性菌株,采用低能氮离子诱变方法,对生物絮凝剂产生菌FJ-7进行诱变选育,筛选得到一株絮凝活性高、遗传稳定性良好的突变株NIM-192.发酵产絮凝剂曲线表明,其菌体生长速度稍慢于原始菌株,但絮凝活性一直高于原始菌株,絮凝率比原始菌株提高了34.26%.进一步研究表明,蔗糖、葡萄糖、半乳糖是NIM-192产生絮凝剂的适宜碳源,酵母膏:尿素为1:1的混合氮源为最佳氮源,培养基起始pH为7～9时,发酵液的絮凝效果最好.     

用核糖体工程技术二次开发海洋微生物菌株资源的研究

经对无活性的海洋来源放线菌B3054进行链霉素抗性筛选,获得了一株具有抗肿瘤活性的链霉素抗性突变株SY-1.从该突变株的发酵物中分离得到了4个活性化合物,其中1个为新天然产物.对该菌株突变前后的rpsL基因(编码核糖体蛋白S12)进行分析未发现发生突变,提示突变位点可能在核糖体的另外亚基上.实验结果表明,核糖体工程技术可用于无活性菌株资源的二次开发利用.     

CD59活性位点相关性基因突变的构建与表达

构建了野生型和突变型CD59重组质粒,建立了高效真核表达系统,探讨了W40位点的生物学活性。采用基因点突变技术使CD59W40基因位置缺失（突变1,M1）及C39W40K41→W39W40W41（突变2,M2）,重叠延伸PCR（overlap extension PCR）定点诱变扩增突变基因,重组入真核表达质粒pIRES,利用阳离子脂质体（Lipfectamine2000）将重组质粒转染中国仓鼠卵巢细胞（CHO）进行表达。酶切鉴定及序列测定证实成功构建了pIRES-MICD59、pIRES—M2CD59和pIRES-WTCD59,突变基因约500bp。G418筛选出了CHO转染细胞的稳定细胞克隆,免疫荧光、ELISA检测筛选MICD59、M2CD59和WTCD59蛋白高表达株,连续传代30代有高表达;补体溶细胞反应显示与野生型CD59相比,突变型M1CD59失去对补体的抑制功能,而M2CD59抗补体活性略增高。证实CD59的W40位点对其功能具有重要作用,封闭此位点可提高补体活性,有望用于肿瘤治疗。     

esaC基因对迟缓爱德华氏菌的毒力及T3SS蛋白分泌的影响

为了确定编码迟缓爱德华氏菌(E.tarda)Ⅲ型分泌系统(T3SS)装置蛋白的esaC基因的功能,采用基因敲除的方法构建了E.tarda野生型致病菌株LSFAO的esaC基因无极性缺失突变株LSE40ΔesaC,该突变株缺失了esaC的46-1452bp。Western blotting分析表明,由于esaC的缺失,E.tarda T3SS的输送器蛋白(EseB,EseC,EseD)不能分泌到细胞外,同时细菌的自凝聚现象消失;毒力检测发现,突变株LSE40ΔesaC对蓝曼龙鱼的半数致死量比野生型菌株提高了11.5倍。结果表明,esaC基因影响T3SS输送器蛋白的分泌和E.tarda的毒力。此研究确定了esaC基因作为E.tarda T3SS装置蛋白的功能,加深了对E.tarda致病机理的了解。     

Simultaneous analysis of amino acids and carboxylic acids by capillary electrophoresis-mass spectrometry using an acidic electrolyte and uncoated fused-silica capillary

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18](+) or [M + 23](+), after protonated molecule detection (m/z = [M + 1](+)) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = [M - 1](-)), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism.     

Effects of gene augmentation on the removal of 2,4-dichlorophenoxyacetic acid in a biofilm reactor under different scales and substrate conditions

With a conjugative plasmid pJP4 carrying strain as the donor, two bioaugmentation experiments were conducted in a microcosm biofilm reactor with 2,4-D as the sole carbon source operated in fed-batch mode, and an enlarged lab-scale sequence batch biofilm reactor with mixed carbon sources of 2,4-D and other easily biodegradable compounds, respectively. In the microcosm study under sole carbon source condition, bioaugmentation led to a persistently increased 2,4-D degradation rate in the five operation cycles with enhancement of 13-64%. For the enlarged lab-scale bioaugmentation experiment under mixed carbon source conditions, no enhancement in 2,4-D removal could be observed during start-up period. After a period of operation, biofilm samples from the bioaugmented reactor demonstrated a stronger degradation capacity than the control and showed the presence of a large number of transconjugants. This study indicates that bioaugmentation based on plasmid horizontal transfer is a feasible strategy to establish functional microbial community in a biofilm reactor, and the strong selective pressure of 2,4-D existing alone and persistently was more favorable for the success of gene augmentation.     

Catalyst-free synthesis of polyesters via conventional melt polycondensation

Most commodity polyesters are synthesized via melt polycondensation of dicarboxylic acid and diol using metallic catalysts; however, the resultant metal residues can pose toxic effects on human and environment. Although polyesters can be synthesized through autocatalysis of dicarboxylic acid without additional catalysts, high molecular weight (HMW) products cannot be obtained by this strategy, which was previously attributed to the low equilibrium constant of esterification and the difficulty of removing water. Herein, we get a new understanding that the kinetic deviation of dicarboxylic acid/diol monomers is the only reason for the low molecular weight of polyesters by autocatalysis. Accordingly, we introduce a dynamic stoichiometric strategy to overcome this difficulty using anhydride-formable dicarboxylic acids as monomers through a tandem mechanism involving proton transfer, anhydride formation and re-esterification. A series of catalyst-free HMW polyesters, including poly(butylene succinate) (PBS), poly(ethylene succinate) (PES), poly(butylene succinate-co-butylene adipate) (PBSA), and poly(ethylene succinate-co-ethylene terephthalate) (PEST), were thereby synthesized. This new approach not only enables large-scale production of HMW polyesters comparable to commercial products, but also avoids the problems associated with catalysts, which is very promising for the applications with high safety requirements.     

洋葱假单胞菌G63脂肪酶基因及其伴侣基因的克隆和同源高效表达

从油污土壤中筛选到一株脂肪酶活性较高的洋葱假单胞菌(Pseudomonas cepacia)G63.运用PCR的方法从该菌株中克隆了脂肪酶基因(lipA)及其伴侣基因(lipB).该脂肪酶基因开放式阅读框(ORF)为1092bp,编码364个氨基酸残基.经KpnⅠ/HindⅢ酶切,将其克隆到广泛宿主质粒pBBR1Tp载体上,构建成质粒pBBR1-lipAB.通过三亲杂交,在辅助质粒pRK2013的帮助下,转入原宿主菌P.cepacia G63中,构建成lipA基因同源高效表达的基因工程菌.该菌株在连续转接5次,培养45h后质粒保持率为82.6%,适用于规模化发酵生产.摇床发酵表明,工程菌在60h时酶活达到150.63 U/mL,较原始菌株提高3.6倍.     

Biodegradation of fenoxaprop-p-ethyl by bacteria isolated from sludge

A mixed bacterial population was isolated using enrichment in a basal medium containing increasing amounts of fenoxaprop-p-ethyl as a sole carbon source from sludge that had been exposed to fenoxaprop-p-ethyl production wastewater for about 2 years. Eight kinds of isolates could utilize fenoxaprop-p-ethyl, but only one was identified belonging to genus Alcaligenes, named Alcaligenes sp. H. In pure culture, there was 45.8, 66.0 and 69.5% loss of fenoxaprop-p-ethyl (initial concentration: 100, 50, 25 ppm, respectively) as the sole carbon source with biodegradation by Alcaligenes sp. H and fenoxaprop-p-ethyl degradation kinetics obeyed the first-order kinetics, the same as the fenoxaprop-p-ethyl biodegradation kinetics in soil. At least five degradation products of fenoxaprop-p-ethyl biodegradation by Alcaligenes sp. H and two degradation products of fenoxaprop-p-ethyl biodegradation by Huv separated by HPTLC. It is possible that the fenoxaprop-p-ethyl biodegradation by Alcaligenes sp. H includes the same pathway as that by Huv comparing with the Rf.     

pH-independent immediate release polymethacrylate formulations – an observational study

Abstract

Using Eudragit® E PO (EudrE) as a polymethacrylate carrier, the aim of the study was to develop a pH-independent dosage form containing ibuprofen (IBP) as an active compound via chemical modification of the polymer (i.e. quaternization of amine function) or via the addition of dicarboxylic acids (succinic, glutaric and adipic acid) to create a pH micro-environment during dissolution. Biconvex tablets (diameter: 10?mm; height: 5?mm) were produced via hot melt extrusion and injection molding. In vitro dissolution experiments revealed that a minimum of 25% of quaternization was sufficient to partially (up to pH 5) eliminate the pH-dependent effect of the EudrE/IBP formulation. The addition of dicarboxylic acids did not alter IBP release in a pH 1 and 3 medium as the dimethyl amino groups of EudrE are already fully protonated, while in a pH 5 solvent IBP release was significantly improved (cf. from 0% to 92% release after 1?h dissolution experiments upon the addition of 20?wt.% succinic acid). Hence, both approaches resulted in a pH-independent (up to pH 5) immediate release formulation. However, the presence of a positively charged polymer induced stability issues (recrystallization of API) and the formulations containing dicarboxylic acids were classified as mechanically unstable. Hence, further research is needed to obtain a pH-independent immediate release formulation while using EudrE as a polmethacrylate carrier.     

Wet oxidation of lignin model compounds and acetic acid production

To investigate the wet oxidation (WO) pathways of acetic acid production from lignin, 2-methoxyphenol, 2,6-dimethoxyphenol, and phenol, as lignin model compounds were oxidized in a batch reactor at a temperature of 300°C, residence times of 10–60 seconds and oxygen supplies of 50–100%. Oxidation experiments using major intermediate products in WO of lignin model compounds, as reactants were also performed.Based on the intermediate products identified by GC/MS and HPLC, WO pathways of lignin model compounds are discussed. Acetic acid production by WO of lignin model compounds is also discussed. It was found that the yield of acetic acid for lignin model compounds was lower, at about 9%. The reason that lignin model compounds cannot produce a large amount of acetic acid may be contributed to the fact that the oxidation of phenols easily forms unsaturated dicarboxylic acids with 4 carbon atoms, which cannot produce a large amount of acetic acid. However, saturated dicarboxylic acids and glutaconic acid can produce a higher yield of acetic acid. Therefore, it is possible to increase the yield of acetic acid from lignin by controlling reaction pathways to increase the formation of saturated dicarboxylic acids and glutaconic acid.