固定化重组大肠杆菌细胞催化合成(R)-环氧氯丙烷

采用包埋法对重组大肠杆菌E.coli BL21进行了固定化研究,确定海藻酸钙是较好的固定化载体。考察了海藻酸钠质量分数、CaCl2质量分数、钙化时间、细胞包埋量以及固定化颗粒直径对固定化细胞活性和机械强度的影响,确定优化的制备条件为:海藻酸钠的质量分数为2.0%,CaCl2的质量分数为2%,钙化10 h,包埋菌体的质量浓度为0.05 g/mL,颗粒直径为2.7 mm。与游离细胞相比,固定化细胞的热稳定性和pH稳定性有明显提高。利用固定化重组大肠杆菌细胞催化拆分体积分数为1.5%的外消旋环氧氯丙烷,得到(R)-环氧氯丙烷的产率和光学纯度(对映体过量)分别为36.3%和100%。固定化细胞重复4批仍保持80%以上的活力,显示了良好的操作稳定性。     

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade

β-Nicotinamide mononucleotide (NMN) has recently gained attention for a nutritional supplement because it is an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD⁺). In this study, we developed NMN synthesis by coupling two modules. The first module is to culture E. coli MG1655 ▵tktA ▵tktB ▵ptsG to metabolize xylose to generate D-ribose in the medium. The supernatant containing D-ribose was applied in the second module which is composed of EcRbsK-EcPRPS-CpNAMPT reaction to synthesize NMN, that requires additional enzymes of CHU0107 and EcPPase to remove feedback inhibitors ADP and pyrophosphate. The second module can be rapidly optimized by comparing NMN production determined by the cyanide assay. Finally, 10 mL optimal biocascade reaction generated NMN with a good yield of 84 % from 1 mM D-ribose supplied from the supernatant of E. coli MG1655 ▵tktA ▵tktB ▵ptsG. Our results can further guide researchers to metabolically engineer E. coli for NMN synthesis.     

大肠杆菌高水平表达脂肪酶BTL2及其催化油脂制备生物柴油

引言随着近年来石化能源的紧缺和日益严重的环境污染问题,作为绿色新型能源之一的生物柴油受到了越来越多的关注。生物柴油是动植物油脂与低碳醇(通常是甲醇或乙醇)发生转酯化反应后生成的脂肪酸低碳醇酯。生物柴油制备工艺按照其催化剂     

Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor β (rh TNF-β) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rh TNF-β from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm^?3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.     

在大肠杆菌K99(中国QH株)菌毛形成亚单位基因(fanC)的克隆和测序中发现IS1 插入序列

目的 构建大肠杆菌K99菌毛表达载体。方法 通过逐级亚克隆的方法克隆k99菌毛形成亚单位基因（fanC），并进行序列测定和fanC亚单位的抗原决定簇模拟，以确定突变部位。结果 克隆了包括K99菌毛形成亚单位基因（fanC）和部分fanD基因在内的一段基因，该基因与国外报道相比明显偏大。序列测定表明：在fanC尾部多出 24bp，在fanC与 fanD，的结合部有一 800bp左右的新基因。经与基因库中大肠杆菌基因作同源性比较发现，该新基因为768bp的插入序列IS1。其他基因为K99自身fonC与fanD片段之间的过渡基因。IS1的插入未改变fanD，基因的阅读框架，只是使fanD片段末端增加了 8个氨基酸。fanC亚单位的抗原决定簇模拟结果显示，K99 fanC片段具有一个非常突出的亲水区域，根据经验它应是抗原决定簇所在的部位。结论 首次发现大肠杆菌K99基因中含有插入序列IS1。     

大肠杆菌E.coli HB101(pBR322) 高密度培养过程质粒的稳定性

通过正交实验考察了大肠杆菌E.coli HB101(pBR322)高密度培养过程中,不同培养条件对质粒的稳定性和β-内酰胺酶活力的影响. 结果表明,当温度在33~39℃、pH为6.4~7.2、DO为 40%~80%、 补料速率在5.4~10.8 g/h范围内变化,以及细胞密度达到27.3 g/L、比生长速率达到0.73 h-1时,质粒没有发生丢失现象,但在培养过程中β-内酰胺酶的活力降低.     

Characterization and Genomic Analysis of Escherichia coli O157:H7 Phage UAE_MI-01 Isolated from Birds

Verotoxin-producing Escherichia coli O157:H7 is responsible for the majority of foodborne outbreaks worldwide and may lead to death. Bacteriophages are natural killers of bacteria. All previously reported E. coli O157:H7 phages were isolated from ruminants or swine. Here, we report for the first time a phage isolated from bird feces in the United Arab Emirates (UAE), designated as UAE_MI-01, indicating birds as a good source of phages. Thus, phages could be a tool for predicting the presence of the host bacteria in an animal or the environment. UAE_MI-01 was found to be a lytic phage that was stable at wide ranges of pH, temperature, and chemical disinfectants, and with a burst size of almost 100 plaque-forming units per host cell after a latent period of 20 min and an adsorption rate constant (K) of 1.25 × 10⁻⁷ mL min⁻¹. The phage genome was found to be 44,281 bp long with an average GC content of 54.7%. The presence of the phage indicates the presence of the host cell E. coli O157:H7 in wild birds. Therefore, other birds, mainly poultry, could be also investigated for the presence of this pathogenic bacterium. To the best of our knowledge, this is the first report of an E. coli O157:H7 bacteriophage isolated from a bird.     

化学法和机械法从大肠杆菌释放人肿瘤坏死因子

对化学法和机械法破碎基因工程大肠杆菌以释放人肿瘤坏死因子的影响因素作了考察。在化学法破菌中研究了破碎时间、试剂浓度、溶剂ｐＨ值、加入表面活性剂等因素的影响。在机械法破碎中研究了停留时间、装珠量、搅拌转速、细胞浓度等的影响，对操作条件进行了优化。通过对二者的比较，发现机械法破碎细胞效率高，但产生细小的碎片不利后分离；化学法释放产物有选择性，但操作时间长。     

Mg~(2+)和氨基酸对重组大肠杆菌BL21(DE3)生长及羧肽酶原B表达的影响

目的研究Mg2+和氨基酸对重组菌株生长及表达pCPB的影响。方法通过摇瓶和发酵罐培养,观察不同Mg2+浓度和氨基酸对重组菌的生长和羧肽酶原B表达的影响。结果添加1g/L Mg2+能提高质粒的稳定性;添加氨基酸能使羧肽酶原B表达量由18·7g/L提高到24g/L。结论添加适量的Mg2+和氨基酸能促进重组菌的生长和提高目的蛋白的表达量。     

Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli

Bovine somatotropin (bST) was secreted from Escherichia coliat moderate levels of 1–2 µg/ml/OD using expressionvectors in which the bST gene was fused to the lamB secretionsignal. To study the secretion properties of bST in E.coli further,two approaches for modifying the secretion signal were employed.In the first case, fusion proteins were constructed with sixalternative bacterial secretion signals: three from E.coli proteins(HisJ, MalE and OmpA), two from bacteriophage proteins (M13coat protein and PA-2 Lc) and one from the chitinase A proteinof Serratia marcescens. The results, as monitored by Westernblot analysis of both total cell protein and the periplasmicfraction, showed that these changes in the secretion signaldid not significantly affect the secretion properties of bST.In the second approach, a library of random mutations was createdin the lamB secretion signal and 200 independent clones werescreened. The level of secreted bST was determined by growingindividual clones in duplicate in microtiter wells, inducingprotein expression and measuring the bST released by osmoticshock using a particle concentration fluorescent immunoassay.The secretion properties of several novel variants in the LamBsignal peptide are presented.     

Production and purification of a fused recombinant protein gp-36 (HIV-2) from Escherichia coli

A fragment of the gp-36 gene of the Human Immunodeficiency Virus type 2 (HIV-2) was fused to a stabilizer sequence, which encodes for the first N-terminal 58 amino acids of the human interleukin-2. The fused protein was expressed under the control of the tryptophan promoter in Escherichia coli, and expressed as 20% of the total cellular protein. Transmission electron microscopy indicated that the fusion protein formed cytoplasmic insoluble inclusion bodies. Inclusion bodies were semipurified by a wash pellet cell procedure, rendering a material with a purity higher than 70% by SDS–polyacrylamide gel electrophoresis. After solubilization with urea, this preparation was further purified by gel-filtration chromatography up to 95% purity.     

E.coli M15 (pQTPL)高效发酵生产酪氨酸酚裂解酶的控制策略

在摇瓶和4 L发酵罐上研究了营养和环境条件对重组菌E. coli M15 (pQTPL)分批发酵生产酪氨酸酚裂解酶(TPL)的影响. 在培养基中添加20 g/L葡萄糖和1.0 g/L玉米浆使TPL酶活提高到63.1 U/g(干重). 在此基础上,维持发酵液中溶氧水平为30%,可使菌体浓度在8 h达到4.78 g/L,酶活为54.6 U/g,比对照组(不控制溶氧)分别提高了21%和31.6%. 采用溶氧反馈调节-限制性补料策略,可使菌体浓度提高到31.5 g/L. 采用两阶段温度和pH控制策略,在发酵前8 h控制pH 7.0、温度37℃,8 h 至发酵结束之间控制pH为8.0、温度为30℃,可使重组菌的TPL酶活达到154.4 U/g,并使TPL在细胞中过量表达,实现了高菌体浓度和高TPL酶活的统一.     

Genotypic Characterization of Escherichia coli O157:H7 Isolates from Different Sources in the North-West Province,South Africa,Using Enterobacterial Repetitive Intergenic Consensus PCR Analysis

In many developing countries, proper hygiene is not strictly implemented when animals are slaughtered and meat products become contaminated. Contaminated meat may contain Escherichia coli (E.coli) O157:H7 that could cause diseases in humans if these food products are consumed undercooked. In the present study, a total of 94 confirmed E. coli O157:H7 isolates were subjected to the enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) typing to generate genetic fingerprints. The ERIC fragments were resolved by electrophoresis on 2% (w/v) agarose gels. The presence, absence and intensity of band data were obtained, exported to Microsoft Excel (Microsoft Office 2003) and used to generate a data matrix. The unweighted pair group method with arithmetic mean (UPGMA) and complete linkage algorithms were used to analyze the percentage of similarity and matrix data. Relationships between the various profiles and/or lanes were expressed as dendrograms. Data from groups of related lanes were compiled and reported on cluster tables. ERIC fragments ranged from one to 15 per isolate, and their sizes varied from 0.25 to 0.771 kb. A large proportion of the isolates produced an ERIC banding pattern with three duplets ranging in sizes from 0.408 to 0.628 kb. Eight major clusters (I–VIII) were identified. Overall, the remarkable similarities (72% to 91%) between the ERIC profiles for the isolate from animal species and their corresponding food products indicated some form of contamination, which may not exclude those at the level of the abattoirs. These results reveal that ERIC PCR analysis can be reliable in comparing the genetic profiles of E.coli O157:H7 from different sources in the North-West Province of South Africa.     

Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue

The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilateisomerase:indole-3-glycerol-phosphate synthase from Escherichiacoli has been crystallized, and the structure has been solvedat 4 Å resolution. Two closely related crystal forms grownfrom ammonium sulphate diffract to 2 Å resolution. Oneform (space group R32, a = 163 Å, = 29.5°) containsthe unliganded synthase domain; the second crystal form (spacegroup P6₃22, a = 144 Å, c = 158 Å) is co-crystallizedwith the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate.The structure of the synthase–inhibitor complex has beensolved by the molecular replacement method. This achievementrepresents the first successful use of a (ß)_g-barrelmonomer as a trial model. The recombinant synthase domain associatesas a trimer in the crystal, the molecules being related by apseudo-crystallographic triad. The interface contacts betweenthe three domains are mediated by those residues that are alsoinvolved in the domain interface of the bifunctional enzyme.This system provides a model for an interface which is usedin both intermolecular and intramolecular domain contacts.