大肠杆菌酸性磷酸酶基因克隆表达及应用

将大肠杆菌K-12的酸性磷酸酶(AphA)完整基因和去信号肽基因分别克隆到pET-28a(+)栽体上,并转化入大肠杆菌BL21( DE3)中.经诱导检测,重组菌均能表达出高活性的可溶性酶蛋白,去信号肽表达更稳定.对重组菌的活性研究表明,相对于野生菌,重组菌酶活力得到大幅度提高,同时,以pNPP、肌苷为底物进行磷酸转移催化反应,在pH4.0-6.0、反应温度37℃条件下,约有30％的肌苷可转化为IMP,但随着反应的进行所形成的IMP又被该酶降解,向反应液中加入EDTA即可明显抑制酶的水解活性,减缓IMP的降解速率.     

三角酵母D-氨基酸氧化酶基因在大肠杆菌中的克隆和表达

从三角酵母中提取总RNA ,反转录后进行PCR扩增得到D 氨基酸氧化酶 (D AminoAcidOxidase ,DAAO)基因 ,经测序可知 ,与文献中三角酵母的DAAO基因序列的同源性在 99%以上。将DAAO基因用NcoⅠ和BamHⅠ双酶切后 ,与相同酶切的大肠杆菌表达载体pET 2 8a连接 ,转化大肠杆菌TOP 1 0F′,并筛选得到重组质粒pET DAAO ,转化BL2 1 (DE3)感受态细胞 ,得到重组大肠杆菌BL2 1 (DE3) pET DAAO。对重组大肠杆菌中的D 氨基酸氧化酶进行了诱导表达 ,考察了诱导温度、菌浓度、诱导剂IPTG用量以及溶氧等因素对酶活的影响。结果表明 ,在 2 8℃、菌浓度 (OD6 0 0 ) 1 0、IPTG浓度 1mmol L时 ,DAAO酶活最高达 2 3 3U mL。研究进一步显示 ,用廉价无毒的乳糖可以替代IPTG进行诱导 ,当乳糖浓度为 2mmol L ,DAAO酶活可达 2 2 7U mL。经过补料分批培养和乳糖诱导 ,DAAO酶活可以达到 1 75U mL。     

formation for phenylethylamine oxidase expressed in Escherichia coli K-12

Abstract Arthrobacter globiformis amine oxidase produced by Escherichia coli cells grown in copper-depleted media was reported to undergo activation due to formation of its topaquinone cofactor in a copper-dependent autocatalytic reaction. Likewise, a mutated E. coli amine oxidase located in the cytoplasm was reported to form topaquinone autocatalytically in an EDTA-sensitive reaction. Here we show unequivocally that formation of an amine oxidase lacking topaquinone is primarily a consequence of the location of the enzyme in the cytoplasm rather than the level of copper in the growth medium. For E. coli , insertion of copper into apoamine oxidase and subsequent topaquinone formation occur after export of the apoenzyme into the periplasm.     

大肠杆菌K-12转醛醇酶基因talB的克隆及在运动发酵单胞菌CP4中的表达

研究了E.coliK-12转醛醇酶基因(talB)在自身启动子和在Z.mobilisCP4eno基因启动子的启动下在E.coliDH5α和Z.mobilisCP4中的表达情况。首先克隆了E.coliK-12talB基因,并连接到穿梭载体pZB1上构建成pZB1-talB;然后利用PCR重叠延伸技术将E.coliK-12talB自身的启动子换成Z.mobilisCP4eno的启动子,构建得到pZB1-Peno-talB。将这两个质粒分别转化E.coliDH5α和Z.mobilisCP4。对转化子粗酶液进行的转醛醇酶酶活力测定结果表明,E.coli talB自身启动子和Z.mobilis eno启动子能以基本相同的效率启动talB基因在E.coli和Z.mobilis中的表达。     

Four critical aspartic acid residues potentially involved in the catalytic mechanism of Escherichia coli K-12 WaaR

Escherichia coli K-12 WaaR is a non-processive alpha-1,2 glucosyltransferase, involved in the synthesis of the R-core of lipopolysaccharide. WaaR possesses the four conserved structural regions I, II, III and IV, each presumably involved in the mechanistic function in catalysis. Regions I and III contain the pair of strictly conserved Asp residues. Asp-129, 131 (region I) and 215, 217 (region III) of WaaR were individually converted to Asn by the site-directed mutagenesis of the waaR gene. All mutated enzymes were inactive, supporting the model for an alpha-glycosyl transfer reaction where the pair of strictly conserved aspartic acid residues in regions I and III play a critical role in the catalytic function.     

Identification of the rhaA, rhaB and rhaD gene products from Escherichia coli K-12

Washed cells of Peptostreptococcus products (strain Marburg), which were incubated in the presence of CO/CO2/N2 (50%/17%/33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis-Menten kinetics. The apparent Km for sodium was determined to be about 2 mmol/l. Sodium also stimulated acetate synthesis from H2 plus CO2. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO2.     

An improved Escherichia coli donor strain for diparental mating

We present a new method for diparental mating with the outstanding advantage that counterselection of the Escherichia coli donor strain is not required. This improved method uses a new donor strain, E. coli ST18, a hemA deletion mutant defective in tetrapyrrole biosynthesis. The hemA mutation can be complemented by addition of 5-aminolevulinic acid. Therefore, counterselection is carried out only using standard media and growth conditions optimal for the recipient strain. Consequently, recipient strains are isolated in a significantly shorter period.     

Induction of cadmium tolerance in Escherichia coli K-12

Abstract Cadmium ions are bacteriocidal, resulting in exponential killing that starts immediately after exposure. We have shown that pretreatment with sublethal concentrations of cadmium induces tolerance. Protection against cadmium killing can also be obtained by preincubation at elevated temperatures, known to induce the heat-shock response. However, in contrast to pretreatment at elevated temperatures, exposure to sublethal cadmium concentrations does not induce thermotolerance.     

'Receptor-independent' infection of Mu G(−) phage in Escherichia coli K-12

Abstract Bacteriophage Mu with its invertible G segment in G(−) orientation does not make plaques on Escherichia coli K-12, due to the absence of a suitable lipopolysaccharide receptor. Plaques formed by Mu G(−) were found, however, when the infected E. coli K-12 strain harbours a plasmid with the cloned DNA inversion function Gin which converts the infecting G(−) phage to G(+). Under overproducing conditions, where Gin expression is placed under the control of the tac promoter, the infectivity of Mu G(−) can be estimated as approximately 1% of that in the presence of the receptor. Furthermore, interaction of Mu G(−) with the E. coli K-12 cell wall leads to interference with the plating of a Mu G(+) variant which has the new phenotype Pen⁻ (penetration-negative).     

大肠杆菌植酸酶基因appA的克隆与高效表达

从猪粪便中分离并筛选出高效生产酸性植酸酶和磷酸酶双功酶(appA植酸酶)的大肠杆菌菌株。通过PCR方法从该菌株基因组中扩增获得了植酸酶基因appA,测序结果显示该基因编码区全长1,299个核苷酸。将该基因克隆到原核表达载体pET-28a( )上,通过转化的大肠杆菌BL21在试管摇床培养条件下得到了高效表达,其表达量达到692U／mL。酶学特性分析表明其反应的最适pH为4．5,最适温度为60℃。     

Purification and Characterization of Aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinyl-glycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni²⁺, Mn²⁺, Co²⁺, and Cd²⁺, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidase B is a metallopeptidase. It was stabilized against heat in the presence of Mn²⁺ or Co²⁺. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The k_cat/K_m for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

大肠杆菌精氨酰tRNA合成酶基因（argS）的上游非编码区存在一个负调控区

含大肠杆菌精氨酰tRNA合成酶（ArgRS）基因(argS）的pUC18重组质粒,在大肠杆菌TG1转化子中能够高表达ArgRS近1000倍。为了研究大肠杆菌argS的表达调控,构建了一系列的缺失突变。分别缺失全部上游序列（argS△1）、Shine-Dalgarno（SD）区（argS△2）和缺失启动子－10区下游（相当于翻译起始位点－65nt,argS△3）前的上游序列后的变种argS都不能表达ArgRS。而缺失－122nt（距翻译起始位点－180nt,argS△8）、－70nt（距翻译起始位点－128nt,argS△7）、－52nt（距翻译起始位点－110nt,argS△6）、－35区9距翻译起始位点－94nt,argS△5）、启动子－10区（距翻译起始位点－71nt,argS△4）前的上游序列后,这些缺失突变基因的表达水平与野生型argS接近。但argS△4、argS△5、argS△6都会形成部分包涵体。通过RNA斑点杂交测定发现,argS△4、argS△5和argS△6的mRNA转录量为argS及argS△7的2到3倍。即－52nt和－70nt之间的19个碱基（AATAGTGAAAACGGCAATA)可能是大肠杆菌argS舞场康负调控区。该元件的缺失将使得ArgRS过快表达并导致部分蛋白质形成包涵体。凝胶阻滞分析也发现在细胞粗抽液中有一个因子可以专一地与这个负调控元件结合,它可能参与该基因的表达调控。精氨酸专一性地诱导argS的转录,其作用与上述转录负调控区相关。     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

Inactivation of the Escherichia coli K-12 twin-arginine translocation system promotes increased hydrogen production

The effect of deleting the genes encoding the twin-arginine translocation (Tat) system on H2 production by Escherichia coli strain MC4100 and its formate hydrogenlyase upregulated mutant (DeltahycA) was investigated. H2 evolution tests using two mutant strains defective in Tat transport (DeltatatC and DeltatatA-E) showed that the rate doubled from 0.88+/-0.28 mL H2 mg dry weight-1 L culture-1 in the parental strain, to 1.70+/-0.15 and 1.75+/-0.18 mL H2 mg dry weight-1 L culture-1, respectively, in the DeltatatC and DeltatatA-E strains. This increase was comparable to that of a previously characterized hydrogen over-producing E. coli strain carrying a DeltahycA allele. Construction of a tatC, DeltahycA double deletion strain did not increase hydrogen production further. Inactivation of the Tat system prevents correct assembly of the uptake hydrogenases and formate dehydrogenases in the cytoplasmic membrane and it is postulated that the subsequent loss of basal levels of respiratory-linked hydrogen and formate oxidation accounts for the observed increases in formate-dependent hydrogen evolution.     

A Simple Method for Multiple Modification of the Escherichia coli K-12 Chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by λ Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.     

On the enthalpy of formation of Escherichia coli K-12 cells

An examination is made of five methods for obtaining values of the enthalpy of formation of a unit mass of living Escherichia coli K-12 cells. The values obtained by these methods ranged from -88.95 kJ to -99.55 kJ, the gross average being 96.01 kJ, per unit carbon formula weight equivalent of living, hydrated cells. Although theoretically the growth of this organism in a microcalorimeter should provide the best value, the value obtained by this method (-88.95 kJ per UCFW equivalent) is not in close agreement with those of the other four methods, the values from which form a cluster averaging -97.8 +/- 1.0 kJ (-23.4 +/- 0.2 kcal)/UCFW equivalent. Calculations using this value indicate that the enthalpy change accompanying anabolism (as this is represented) is zero, or very nearly so, and that the heat of growth is that from catabolism alone.     

Cloning of the ethidium efflux gene from Escherichia coli

The gene specifying the ethidium efflux system of Escherichia coli has been cloned on a 3.2 kbp HindIII fragment and located on a 1.2 kbp fragment within this. Cross-resistance studies indicate that the system has a broad specificity for monovalent cations and the gene shows no hybridisation with similar genes found in Staphylococci.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12

Abstract Five different c -type cytochromes have been detected during anaerobic growth of various Escherichia coli strains in different media. None of these cytochromes was detectable in aerobically-grown cultures. Only a single, 43 kDa cytochrome was synthesized in response to the presence of trimethylamine-N-oxide: synthesis of this cytochrome was unaffected by the presence of nitrate or nitrite, was repressed by oxygen, but was dependent upon a funtional tor operon located at minute 22 (coordinate 1070 kb) on the E. coli chromosome. The other four cytochromes, masses 16, 18, 24 and 50 kDa, were induced by nitrite coordinately with formate-dependent nitrite reductase activity, but repressed by oxygen and nitrate. As only the 18 kDa and 50 kDa cytochromes are encoded by the nrf operon located at minute 92 (coordinate 4366 kb), there must be other loci, possibly essential for formate-dependent nitrite reduction, encoding the 16 kDa and 24 kDa cytochromes. No other c -type cytochrome was detected under any growth condition tested.