Pseudomonas putida S1海藻糖合成酶基因在大肠杆菌中的克隆表达

利用PCR和TA克隆方法扩增和克隆得到了恶臭假单胞菌Pseudomonas putida S1的海藻糖合成酶基因treS.对其进行序列分析表明,其编码区含有2067bp,编码含688个氨基酸残基的蛋白质,其核苷酸序列和蛋白质序列与来源于其它假单胞菌属细菌的海藻糖合成酶的序列表现出了较高同源性.将该基因序列与表达载体pQE30T连接,构建重组质粒pQE30T-TS,并将其转化至E.coli M15菌株中.重组菌株经诱导表达后SDS-聚丙烯酰胺凝胶电泳结果显示有明显的分子量约77.5kD的特异蛋白条带出现.经测定酶活力达19U/mL,约是原始菌株P.putida S1的50倍.     

热激对恶臭假单胞菌S1胞内海藻糖合成酶的影响

研究了热激对恶臭假单胞菌S1胞内海藻糖合成酶的影响。通过正交试验确定了热激的最佳条件:热激在产酶初期(培养20 h)进行,热激时培养温度由20℃升高至30℃,处理时间30 min。在此条件下,海藻糖合成酶的酶活提高了76%。同时在热激后菌体的耐盐性也有较大增强。     

Pseudomonas putida S1海藻糖合成酶表达质粒的构建及诱导条件优化

采用PCR方法从Pseudomonas putida S1中克隆出编码海藻糖合成酶的基因treS,并与质粒pQE30T相连,构建了表达质粒pQE—TS2。将此重组质粒转化宿主菌E．coliM15进行诱导表达。十二烷基磺酸钠-聚丙烯酰胺凝胶SDS—PAGE电泳结果表明,treS基因在大肠杆菌中获得了高效表达。通过对诱导温度、诱导剂浓度、加诱导剂时间和诱导时间的优化研究,在菌液生长至OD600值为0．6时,加入诱导剂IPTG至终浓度0．01mmol／L,20℃诱导20h,蛋白的表达量达到每克干细胞89mg的蛋白,粗酶液酶活达到19U／mL。     

Transduction and Genetic Homology Between Pseudomonas Species putida and aeruginosa

Genetic crosses occur by transduction between the species Pseudomonas aeruginosa and P. putida. The frequency relative to intraspecific transfer is reduced and varies among markers, suggesting that these genomes contain discrete regions of homology and nonhomology.     

Production and characterization of homopolymer polyhydroxyheptanoate (P3HHp) by a fadBA knockout mutant Pseudomonas putida KTOY06 derived from P. putida KT2442

Pseudomonas putida KT2442 commonly produces medium-chain-length polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (C6) to 3-hydroxydodecanoate (C12) when grown in glucose or even number fatty acid. When two of the beta-oxidation genes fadBA were deleted, the P. pudida KT2442 mutant named KTOY06 accumulated a homopolymer of poly-3-hydroxyheptanoate (P3HHp) up to 71 wt% of its cell dry weight in the presence of heptanoate as a single carbon source. P3HHp contents in the cell dry weight were in direct proportional to Na-heptanoate concentration up to 10 g/L. In contrast, under the same cultivation conditions, the wild type P. putida KT2442 produced a copolymer consisting of 3-hydroxyheptanoate (3HHp) and 5.3–8.4 mol% 3-hydroxynonanoate (3HN). Gas chromatography (GC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and gel permeation chromatography (GPC) were used to characterize the homopolymer P3HHp, respectively. It was found that the P3HHp with an average molecular weight of 455 kDa was a completely amorphous homopolymer without crystallinity. P3HHp is thermo-degradable at around 250 °C.     

Properties and function of malate enzyme from Pseudomonas putida

Malate enzyme (L-malate: NADP+ oxidoreductase (oxalacetate-decarboxylating, EC 1.1.1.40)) has been purified from Pseudomonas putida to 99 per cent homogeneity by heat, ammonium suphate fractionation, gel filtration and anion exchange chromatography. Sodium dodecylsulphate-(SDS)-polyacrylamide disc gel electrophoresis analysis showed an approximate tetrameric subunit with a molecular weight of 52,000. The purified enzyme showed a pH optimum between 8.0 and 8.5 (for Tris-HCl buffer) and required bivalent cations for catalysis; monovalent ions like K+ and NH4+ acted as very effective activators. The temperature-activity relationship for the malate enzyme from 35-80 degrees C showed broken Arrhenius plots with an inflexion at 65 degrees C. The enzyme halflife was 30s at 85 degrees C. The enzyme showed hyperbolic kinetics for both substrates with apparent Km values of 4.0 X 10(-4) M and 2.3 X 10(-5) M for L-malate and NADP+ respectively. From the study of the effects of some compounds on the enzyme, the physiological significance of those produced by fumarate, succinate and oxalacetate can be emphasized.     

Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose. A novel treS gene with a length of 3,369 bp, encoding a protein of 1,122 amino acid residues with a predicted molecular mass of 126 kDa, was cloned from a marine Pseudomonas sp. P8005 (CCTCC: M2010298) and expressed in Escherichia coli. The amino acid sequence identities between this novel TreS and other reported TreS is relatively low. The purified recombinant TreS showed an optimum pH and temperature of 7.2 and 37 °C, respectively. The enzyme displayed a high conversion rate (70 %) of maltose to trehalose during equilibrium and had a higher catalytic efficiency (k _cat/K _m) for maltose than for trehalose, suggesting its application in the production of trehalose. In addition to maltose and trehalose, this enzyme can also act on sucrose, although this activity is relatively low. Mutagenesis studies demonstrated that enzymatic activity was reduced dramatically by individually substitution with alanine for D78, Y81, H121, D219, E261, H331 or D332, which implied that these residues might be important in P8005-TreS. Experiments using isotope-labeled substrates showed that [²H₂]trehalose combined with unlabeled trehalose was converted to [²H₂]maltose and maltose, but without any production of [²H]maltose or [²H]trehalose and with no incorporation of exogenous [²H₇]glucose into the disaccharides during the conversion catalyzed by this enzyme. This finding indicated the involvement of an intramolecular mechanism in P8005-TreS catalyzing the reversible interconversion of maltose and trehalose.     

Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose

A novel strain was isolated, Pseudomonas stutzeri CJ38, that enabled direct transformation of maltose to trehalose. In comparison with others reported to date, CJ38 provided a novel trehalose synthase (TSase) without any byproduct, including glucose. Activity analysis, using either maltose or trehalose as a substrate, showed a reversible reaction. There was also no detectable activity of related enzymes with liquid starch and maltooligosaccharides as substrates. Using a malPQ-negative host and MacConkey medium, the TSase gene was cloned in Escherichia coli from CJ38. The resulting sequence contained an open reading frame consisted of 689 amino acids with a calculated molecular mass of 76 kDa. A search for related sequences in various gene and protein data banks revealed a novel family of enzymes that was predicted putatively as a glycosidase or TSase family, with no biochemical evidence. The recombinant enzyme exhibited a high activity toward the substrate maltose, about 50-fold higher than the parent strain and resulted in a high conversion yield (72%) at a relatively high substrate concentration (20%). These results provided the possibility that the strain was effectively used as a potential biocatalyst for the production of trehalose from maltose in a one-step reaction.     

Hydroxyectoine is superior to trehalose for anhydrobiotic engineering of Pseudomonas putida KT2440

Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.     

DNA sequence of the tryptophan synthase genes of Pseudomonas putida

Genes encoding the 2 subunits of tryptophan synthase in Pseudomonas putida have been identified and cloned by their similarity to the corresponding genes in Pseudomonas aeruginosa. The deduced amino acid sequences were confirmed by comparison with regions ascertained earlier by protein sequencing. The Pseudomonas amino acid sequences are 85% identical for the beta subunit and 70% identical for the alpha subunit. These sequences are compared to those of Salmonella typhimurium, where the structure is known from X-ray crystallography. Although amino acid conservation drops to 54% and 36% for the beta and alpha subunits, only 3 single residue gaps are required to maintain alignment throughout and most of the residues identified as important for catalysis or cofactor binding are conserved. The 23 residues surrounding the beta chain lysine that enters into a Schiff base linkage with the pyridoxal phosphate cofactor are compared in 13 species, including representatives from the eukaryotic and both prokaryotic kingdoms; appreciable conservation is apparent. The approximately 100 base pairs separating the trpB gene from its divergently transcribed activator gene are similar in the 2 pseudomonads, but do not resemble those of any other bacterium or fungus studied to date.     

Homology modeling of an antifungal metabolite plipastatin synthase from the Bacillus subtilis 168

Lipopeptides have a widespread role in different pathways of Bacillus subtilis; they can act as antagonists, spreader and immunostimulators. Plipastatin, an antifungal antibiotic, is one of the most important lipopeptide nonribosomly produced by Bacillus subtilis. Plipastatin has strong fungitoxic activity and involve in inhibition of phospholipase A2 and biofilm formation. For better understanding of the molecule and pathway by which lipopeptide plipastatin is synthesized, we present a computationally predicted structure of plipastatin using homology modeling. Primary and secondary structure analysis suggested that ppsD is a hydrophilic protein containing a significant proportion of alpha helices, and subcellular localization predictions suggested it is a cytoplasmic protein. The tertiary structure of protein (plipastatin synthase subunit D) was predicted by homology modeling. The results suggest a flexible structure which is also an important characteristic of active enzymes enabling them to bind various cofactors and substrates for proper functioning. Validation of 3D structure was done using Ramachandran plot ProsA-web and QMEAN score.This predicted information will help in better understanding of mechanisms underlying plipastatin synthase subunit D synthesis. Plipastatin can be used as an inhibitor of various fungal diseases in plants.     

Homology modeling of plant cytochrome P450s

Plant P450 monooxygenases represent the largest family of plant proteins and the largest collection of P450s available for comparative studies and biotechnological applications. They have been shown to catalyze a diverse array of difficult chemical reactions and have demonstrated potential to be used in pharmacological, agronomic and phytoremediative applications. Central to our use of these catalytically competent enzymes is the need to understand their interactions with substrates. Because most characterized plant P450s are membrane-bound proteins that are resistant to standard X-ray and NMR structure determinations, homology modeling represents a reliable and relatively rapid alternative method for analyzing structure-function relationships and predicting substrates for many P450s that are only now being characterized. These methods, which are being widely used in mammalian P450 structure-function studies, can allow plant biologists to define critical residues interacting with substrates and, in a directed fashion, alter the reactivities of individual monooxygenases. The homology modelings that have been done on a limited number of plant P450s and the site-directed mutations that validate them indicate that current modeling and substrate docking procedures are capable of providing structural explanations for sequence variants as well as for predicting functional characteristics of undefined P450s.     

假单胞菌YZ-26来源环酰亚胺水解酶中半胱氨酸残基的反应性能

【目的】研究环酰亚胺水解酶(Imidase,CIH)中的两个半胱氨酸残基的反应性及功能。【方法】设计了3个半胱氨酸突变酶:CIH7,108、CIH7、CIH108。将天然酶以及突变酶基因分别与麦芽糖结合蛋白(MBP)基因在大肠杆菌(Escherichia coli)中进行融合表达,融合蛋白经纯化后得到了电泳纯的样品。使用5,5’-二硫代双(2-硝基苯甲酸)(DTNB)对天然酶CIH的巯基基团进行修饰,并分析了DTT对分子状态的影响。进一步研究了经H2O2处理后CIH及其突变酶的锌离子结合能力及分子状态。【结果】酶活测定表明CIH7,108和CIH7的活力基本丧失,而CIH108仍保持了72%的酶活性。CIH中的两个半胱氨酸残基以游离形式存在,不形成链内或链间二硫键。CIH与CIH108为四聚体结构且具有一定的锌离子结合能力,CIH7,108为多聚体,CIH7为单体及多体的混合物且都不具备锌离子结合能力,随着H2O2浓度的增加,CIH中的链内二硫键及CIH108中的链间二硫键逐渐增加。【结论】说明Cys7是结合锌离子和稳定CIH分子结构的必要残基。     

Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8

The gene encoding cardiolipin synthase ( cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced. The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents. Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin. As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids. Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production. Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis. As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.     

Regulation of the cyclopropane synthase cfaB gene in Pseudomonas putida KT2440

In Pseudomonas putida, as in many other eubacteria, cyclopropane fatty acids (CFAs) accumulate in the membrane during the stationary phase of growth. Here, we show that cfaB gene expression in P. putida KT2440 is dependent on the RpoS sigma factor that recognizes the sequence 5'-CTACTCT-3' between -8 and -14. We have carried out a mutational study of the cfa promoter and have determined that positions -9, -12, -13 and -14 are the most critical for maximal activity. In P. putida, the substrates of the CFA synthase, cis-unsaturated fatty acids (cis-UFAs), are also substrates for another stress-related enzyme, the cis-trans isomerase (CTI). Despite using the same substrates, we have found that the activity of the CTI is not limited by the CFA synthase activity and vice versa. For instance, in a cfaB knockout mutant, the amount of trans-UFAs synthesized after a specific stress was no higher than in the parental background despite the fact that there are more cis-UFAs available to be used by the CTI as substrates. In this regard, in a cti-deficient mutant background, the levels of CFAs were similar to those in the parental one under the same conditions.