优化细胞表面组分与结构以提高P.Putida 5-x细胞的重金属吸附能力

研究了生物吸附剂Pseudomonas putida 5-X细胞的最佳制备条件,以提高其对重金属的吸附能力。并用Pseudomonas putida 5-X细胞为生物吸附剂处理含铜离子城市污水。实验结果表明,在优化的条件下,Pseudomonas putida 5-x细胞对铜离子的吸附容量可达87．3mg／g,经生物吸附过程处理后,废水中Cu^2 的含量降低至微量级,而且该吸附剂可有效循环使用5次。     

Production of chiral R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters via hydrolytic degradation of polyhydroxyalkanoate synthesized by pseudomonads.

A novel and efficient method for the production of enantiomericaly pure R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters was developed. The described method is based on hydrolysis of poly(hydroxyalkanoate) copolymers synthesized by Pseudomonas putida. The polymer was isolated via solvent recovery and hydrolyzed by acid methanolysis. The obtained 3-hydroxyalkanoic acid methylester mixture was distilled into several fractions with an overall yield of 96.6% (w/w). Gas chromatography-mass spectrometry analysis of the fractions showed that 3-hydroxyhexanoic-, 3-hydroxyoctanoic-, 3 hydroxydecanoic-, and 3-hydroxydodecanoic acid methylesters were enriched to purities exceeding 96 mol%, with distillation yields of 99.9, 99.8, 88.4, and 56.8% (w/w), respectively. Subsequent saponification of the purified methylester fractions yielded the corresponding 3-hydroxyalkanoic acids, which were recovered up to 92.8% (w/w). Chiral gas chromatography analysis confirmed that both 3-hydroxyoctanoic acid and 3-hydroxyoctanoic acid methylester are present in the R-form at a very high enantiomeric excess (>99.9%).     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

假单胞菌海因酶基因在大肠杆菌中的高效表达(英文)

为实现利用生物酶转化法进行D 对羟基苯甘氨酸的工业化生产 ,构建了 3株海因酶基因工程菌 .利用PCR技术从恶臭假单胞菌 (Pseudomonasputida)CPU 980 1染色体DNA中扩增得到长约1.8kb的含编码区和自身启动子的海因酶全基因 .通过将海因酶全基因插入pMD18 T质粒、海因酶基因的编码区与pET 17 b质粒重组、海因酶基因编码区和T7强启动子一起插入pMD18 T质粒分别得到重组质粒pMD dht、pET dht和pMD T7 dht.将上述重组质粒分别转化大肠杆菌 (Escherichiacoli) ,通过地高辛标记菌落原位杂交和海因酶活力测定两种方法 ,筛选出具有海因酶活力的阳性转化子 .结果表明 ,大肠杆菌的RNA聚合酶能够识别和结合来自恶臭假单胞菌海因酶基因的自身启动子 ,该启动子在大肠杆菌中能够工作 .基因工程菌E .coliBL2 1 pMD dht、E .coliBL2 1 pET dht和E .coliBL2 1 pMD T7 dht的海因酶活力分别为 170 0U L、190 0U L和 2 5 0 0U L ,比野生菌P .putidaCPU 980 1的海因酶活力分别提高了 8倍、9倍和 12倍 .薄层扫描结果显示 ,这些工程菌的海因酶表达量分别约占菌体总可溶性蛋白质的 2 0 %、31%和 5 7%.SDS PAGE显示 ,海因酶的单体分子量约为 5 0kD .经工程菌E .coliBL2 1 pMD T7 dht催化 ,底物对羟基苯海因的转化率在 13h内可达到 9     

Biotransformation of isoeugenol catalyzed by growing cells of Pseudomonas putida

Abstract

Growing cells of Pseudomonas putida transformed isoeugenol after 5 days of incubation to give mainly vanillin, eugenol, 4-(E)-(3-hydroxyprop-1-enyl)-2-methoxyphenol and the dimeric molecule (+)-4-[2,3-dihydro-7-methoxy-3-methyl-5-(E)-(1-propenyl)-2-benzofuranyl]-2-methoxyphenol (licarin A). The formation of the latter compound from isoeugenol by biotransformation with P. putida is reported here for the first time.     

Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250

Abstract Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho -substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.     

Degradation and Detoxification of Nicosulfuron by a Pseudomonas Strain Isolated from a Contaminated Cornfield Soil

ABSTRACT

The dissipation and detoxification of nicosulfuron (NS) by Pseudomonas aeruginosa B9 isolated from a cornfield soil was investigated. The fastest decline of NS occurred at 40 µg ml^?1 in liquid media with 0.25% glucose plus 0.05% yeast extract (DT₅₀ = 4 days) with a notable pH reduction (pH ? 5). Bioassay tests showed considerable phytotoxicity of NS for Cress (Lepidium sativum L.) with 50% shoot growth inhibition (SGI) at 40 µg ml^?1. The dissipation of NS (40 µg ml^?1) by the B9 isolate reduced the SGI significantly (SGI: up to 45 ± 3%) compared to the non-inoculated media (SGI: up to 58 ± 4%). In soils with the B9 isolate, NS dissipation, especially at 0.3 µg g^?1, was faster with a more significant SGI reduction (k = 0.08 ± 0.00 day^?1; SGI = 2 ± 1%) compared to non-inoculated samples (k = 0.03 ± 0.00 day^?1; SGI = 8 ± 1%). NS initially inhibited soil respiration, microbial biomass carbon, and dehydrogenase activity. The effect was however transient, and these parameters recovered within 10 days, especially in the presence of the isolate. Overall, this study proves Pseudomonas aeruginosa B9 as a suitable candidate for bioremediation of NS in contaminated sites.