Expression and characterization of a novel halohydrin dehalogenase from Rhodospirillaceae bacterium / 生物工程学报

As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral β-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0-8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.

A purple non-sulfur bacterium producing polyhydroxybutyrate and the conserved region of pha synthase gene / Bactéria púrpura não sulfurosa produtora de polihidroxibutirato e a região conservada do gene pha sintase

This study aimed to screen purple non-sulfur bacteria capable of accumulating granules or polyhydroxybutyrate (PHB) inside the cells, identify the potent strain, assay the enzyme or PHA synthase, and compare the PHB synthase gene with that of related strains. A total of 58 strains of purple non-sulfur bacteria were isolated from 108 samples of chicken feces in the chicken-egg farm of the Department of Animal Science, Faculty of Natural Resources at Prince of Songkla University, Hat Yai, Thailand. After cultivating the bacteria in glutamate malate (GM) medium without added glutamic acid under light (3,000 Lux) at 35oC for 5 days, the intracellular biopolymer granules of the bacteria were observed by using a Confocal Laser Scanning Microscope (CLSM) with excitation and emission wavelength of 530 and 605 nm, respectively. Gas chromatography (GC) was carried out for quantitative analysis of PHB. There were five strains, CH12, CH52, CH72, CH90 and CH92, showed biopolymer granules under CLSM, and accumulated PHB 5, 1.7, 1.5, 1.4 and 1.8% (w w-1) of the cell dry weight (CDW), respectively. The 16S rDNA sequence analysis of CH12 strain showed a high homology of 100% correlation to that of Rhodopseudomonas palustris strain NCIB8288. Regarding the taxonomic characteristics and 16S rDNA sequence analysis, CH12 strain was identified as Rps. palustris NCIB8288. The PHA synthase activity of the crude extract from CH12 strain was 25 units/mL. The conserved regions could be aligned and selected among 5 strains of Rhodopseudomonas palustris (strains BisA53, TIE-1, CGA009, HaA2 and BisB18). The purified PCR product was obtained for further studies.

Este estudo teve como objetivo rastrear bactérias púrpuras não sulfurosas capazes de acumular grânulos ou polihidroxibutirato (PHB) dentro das células, identificar a estirpe potente, ensaiar a enzima ou PHA sintaxe, e comparar com o gene PHB sintase com aquele de estirpes relacionadas. Um total de 58 estirpes de bactérias púrpuras não sulfurosas foram isoladas a partir de 108 amostras de fezes de galinhas na granja produtora de ovos do Departamento de Ciência Animal, Faculdade de Recursos Naturais da Universidade Prince of Songkla, Hat Yai, Tailândia. Depois de cultivar as bactérias em um substrato de glutamato/malato (GM), sem ácido glutâmico adicionado, sob luz (3000 lux) a 35 ºC durante 5 dias, os grânulos de biopolímeros intracelulares das bactérias foram observados utilizando um microscópio confocal (do inglês Confocal Laser Scanning Microscope - CLSM) com comprimentos de onda de excitação e emissão de 530 e 605 nm, respectivamente. A cromatografia gasosa (do inglês Gas chromatography - GC) foi realizada para uma análise quantitativa de PHB. Havia 5 estirpes, CH12, CH52, CH72, CH90 e CH92, que mostraram grânulos biopoliméricos quando submetidos ao CLSM, e PHB-5 acumulado de 1.7, 1.5, 1.4 and 1.8% (w w-1) do peso celular seco (do inglês cell dry weight - CDW), respectivamente. A análise da sequência do rDNA 16S da estirpe CH12 demonstrou uma alta correlação de homologia de 100% para aquela da estirpe NCIB8288 da Rhodopseudomonas palustris. Em relação às características taxonômicas e da análise da sequência do rDNA 16S, a estirpe CH12 foi identificada como Rps. palustris NCIB8288. A atividade da PHA sintase do extrato bruto da estirpe CH12 foi de 25 unidades/mL. As regiões conservadas puderam ser alinhadas e selecionadas entre 5 estirpes de Rhodopseudomonas palustris (BisA53, TIE-1, CGA009, HaA2 e BisB18). O produto purificado da reação em cadeia da polimerase - PCR foi obtido para estudos futuros.

Draft genome sequence of Inquilinus limosus strain MP06, a multidrug-resistant clinical isolate

The bacterium, Inquilinus limosus, with its remarkable antimicrobial multiresistant profile, has increasingly been isolated in cystic fibrosis patients. We report draft genome sequence of a strain MP06, which is of considerable interest in elucidating the associated mechanisms of antibiotic resistance in this bacterium and for an insight about its persistence in airways of these patients.

Expression and characterization of a novel halohydrin dehalogenase from Tistrella mobilis KA081020-065 / 生物工程学报

Halohydrin dehalogenase is of great significance for biodegradation of the chlorinated pollutants, and also serves as an important biocatalyst in the synthesis of chiral pharmaceutical intermediates. A putative halohydrin dehalogenase (HheTM) gene from Tistrella mobilis KA081020-065 was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified by Ni-NTA column and characterized. Gel filtration and SDS-PAGE analysis showed that the native form of HheTM was a tetramer. It exhibited the highest activity at 50 degrees C. The nature and pH of the buffer had a great effect on its activity. The enzyme maintained high stability under the alkaline conditions and below 30 degrees C. HheTM catalyzed the transformation of ethyl(S)-4-chloro-3-hydroxybutyrate in the presence of cyanide, to give ethyl (R)-4-cyano-3-hydroxybutyrate, a key intermediate for the synthesis of atorvastatin.

The use of rice straw broth as an appropriate medium to isolate purple nonsulfur bacteria from paddy fields

The aims were to explore an appropriate isolating medium for obtaining purple nonsulfur bacteria (PNSB) for use as biofertilizers in saline paddy fields and to obtain pure cultures. We therefore chose a defined isolating medium containing 0.25 percent NaCl, (Glutamate-Acetate broth, GA) and a rice straw broth to compare them for numbers of PNSB obtained, time to obtain pure cultures, diversity and costs. A total of 30 water and 30 sediment samples were collected from saline paddy fields in southern Thailand and used to isolate PNSB in both the isolating media. Based on 60 samples and a period of 13 days incubation under anaerobic light conditions, a greater number of samples produced PNSB growth in GA broth after only day 3; however, after that the rice straw broth provided about a 2 fold increase in the number of samples that produced PNSB growth. Colonies isolated from GA broth required a significantly higher number of repeated streaking to obtain a pure culture (average 3.5) than those from rice straw broth (average 2.7) and the latter medium also produced significantly (P < 0.05) more isolates per sample. Sixty samples of water and sediment, from rice paddies with salinity (average, 3.43 +/- 0.67 mS/cm) and slight acidity (average, pH 5.84 +/- 0.42) provided 62 PNSB isolates by GA broth and 210 isolates by rice straw broth, and rice straw broth also produced a greater prevalence of PNSB. Estimates of the costs based on current prices of media, Gas Pak and electricity to obtain PNSB with the use of GA broth was roughly 6 times higher than for the rice straw broth.

Isolation of purple nonsulfur bacteria for the removal of heavy metals and sodium from contaminated shrimp ponds

In order to determine whether waters used for the shrimp cultivation contained toxic levels of heavy metals (HMs) and sodium (Na), analysis was carried out on 31 shrimp ponds in areas of southern Thailand. Purple nonsulfur bacteria (PNB) were also isolated from the same ponds to investigate if they could be used for bioremediation of the above contaminants. The highest HMs concentrations of the sediment samples in mg/kg dry weight were found as follows: 0.75 cadmium (Cd), 62.63 lead (Pb), 34.60 copper (Cu) and 58.50 zinc (Zn). However, all sediment samples met Hong Kong standards for dredged sediment. In contrast, contamination of Cu (9-30 ug/L) and Zn (140-530 ug/L) exceeding the standard guidelines for marine aquatic animal set by the Pollution Control Department, Thailand, were found in 32 and 61 percent of water samples, respectively. Two metal resistant PNB isolates, NW16 and KMS24, were selected from the 120 PNB isolates obtained. Both isolates reduced the levels of HMs by up to 39 percent for Pb, 20 percent for Cu, 7 percent for Cd, 5 percent for Zn and 31 percent for Na from water that contained the highest levels of HMs found and 3 percent NaCl when cultured with either microaerobic-light or aerobic-dark conditions. The isolate NW16 removed a greater percentage of the HMs than the isolate KMS24, but the isolate KMS24 was able to survive better under a greater variety of environmental conditions. Both strains were therefore suitable to use for further investigating their abilities to remediate water contaminated with HMs and Na.