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1.
Sci Rep ; 11(1): 11496, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075178

RESUMO

On recreational sandy beaches, there are guidelines for the management of bacterial pollution in coastal waters regarding untreated sewage, urban wastewater, and industrial wastewater. However, terrestrial plant debris on coastal beaches can be abundant especially after floods and whilst it has rarely been considered a concern, the bacterial population associated with this type of pollution from the viewpoint of public health has not been adequately assessed. In this study, microbes associated with plant debris drifting onto Kizaki Beach in Japan were monitored for 8 months throughout the rainy season, summer, typhoon season, and winter. Here we show that faecal-indicator bacteria in the plant debris and sand under the debris were significantly higher than the number of faecal bacteria in the sand after a 2015 typhoon. When we focused on specific pathogenic bacteria, Brevundimonas vesicularis and Pseudomonas alcaligenes were commonly detected only in the plant debris and sand under the debris during the survey period. The prompt removal of plant debris would therefore help create safer beaches.


Assuntos
Praias , Caulobacteraceae , Monitoramento Ambiental , Plantas/microbiologia , Pseudomonas alcaligenes , Microbiologia da Água , Caulobacteraceae/crescimento & desenvolvimento , Caulobacteraceae/isolamento & purificação , Humanos , Pseudomonas alcaligenes/crescimento & desenvolvimento , Pseudomonas alcaligenes/isolamento & purificação
2.
Waste Manag ; 131: 268-276, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34175751

RESUMO

Bioconversion of food waste oil (FWO) into biodegradable plastic is a promising method for converting waste into high-value products. In this study, a strain (Pseudomonas sp. H3) was isolated for polyhydroxyalkanoate (PHA) synthesis from FWO. After 72 h of cultivation with 20 g/L of FWO, the high cell dry weight (CDW) of 3.6 g/L, PHA yield of 2.4 g/L, and PHA content of 65 wt% were obtained under the optimal temperature (25 °C) and inoculum amount (6% (v/v)). Fed-batch fermentation was conducted in a 5 L bioreactor with a maximum CDW of 16 g/L, PHA content of 54 wt%, and PHA productivity of 0.23 g/(L·h) after 36 h. The PHA had a molecular weight of 54 782 Da and a low polydispersity index of 1.41 with glass transition, melting, and degradation temperatures of -20 °C, 34 °C, and 210 °C, respectively. To further utilize the wastewater after PHA production, anaerobic digestion was employed for CH4 production, and the CH4 yield was 284 mL/g volatile solids. Microbial community analysis showed that the abundance of acetate-oxidizing bacteria and Methanobacterium significantly increased during anaerobic digestion. This study describes a new strain for the economical synthesis of biodegradable plastics and presents a novel framework for fully utilizing FWO with the production of PHA and CH4.


Assuntos
Poli-Hidroxialcanoatos , Pseudomonas alcaligenes , Eliminação de Resíduos , Reatores Biológicos , Fermentação , Alimentos , Águas Residuárias
3.
Protein Expr Purif ; 175: 105721, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32763465

RESUMO

Lipomax is a commercialized foldase-dependent Pseudomonas lipase that was previously expressed only in Pseudomonas strains. Here, using Pichia pastoris as the host, we report a new co-expression method that leads to the successful production of Lipomax. The active Lipomax is extracellularly co-expressed with its cognate foldase (LIM); and the purified enzyme mix has the optimum pH at pH 8.0 and an optimal temperature around 40 °C. N-glycosylation was observed for Pichia produced Lipomax, and its reduction was shown to increase the lipolytic activity. With different p-nitrophenyl esters as the substrates, the substrate profiling analyses further indicate that Lipomax prefers esters with middle-long chain fatty acids, showing the highest specific activity to p-nitrophenyl caprylate (C8). The extracellular co-expression of Lipomax and LIM in Pichia will not only increase our ability to investigate additional eukaryotic hosts for lipase expression, but also be of considerable value in analyzing other foldase-dependent lipases.


Assuntos
Proteínas de Bactérias , Expressão Gênica , Lipase , Pseudomonas alcaligenes/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Estabilidade Enzimática , Lipase/biossíntese , Lipase/química , Lipase/genética , Lipase/isolamento & purificação , Pseudomonas alcaligenes/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/genética , Saccharomycetales/metabolismo
4.
Curr Microbiol ; 76(4): 415-424, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30747257

RESUMO

The platform chemicals 1,3-propanediol (1,3-PD) and 2,3-butanediol (2,3-BD) are important raw materials for polyesters and biofuels. However, the biosynthesis of the compounds relies on massive consumption of glucose or glycerol, leading to the uneconomical production in industrial scale. In this work, we developed a new method for co-production of 1,3-PD and 2,3-BD from waste lard to reduce the cost in carbon source supply. A waste lard utilizing Pseudomonas alcaligenes PA-3 and a 1,3-PD producing Klebsiella pneumoniae AA405 were co-cultivated by using waste lard as the sole carbon source. In a shake flask, 1.05 g/L 1,3-PD and 0.35 g/L 2,3-BD were produced from waste lard within 24 h. The addition of nitrogen source significantly increased the relative ratio of K. pneumoniae AA405 in the medium, which further favored to the higher titers of the two products. In bioreactor, the co-cultivation system produced 5.98 g/L 1,3-PD and 4.29 g/L 2,3-BD from 100 g/L waste lard within 72 h, and the conversion rate of 1,3-PD and 2,3-BD from waste lard were 62.95% and 0.75%, respectively. In all, this is the first work on 1,3-PD and 2,3-BD production from waste triglyceride, which will favor the utilization of low-cost carbon source in industrial production of chemicals.


Assuntos
Butileno Glicóis/metabolismo , Gorduras na Dieta/metabolismo , Microbiologia Industrial , Klebsiella pneumoniae/metabolismo , Propilenoglicóis/metabolismo , Pseudomonas alcaligenes/metabolismo , Biodegradação Ambiental , Biomassa , Reatores Biológicos , Técnicas de Cocultura , Ácidos Graxos/metabolismo , Glicerol/metabolismo , Klebsiella pneumoniae/crescimento & desenvolvimento , Redes e Vias Metabólicas , Nitrogênio/metabolismo , Pseudomonas alcaligenes/crescimento & desenvolvimento
5.
Artigo em Inglês | MEDLINE | ID: mdl-29278978

RESUMO

Bioremediation of arsenic using green technology via microbial enzymes has attracted scientists due to its simplicity and cost effectiveness. Statistical optimization of arsenate bioremediation was conducted by the enzyme arsenate reductase extracted from arsenic tolerant bacterium Pseudomonas alcaligenes. Response surface methodology based on Box-Behnken design matrix was performed to determine the optimal operational conditions of a multivariable system and their interactive effects on the bioremediation process. The highest biosorptive activity of 96.2 µg gm-1 of beads was achieved under optimized conditions (pH = 7.0; As (V) concentration = 1000 ppb; time = 2 h). SEM analysis showed the morphological changes on the surface of enzyme immobilized gluteraldehyde crosslinked Ca-alginate beads. The immobilized enzyme retained its activity for 8 cycles. ANOVA with a high correlation coefficient (R2 > 0.99) and lower "Prob > F"value (<0.0001) corroborated the second-order polynomial model for the biosorption process. This study on the adsorptive removal of As (V) by enzyme-loaded biosorbent revealed a possible way of its application in large scale treatment of As (V)-contaminated water bodies.


Assuntos
Alginatos/metabolismo , Arsênio/farmacocinética , Microesferas , Pseudomonas alcaligenes/enzimologia , Poluentes Químicos da Água/farmacocinética , Purificação da Água , Adsorção , Alginatos/química , Arseniatos/análise , Arseniatos/isolamento & purificação , Arseniatos/farmacocinética , Arsênio/análise , Arsênio/isolamento & purificação , Biodegradação Ambiental , Cálcio/química , Cálcio/metabolismo , Calibragem/normas , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/química , Ácidos Hexurônicos/metabolismo , Concentração de Íons de Hidrogênio , Pseudomonas alcaligenes/metabolismo , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/isolamento & purificação , Purificação da Água/métodos , Purificação da Água/normas , Purificação da Água/estatística & dados numéricos
6.
Biochem Biophys Res Commun ; 490(2): 528-534, 2017 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-28624459

RESUMO

The phytoremediation by using of green plants in the removal of environmental pollutant is an environment friendly, green technology that is cost effective and energetically inexpensive. By using Agrobacterium-mediated gene transfer, we generated transgenic Arabidopsis plants ectopically expressing mercuric transport protein gene (merT) from Pseudomonas alcaligenes. Compared with wild-type (WT) plants, overexpressing PamerT in Arabidopsis enhanced the tolerance to HgCl2. Further results showed that the enhanced total activities or corresponding transcripts of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) were observed in transgenic Arabidopsis under HgCl2 stress. These results were confirmed by the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) contents and reactive oxygen species (ROS) accumulation. In addition, localization analysis of PaMerT in Arabidopsis protoplast showed that it is likely to be associated with vacuole. In all, PamerT increased mercury (Hg) tolerance in transgenic Arabidopsis, and decreased production of Hg-induced ROS, thereby protecting plants from oxidative damage. The present study has provided further evidence that bacterial MerT plays an important role in the plant tolerance to HgCl2 and in reducing the production of ROS induced by HgCl2.


Assuntos
Arabidopsis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Técnicas de Transferência de Genes , Mercúrio/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Pseudomonas alcaligenes/genética , Arabidopsis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Genes Bacterianos , Plantas Geneticamente Modificadas/genética , Pseudomonas alcaligenes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico
7.
Mol Ecol ; 26(7): 1720-1733, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28029196

RESUMO

Environmental heterogeneity is considered a general explanation for phenotypic diversification, particularly when heterogeneity causes populations to diverge via local adaptation. Performance trade-offs, such as those stemming from antagonistic pleiotropy, are thought to contribute to the maintenance of diversity in this scenario. Specifically, alleles that promote adaptation in one environment are expected to promote maladaptation in alternative environments. Contrary to this expectation, however, alleles that underlie locally adaptive traits often fail to exhibit fitness costs in alternative environments. Here, we attempt to explain this paradox by reviewing the results of experimental evolution studies, including a new one of our own, that examined the evolution of trade-offs during adaptation to homogeneous versus heterogeneous environments. We propose that when pleiotropic effects vary, whether or not trade-offs emerge among diverging populations will depend critically on ecology. For example, adaptation to a locally homogeneous environment is more likely to occur by alleles that are antagonistically pleiotropic than adaptation to a locally heterogeneous environment, simply because selection is blind to costs associated with environments that are not experienced locally. Our literature review confirmed the resulting prediction that performance trade-offs were more likely to evolve during selection in homogeneous than heterogeneous environments. The nature of the environmental heterogeneity (spatial versus temporal) and the length of the experiment also contributed in predictable ways to the likelihood that performance trade-offs evolved.


Assuntos
Adaptação Fisiológica/genética , Meio Ambiente , Evolução Molecular , Pleiotropia Genética , Alelos , Bacteriófago phi 6/genética , Aptidão Genética , Mutação , Pseudomonas alcaligenes/virologia , Pseudomonas syringae/virologia
8.
Appl Microbiol Biotechnol ; 101(6): 2397-2413, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27942753

RESUMO

One of the major constraints in pig and poultry farming is the supply of protein-rich forage, containing sufficient amounts of key amino acids such as arginine (Ufaz and Galili 2008). Since these are underrepresented in plant proteins, the usage of plants as feed is limited. The heterologous production of the cyanobacterial storage polymer cyanophycin granule polypeptide (CGP) in plastids increases the amount of arginine substantially (Huhns et al. 2008; Huhns et al. 2009; Nausch et al. 2016a). CGP degradation releases arginine-aspartate dipeptides. CGP is stable in plants because its degradation is exclusively restricted to bacterial cyanophycinases (CGPases; Law et al. 2009). Since animals are also unable to digest CGP, CGPases need to be co-delivered with CGP-containing plant feed in order to release the dipeptides in the gastrointestinal tract of animals during digestion. Therefore, an extracellular CGPase, CphE from Pseudomonas alcaligenes DIP-1, was targeted to the cytosol, ER, and apoplasm of Nicotiana benthamiana. Translocation to the chloroplast was not successful. Although CphE accumulated in high amounts in the cytosol, only moderate levels were present in the ER, while the enzyme was nearly undetectable in the apoplasm. This correlates with the higher instability of post-translationally modified CphE in crude plant extracts. In addition, the production in the ER led to an increased number and size of necroses compared with cytosolic expression and might therefore interfere with the endogenous metabolism in the ER. Due to the high and robust enzyme activity, even moderate CphE concentrations were sufficient to degrade CGP in plant extracts.


Assuntos
Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Extratos Vegetais/química , Pseudomonas alcaligenes/enzimologia , Tabaco/metabolismo , Sequência de Aminoácidos , Ração Animal , Arginina/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Citosol/metabolismo , Proteínas na Dieta/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Hidrólise , Modelos Moleculares , Peptídeo Hidrolases/genética , Células Vegetais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Conformação Proteica , Pseudomonas alcaligenes/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/genética , Transgenes
9.
Colloids Surf B Biointerfaces ; 142: 207-213, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26954087

RESUMO

The introduction of high toxicity petroleum contaminants to the natural environment causes damage to ecosystems and the aesthetics of the surroundings. Therefore it is critical to enhance microbial community performance to manage the degradation process. This paper analyses the effect of natural surfactants from the tree Sapindus mukorossi on biodegradation of hydrocarbons. Analysis of cell surface hydrophobicity and zeta potential confirmed effective modifications of the cell surface parameters essential for the bioavailability of contaminants to microorganisms. Interestingly, favorable differences were observed only for microorganisms from non-contaminated soil. There was also recorded an increase in diesel oil biodegradation to 41% for Sphingomonas sp. and 56% for Pseudomonas alcaligenes on addition of 100mgL(-1) of Sapindus saponins. The addition of natural surfactants has no significant impact on bacterial strains isolated from long-term contaminated soil. This research demonstrates that the addition of Sapindus extract could be a useful tool to improve the effectiveness of microbial degradation of hydrocarbon pollutants by environmental strains in recently contaminated.


Assuntos
Hidrocarbonetos/metabolismo , Pseudomonas alcaligenes/efeitos dos fármacos , Sapindus/química , Saponinas/farmacologia , Poluentes do Solo/metabolismo , Sphingomonas/efeitos dos fármacos , Tensoativos/farmacologia , Biodegradação Ambiental , Técnicas Eletroquímicas , Poluição Ambiental , Gasolina , Humanos , Interações Hidrofóbicas e Hidrofílicas , Consórcios Microbianos/efeitos dos fármacos , Pseudomonas alcaligenes/crescimento & desenvolvimento , Pseudomonas alcaligenes/metabolismo , Saponinas/isolamento & purificação , Sphingomonas/crescimento & desenvolvimento , Sphingomonas/metabolismo , Propriedades de Superfície , Tensoativos/isolamento & purificação
10.
Appl Environ Microbiol ; 81(17): 5753-60, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26070679

RESUMO

In contrast to the well-characterized and more common maleylpyruvate isomerization route of the gentisate pathway, the direct hydrolysis route occurs rarely and remains unsolved. In Pseudomonas alcaligenes NCIMB 9867, two gene clusters, xln and hbz, were previously proposed to be involved in gentisate catabolism, and HbzF was characterized as a maleylpyruvate hydrolase converting maleylpyruvate to maleate and pyruvate. However, the complete degradation pathway of gentisate through direct hydrolysis has not been characterized. In this study, we obtained from the NCIMB culture collection a Pseudomonas alcaligenes spontaneous mutant strain that lacked the xln cluster and designated the mutant strain SponMu. The hbz cluster in strain SponMu was resequenced, revealing the correct location of the stop codon for hbzI and identifying a new gene, hbzG. HbzIJ was demonstrated to be a maleate hydratase consisting of large and small subunits, stoichiometrically converting maleate to enantiomerically pure d-malate. HbzG is a glutathione-dependent maleylpyruvate isomerase, indicating the possible presence of two alternative pathways of maleylpyruvate catabolism. However, the hbzF-disrupted mutant could still grow on gentisate, while disruption of hbzG prevented this ability, indicating that the direct hydrolysis route was not a complete pathway in strain SponMu. Subsequently, a d-malate dehydrogenase gene was introduced into the hbzG-disrupted mutant, and the engineered strain was able to grow on gentisate via the direct hydrolysis route. This fills a gap in our understanding of the direct hydrolysis route of the gentisate pathway and provides an explanation for the high yield of d-malate from maleate by this d-malate dehydrogenase-deficient natural mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Gentisatos/metabolismo , Hidroliases/metabolismo , Pseudomonas alcaligenes/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Hidroliases/química , Hidroliases/genética , Hidrolases/genética , Hidrolases/metabolismo , Hidrólise , Malatos/metabolismo , Maleatos/metabolismo , Pseudomonas alcaligenes/química , Pseudomonas alcaligenes/genética , Pseudomonas alcaligenes/metabolismo
11.
Appl Environ Microbiol ; 81(8): 2852-60, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25681184

RESUMO

Inorganic arsenic (As) is highly toxic and ubiquitous in the environment. Inorganic As can be transformed by microbial methylation, which constitutes an important part of the As biogeochemical cycle. In this study, we investigated As biotransformation by Pseudomonas alcaligenes NBRC14159. P. alcaligenes was able to methylate arsenite [As(III)] rapidly to dimethylarsenate and small amounts of trimethylarsenic oxide. An arsenite S-adenosylmethionine methyltransferase, PaArsM, was identified and functionally characterized. PaArsM shares low similarities with other reported ArsM enzymes (<55%). When P. alcaligenes arsM gene (PaarsM) was disrupted, the mutant lost As methylation ability and became more sensitive to As(III). PaarsM was expressed in the absence of As(III) and the expression was further enhanced by As(III) exposure. Heterologous expression of PaarsM in an As-hypersensitive strain of Escherichia coli conferred As(III) resistance. Purified PaArsM protein methylated As(III) to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases. We propose that PaArsM plays a role in As methylation and detoxification of As(III) and could be exploited in bioremediation of As-contaminated environments.


Assuntos
Arsenitos/metabolismo , Proteínas de Bactérias/genética , Metiltransferases/genética , Pseudomonas alcaligenes/enzimologia , Pseudomonas alcaligenes/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biotransformação , Metiltransferases/química , Metiltransferases/metabolismo , Dados de Sequência Molecular , Pseudomonas alcaligenes/metabolismo , Alinhamento de Sequência
12.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 32(5): 1044-9, 2015 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-26964309

RESUMO

In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.


Assuntos
Proteínas de Bactérias/química , Dexametasona/química , Pseudomonas alcaligenes/enzimologia , Sequência de Aminoácidos , Cromatografia , Peso Molecular
13.
J Basic Microbiol ; 55(2): 262-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25284640

RESUMO

Glucocorticosteroids such as dexamethasone have polluted hospital wastewater, urban sewage, and river water in varying degrees. However, dexamethasone degradation by bioremediation technology is less understood. This study aims to isolate bacteria that could degrade dexamethasone and to identify their degradation characteristics. Hospital wastewater contaminated by dexamethasone was collected. After culturing in inorganic salt medium and in carbon deficient medium containing dexamethasone sodium phosphate, a bacterial strain with dexamethasone sodium phosphate as the sole carbon and energy source was enriched and isolated from the contaminated wastewater. The strain was identified as Pseudomonas alcaligenes by morphology, Gram staining, biochemical test, and 16S rDNA sequencing. Isolated bacteria were domesticated. Then its degradation characteristic was determined by high-performance liquid chromatography method. The degradation rate of P. alcaligenes on dexamethasone sodium phosphate was 50.86%. Of the degraded dexamethasone sodium phosphate, 75.23% of dexamethasone sodium phosphate was degraded to dexamethasone and 23.63% was degraded to other metabolites. In conclusion, the isolated P. alcaligenes in this study would provide experimental evidence for further research on the bioremediation technology to treat dexamethasone sodium phosphate and dexamethasone polluted water and further for the elimination of side effects of dexamethasone.


Assuntos
Dexametasona/análogos & derivados , Glucocorticoides/metabolismo , Pseudomonas alcaligenes/isolamento & purificação , Pseudomonas alcaligenes/metabolismo , Poluentes Químicos da Água/metabolismo , Biodegradação Ambiental , Cromatografia Líquida de Alta Pressão , DNA Ribossômico/genética , Dexametasona/metabolismo , Filogenia , Pseudomonas alcaligenes/citologia , Pseudomonas alcaligenes/genética , RNA Ribossômico 16S/genética
14.
Appl Environ Microbiol ; 80(8): 2381-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24509932

RESUMO

Soluble cyanobacterial granule polypeptide (CGP), especially that isolated from recombinant Escherichia coli strains, consists of aspartic acid, arginine, and a greater amount of lysine than that in insoluble CGP isolated from cyanobacteria or various other recombinant bacteria. In vitro guanidination of lysine side chains of soluble CGP with o-methylisourea (OMIU) yielded the nonproteinogenic amino acid homoarginine. The modified soluble CGP consisted of 51 mol% aspartate, 14 mol% arginine, and 35 mol% homoarginine. The complete conversion of lysine residues to homoarginine was confirmed by (i) nuclear magnetic resonance spectrometry, (ii) coupled liquid chromatography-mass spectrometry, and (iii) high-performance liquid chromatography. Unlike soluble CGP, this new homoarginine-containing polyamide was soluble only under acidic or alkaline conditions and was insoluble in water or at a neutral pH. Thus, it showed solubility behavior similar to that of the natural insoluble polymer isolated from cyanobacteria, consisting of aspartic acid and arginine only. Polyacrylamide gel electrophoresis revealed similar degrees of polymerization of the native (12- to 40-kDa) and modified (10- to 35-kDa) polymers. This study showed that the chemical structure and properties of a biopolymer could be changed by in vitro introduction of a new functional group after biosynthesis of the native polymer. In addition, the modified CGP could be digested in vitro using the cyanophycinase from Pseudomonas alcaligenes strain DIP1, yielding a new dipeptide consisting of aspartate and homoarginine.


Assuntos
Proteínas de Bactérias/síntese química , Guanidina/metabolismo , Homoarginina/síntese química , Nylons/síntese química , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Homoarginina/metabolismo , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Compostos de Metilureia/metabolismo , Peso Molecular , Nylons/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Pseudomonas alcaligenes/enzimologia , Solubilidade
16.
Biochim Biophys Acta ; 1834(12): 2494-501, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24012560

RESUMO

A combination of site-directed mutagenesis and chemical modification was employed to alter protein structure with the objective of improving diastereopreference over that achieved by simple site-directed mutagenesis. Conformational analysis using molecular dynamic (MD) simulation of Pseudomonas alcaligenes lipase (PAL) indicated that stronger steric exclusion and structural rigidity facilitated diastereopreference. A cysteine (Cys) residue was introduced using site-directed mutagenesis to construct variant A272C. The modifier 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was then reacted with the introduced Cys residue to provide stronger steric exclusion and structural rigidity. The modification was verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Diastereopreference was improved significantly. The diastereomeric excess (dep) of l-menthol increased from 35% with wild type PAL to 90% with A272C-DTNB modified PAL when the conversion ratio of l-menthyl propionate was nearly 100%. Conformation and kinetic parameter analysis showed that A272C-DTNB modified PAL exhibited stronger steric exclusion and increased structural rigidity around the modification site that inhibited the hydrolysis of non-targeted substrates. The combination of site-directed mutagenesis and chemical modification could be an effective method to alter protein properties and enhance diastereopreference through the combined effect of steric exclusion and structural rigidity.


Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Lipase/química , Pseudomonas alcaligenes/enzimologia , Proteínas de Bactérias/genética , Estabilidade Enzimática/genética , Lipase/genética , Mentol/química , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Pseudomonas alcaligenes/genética , Especificidade por Substrato/genética
17.
J Basic Microbiol ; 53(4): 318-26, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22915081

RESUMO

The effects of root-associated fungi (Aspergillus awamori and Glomus mosseae) and plant growth promoting rhizobacteria (PGPR) (Pseudomonas putida, Pseudomonas alcaligenes and Paenibacillus polymyxa) were studied alone and in combination in glasshouse experiments on the growth of pea, enzyme activity (peroxidase and catalase) and reproduction of root-knot nematode Meloidogyne incognita. Application of A. awamori, G. mosseae and PGPR caused a significant increase in pea growth and enzyme activities of both nematode inoculated and uninoculated plants. A. awamori was more effective in reducing galling and improving the growth of nematode inoculated plants than P. alcaligenes or P. polymyxa. The greatest increase in growth, enzyme activities of nematode-inoculated plants and reduction in galling and nematode multiplication was observed when A. awamori was used with P. putida or G. mosseae as compared to the other combinations tested. Percentage root colonization was higher when AM fungus inoculated plants were treated with P. putida both in presence and absence of nematode.


Assuntos
Ervilhas/microbiologia , Ervilhas/parasitologia , Raízes de Plantas/microbiologia , Microbiologia do Solo , Tylenchoidea/crescimento & desenvolvimento , Animais , Aspergillus/crescimento & desenvolvimento , Glomeromycota/crescimento & desenvolvimento , Interações Microbianas , Paenibacillus/crescimento & desenvolvimento , Ervilhas/enzimologia , Ervilhas/crescimento & desenvolvimento , Pseudomonas alcaligenes/crescimento & desenvolvimento , Pseudomonas putida/crescimento & desenvolvimento
18.
J Basic Microbiol ; 53(8): 645-56, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22961241

RESUMO

The killer phenomenon of yeast may have technological implications in many areas like beverage fermentation, food technology, biological control in agriculture, and in medicine. In the present study the killer phenomenon in Pichia kudriavzevii (P. kudriavzevii RY55) is being reported for the first time. The P. kudriavzevii RY55 toxin exhibited excellent antibacterial activity against several pathogens of human health significance such as Escherichia coli, Enterococcus faecalis, Klebsiella sp., Staphylococcus aureus, Pseudomonas aeruginosa and Pseudomonas alcaligenes. Killer toxin was purified to homogeneity by using ammonium sulphate precipitation and ion exchange chromatography and characterized for few properties. P. kudriavzevii RY55 killer toxin may be of vast significance in the development of novel antimicrobial chemotherapeutic agents, new bio-based safer candidates for food preservation and biocontrol, and starter cultures for fermentation industries.


Assuntos
Antibacterianos/farmacologia , Micotoxinas/farmacologia , Pichia/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fermentação , Concentração de Íons de Hidrogênio , Klebsiella/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Micotoxinas/isolamento & purificação , Micotoxinas/metabolismo , Pichia/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas alcaligenes/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Temperatura , Fatores de Tempo
19.
Appl Environ Microbiol ; 79(3): 1044-7, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23204427

RESUMO

HbzF from Pseudomonas alcaligenes NCIMB 9867 was purified to homogeneity as a His-tagged protein and likely a dimer by SDS-PAGE and gel filtration. This protein was demonstrated to be a novel maleylpyruvate hydrolase, catalyzing direct hydrolysis of maleylpyruvate to maleate and pyruvate, and belongs to the fumarylacetoacetate hydrolase superfamily. This study reveals the genetic determinate for the direct maleylpyruvate hydrolysis in the gentisate pathway, complementary to the well-studied maleylpyruvate isomerization route.


Assuntos
Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Redes e Vias Metabólicas/genética , Ácidos Pimélicos/metabolismo , Pseudomonas alcaligenes/enzimologia , Pseudomonas alcaligenes/metabolismo , Cromatografia em Gel , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Hidrolases/genética , Hidrólise , Maleatos/metabolismo , Dados de Sequência Molecular , Peso Molecular , Multimerização Proteica , Pseudomonas alcaligenes/genética , Ácido Pirúvico/metabolismo , Análise de Sequência de DNA
20.
Bioresour Technol ; 121: 47-53, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22858467

RESUMO

The potential of non-ionic polysorbate surfactants as alternative inducers of medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHAs) for the production of diverse bacterial MCL-PHA depolymerases was evaluated. When grown with corn oil as the sole carbon substrate, Pseudomonas alcaligenes LB19 preferentially produced lipolytic enzymes, but its MCL-PHA depolymerase was not induced by the substrate. However, the results of activity staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis clearly revealed that Tween 20 induced simultaneous production of lipolytic enzymes and the MCL-PHA depolymerase with the molecular mass (26.5 kDa) of P. alcaligenes LB19, which has been previously identified. Moreover, the co-production of two functionally distinct hydrolytic enzymes induced by Tween 20 was commonly observed in various Gram-positive and Gram-negative bacteria that were fed the substrate. Thus, it is expected that non-ionic polysorbate surfactants including Tween 20 can be widely exploited as promising universal substrates for the facile and efficient production of diverse MCL-PHA depolymerases.


Assuntos
Biotecnologia/métodos , Hidrolases de Éster Carboxílico/biossíntese , Poli-Hidroxialcanoatos/metabolismo , Polissorbatos/metabolismo , Pseudomonas alcaligenes/metabolismo , Tensoativos/metabolismo
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