RESUMEN
Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.
Asunto(s)
Antiinflamatorios , Neoplasias de la Mama , Clonación Molecular , Escherichia coli , Proteínas Recombinantes , Serratia , Humanos , Animales , Femenino , Antiinflamatorios/farmacología , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Células MCF-7 , Neoplasias de la Mama/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Serratia/genética , Serratia/enzimología , Metaloproteasas/genética , Metaloproteasas/metabolismo , Metaloproteasas/aislamiento & purificación , Antineoplásicos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Quorum sensing can induce density-dependent gene expressions that cause various problems. For quorum-sensing inhibition, fundamental solutions such as gene manipulation are required, and acyl-homoserine lactone synthase (AHL synthase), which synthesizes the universal quorum-sensing signal of gram-negative bacteria, can be used as a target. In this study, researchers synthesized His-tagged AHL synthase and its deletion mutant that lacks the active site and compared their biochemical characteristics. His-YpeI, the 6x His-tagged AHL synthase of Serratia fonticola, and His-ΔYpeI, its deletion mutant, were designed, and their property conservation were examined using in silico projection tools. For in vitro synthesis of enzymes, the His-YpeI CFPS template was synthesized by in vitro gene synthesis, and the His-ΔYpeI CFPS template was obtained by deletion PCR. CFPS was performed and the products were purified with the 6x His-tag. The enzymes' properties were compared using an enzymatic assay. The bioinformatic analysis confirmed the conservation of biochemical properties between 6x His-tagged and untagged enzymes, including helix-turn-helix interactions, hydropathy profiles, and tertiary structure between His-YpeI and YpeI and between His-ΔYpeI and ΔYpeI. His-YpeI and His-ΔYpeI synthesized by CFPS were found to have the expected molecular weights and demonstrated distinct differences in enzyme activity. The analyzed enzymatic constants supported a significant decrease in substrate affinity and reaction rate as a result of YpeI's enzyme active site deletion. This result showed that CFPS could be used for in vitro protein synthesis, and quorum sensing could be inhibited at the enzymatic level due to the enzyme active site's deletion mutation.
Asunto(s)
Percepción de Quorum , Percepción de Quorum/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Aciltransferasas/química , Eliminación de Secuencia , Serratia/enzimología , Serratia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Dominio Catalítico , Secuencia de Aminoácidos , LigasasRESUMEN
Bacterial multimodular polyketide synthases (PKSs) are large enzymatic assembly lines that synthesize many bioactive natural products of therapeutic relevance. While PKS catalysis is mostly based on fatty acid biosynthetic principles, polyketides can be further diversified by post-PKS enzymes. Here, we characterized a remarkably versatile trans-acyltransferase (trans-AT) PKS from Serratia that builds structurally complex macrolides via more than ten functionally distinct PKS modules. In the oocydin PKS, we identified a new oxygenation module that α-hydroxylates polyketide intermediates, a halogenating module catalyzing backbone γ-chlorination, and modular O-acetylation by a thioesterase-like domain. These results from a single biosynthetic assembly line highlight the expansive biochemical repertoire of trans-AT PKSs and provide diverse modular tools for engineered biosynthesis from a close relative of E.â coli.
Asunto(s)
Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Acilación , Biocatálisis , Halogenación , Hidroxilación , Sintasas Poliquetidas/química , Policétidos/química , Serratia/enzimologíaRESUMEN
The stalling global progress in the fight against malaria prompts the urgent need to develop new intervention strategies. Whilst engineered symbiotic bacteria have been shown to confer mosquito resistance to parasite infection, a major challenge for field implementation is to address regulatory concerns. Here, we report the identification of a Plasmodium-blocking symbiotic bacterium, Serratia ureilytica Su_YN1, isolated from the midgut of wild Anopheles sinensis in China that inhibits malaria parasites via secretion of an antimalarial lipase. Analysis of Plasmodium vivax epidemic data indicates that local malaria cases in Tengchong (Yunnan province, China) are significantly lower than imported cases and importantly, that the local vector A. sinensis is more resistant to infection by P. vivax than A. sinensis from other regions. Analysis of the gut symbiotic bacteria of mosquitoes from Yunnan province led to the identification of S. ureilytica Su_YN1. This bacterium renders mosquitoes resistant to infection by the human parasite Plasmodium falciparum or the rodent parasite Plasmodium berghei via secretion of a lipase that selectively kills parasites at various stages. Importantly, Su_YN1 rapidly disseminates through mosquito populations by vertical and horizontal transmission, providing a potential tool for blocking malaria transmission in the field.
Asunto(s)
Anopheles/microbiología , Proteínas Bacterianas/inmunología , Lipasa/inmunología , Mosquitos Vectores/microbiología , Serratia/enzimología , Serratia/aislamiento & purificación , Animales , Anopheles/inmunología , Anopheles/parasitología , Anopheles/fisiología , Proteínas Bacterianas/genética , China , Femenino , Tracto Gastrointestinal/microbiología , Humanos , Lipasa/genética , Malaria Vivax/transmisión , Masculino , Mosquitos Vectores/inmunología , Mosquitos Vectores/parasitología , Mosquitos Vectores/fisiología , Plasmodium falciparum/fisiología , Plasmodium vivax/fisiología , Serratia/genética , Serratia/fisiología , SimbiosisRESUMEN
In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enzimas Inmovilizadas/metabolismo , Extremófilos/enzimología , Lipasa/metabolismo , Serratia/enzimología , Basidiomycota/enzimología , Biocatálisis , Estabilidad de Enzimas , Proteínas Fúngicas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Sefarosa/análogos & derivados , Sefarosa/químicaRESUMEN
Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure-function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria Serratia plymuthica strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between ß9 and α9 (aa228-246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272-275) made the SpPLD unable to form the gate-like structure which existed specically in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between ß11 and ß12 was also different from the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from Serratia. The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.
Asunto(s)
Dominio Catalítico , Modelos Moleculares , Fosfolipasa D/química , Conformación Proteica , Serratia/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Catálisis , Biología Computacional/métodos , Secuencia Conservada , Cristalografía por Rayos X , Fosfolipasa D/genética , Fosfolipasa D/aislamiento & purificación , Fosfolipasa D/metabolismo , Filogenia , Plantas/microbiología , Serratia/clasificación , Serratia/genéticaRESUMEN
In a cohort of 257 infants with congenital heart disease admitted to the pediatric intensive care unit, 22 infants had positive cultures for extended-spectrum beta-lactamase or AmpC Gram-negative bacteria. These infants had longer exposure to broad-spectrum antibiotics, greater support with invasive devices and longer intensive care and hospital lengths of stay.
Asunto(s)
Proteínas Bacterianas , Infección Hospitalaria/microbiología , Infecciones por Bacterias Gramnegativas/complicaciones , Cardiopatías Congénitas/complicaciones , Resistencia betalactámica , beta-Lactamasas , Estudios de Casos y Controles , Citrobacter/enzimología , Estudios de Cohortes , Enfermedad Crítica , Enterobacter/enzimología , Escherichia coli/enzimología , Femenino , Hospitales Pediátricos , Humanos , Lactante , Unidades de Cuidado Intensivo Pediátrico , Klebsiella/enzimología , Masculino , Prevalencia , Estudios Retrospectivos , Factores de Riesgo , Serratia/enzimologíaRESUMEN
Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a SN2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.
Asunto(s)
Proteínas Bacterianas/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Metiltransferasas/metabolismo , Octanos/metabolismo , Serratia/enzimología , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Biocatálisis , Compuestos Bicíclicos con Puentes/química , Clonación Molecular , Ciclización , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Octanos/química , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serratia/química , Serratia/genética , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Especificidad por SustratoRESUMEN
Protealysin is a Serratia proteamaculans metalloproteinase of the M4 peptidase family and the prototype of a large group of protealysin-like proteases (PLPs). PLPs are likely involved in bacterial interaction with plants and animals as well as in bacterial pathogenesis. We demonstrated that the PLP genes in bacteria colocalize with the genes of putative conserved proteins. In S. proteamaculans, these two genes form a bicistronic operon. The putative S. proteamaculans protein that we called emfourin (M4in) was expressed in Escherichia coli and characterized. M4in forms a complex with protealysin with a 1:1 stoichiometry and is a potent slow-binding competitive inhibitor of protealysin (Ki = 52 ± 14 pM); besides, M4in is not secreted from S. proteamaculans constitutively. A comparison of amino acid sequences of M4in and its homologs with those of known inhibitors suggests that M4in is the prototype of a new family of protein inhibitors of proteases.
Asunto(s)
Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/genética , Serratia/enzimología , Serratia/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Metaloproteasas/química , Metaloproteasas/metabolismo , Operón/genética , Péptido Hidrolasas/metabolismo , Serratia/metabolismoRESUMEN
Five novel strains of Serratia fonticola that produce FONA, a minor extended-spectrum beta-lactamase (ESBL), were isolated during routine surveillance of ESBL-producing Enterobacteriaceae in imported chicken meat in Japan in 2017 and 2018. These strains exhibited a clear ESBL phenotype in susceptibility tests carried out in the presence of clavulanic acid; however, all strains tested negative in a multiplex polymerase chain reaction assay used to detect TEM, SHV, and CTX-M ß-lactamase genes. After identification of the bacterial species as S. fonticola, full length blaFONA genes were amplified and the DNA sequences were determined. The blaFONA genes from all 5 strains were different from those previously reported (blaFONA-1 to blaFONA-6); they clustered close to one another but were distinct from previously reported blaFONA genes in a phylogenic analysis based on amino acid sequences.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carne/microbiología , Serratia/aislamiento & purificación , beta-Lactamasas/metabolismo , Animales , Proteínas Bacterianas/genética , Pollos , Enterobacteriaceae/enzimología , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Contaminación de Alimentos/análisis , Microbiología de Alimentos/métodos , Humanos , Japón , Pruebas de Sensibilidad Microbiana , Serratia/enzimología , Serratia/genéticaRESUMEN
In the present study, the nematicidal activity of an isolated strain of Mimosa pudica nodules was evaluated against the Nacobbus aberrans (J2) phytonymatodes with a mortality of 88.8%, while against the gastrointestinal nematode Haemonchus contortus (L3) and free-living Panagrellus redivivus was 100%. The ability to inhibit the growth of phytopathogenic fungi Fusarium sp., and Alternaria solani, as well as the oomycete Phytophthora capsici, this antifungal activity may be related to the ability to produce cellulases, siderophores and chitinases by this bacterial strain. Another important finding was the detection of plant growth promoter characteristics, such as auxin production and phosphate solubilization. The strain identified by sequences of the 16S and rpoB genes as Serratia sp. is genetically related to Serratia marcescens and Serratia nematodiphila. The promoter activity of plant growth, antifungal and nematicide of the Serratia sp. strain makes it an alternative for the biocontrol of fungi and nematodes that affect both the livestock and agricultural sectors, likewise, candidate as a growth-promoting bacterium.
Asunto(s)
Hongos/efectos de los fármacos , Mimosa/microbiología , Nematodos/efectos de los fármacos , Nódulos de las Raíces de las Plantas/microbiología , Serratia/química , Alternaria/efectos de los fármacos , Animales , Antifúngicos/farmacología , Proteínas Bacterianas/genética , Quitinasas/metabolismo , Endófitos/química , Endófitos/fisiología , Fusarium/efectos de los fármacos , Ácidos Indolacéticos/metabolismo , Mimosa/efectos de los fármacos , Phytophthora/efectos de los fármacos , ARN Ribosómico 16S/genética , Serratia/clasificación , Serratia/enzimología , Serratia/genética , Especificidad de la EspecieRESUMEN
Serratia fonticola is a human pathogen widely found in the environment, with birds being reported as possible natural hosts. During an epidemiological and genomic surveillance study conducted to monitor the occurrence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in South American wild birds, we identified an ESBL-positive S. fonticola in a fecal sample collected from a Hudsonian Whimbrel, during its non-breeding range on the Pacific Coast of Chile. Whole genome sequencing analysis and "in silico" modeling revealed a novel variant of the class A ESBLs FONA family, designated FONA-7, which shows 96.28% amino acid identity with FONA-6; with amino acid substitutions occurring in the signal peptide sequence (Thr22âSer), and in the mature protein (Ser39âAsn and Thr227âIle). This finding denotes that migratory birds can be potential vectors for the transboundary spread of ESBL-producing bacteria, creating a further theoretical risk for the origin of novel plasmid-encoded ß-lactamases.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Enfermedades de las Aves/microbiología , Serratia/efectos de los fármacos , beta-Lactamasas/genética , Animales , Aves/microbiología , Chile/epidemiología , Vectores de Enfermedades , Heces , Polimorfismo Genético , Serratia/enzimología , Serratia/genética , Secuenciación Completa del GenomaRESUMEN
Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CHOS is not straightforward, chemo-enzymatic approaches have shown some promise. We have used engineered glycoside hydrolases to catalyze oligomerization of activated DA building blocks through transglycosylation reactions. The building blocks were generated from readily available (GlcNAc)2-para-nitrophenol through deacetylation of the nonreducing end sugar with a recombinantly expressed deacetylase from Aspergillus niger (AnCDA9). This approach, using a previously described hyper-transglycosylating variant of ChiA from Serratia marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to production of CHOS containing up to ten alternating D and A units [(DA)2, (DA)3, (DA)4, and (DA)5]. The most abundant compounds were purified and characterized. Finally, we demonstrate that (DA)3 generated in this study may serve as a specific inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic sclerosis.
Asunto(s)
Quitina/análogos & derivados , Oligosacáridos/biosíntesis , Oligosacáridos/síntesis química , Acetilglucosamina/química , Aspergillus niger/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Carbohidratos , Quitina/biosíntesis , Quitina/síntesis química , Quitinasas/genética , Quitinasas/metabolismo , Cristalografía por Rayos X , Glucosamina/química , Hexosaminidasas/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligosacáridos/química , Serratia/enzimología , Serratia/genética , Serratia marcescens/enzimología , Serratia marcescens/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The unintentional crystallization of contaminant proteins in the place of target recombinant proteins is sporadically reported, despite the availability of stringent expression/purification protocols and of software for the detection of contaminants. Typically, the contaminant protein originates from the expression organism (for example Escherichia coli), but in rare circumstances contaminants from different sources have been reported. Here, a case of contamination from a Serratia bacterial strain that occurred while attempting to crystallize an unrelated protein from Burkholderia pseudomallei (overexpressed in E. coli) is presented. The contamination led to the unintended crystallization and structure analysis of a cyanase hydratase from a bacterial strain of the Serratia genus, an opportunistic enterobacterium that grows under conditions similar to those of E. coli and that is found in a variety of habitats, including the laboratory environment. In this context, the procedures that were adopted to identify the contaminant based on crystallographic data only are presented and the crystal structure of Serrata spp. cyanase hydratase is briefly discussed.
Asunto(s)
Artefactos , Cristalografía por Rayos X/normas , Cianatos/química , Escherichia coli/genética , Hidroliasas/ultraestructura , Sitios de Unión , Burkholderia pseudomallei/enzimología , Burkholderia pseudomallei/genética , Cianatos/metabolismo , Escherichia coli/enzimología , Expresión Génica , Humanos , Hidroliasas/genética , Hidroliasas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Serratia/enzimología , Serratia/genética , TransgenesRESUMEN
The sucrose isomerase SmuA from Serratia plymuthica efficiently catalyses the isomerisation of sucrose into isomaltulose, an artificial sweetener used in the food industry. However, the formation of a hygroscopic by-product, trehalulose, necessitates additional separation to obtain a crystalline product. Therefore, we have improved the product specificity of SmuA by first introducing a few exploratory amino acid exchanges around the active site and investigating their influence. Then, we devised a second set of mutations, either at promising positions from the preceding cycle, but with a different side chain, or at alternative positions in the vicinity. After seven iterative cycles involving just 55 point mutations, we obtained the triple mutant Y219L/D398G/V465E which showed 2.3 times less trehalulose production but still had high catalytic efficiency (kcat /KM =11.8â mM-1 s-1 ). Not only does this mutant SmuA appear attractive as an industrial biocatalyst, but our semirational protein-engineering strategy, which resembles the battleship board game, should be of interest for other challenging enzyme optimization endeavours.
Asunto(s)
Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Estabilidad de Enzimas , Glucosiltransferasas/química , Cinética , Modelos Moleculares , Conformación Proteica , Serratia/enzimología , Serratia/genética , Especificidad por SustratoRESUMEN
The use of an immobilized Serratia. sp W3 lipase as a replacement for the standard pancreatic lipases in the hydrolysis of liver oil from the Musteleus mustelus was studied. Monoacylglycerols (MAGs) and diacylglycerols (DAGs) containing ω-3 polyunsaturated fatty acids, namely eicosapentaenoic and docosahexaenoic acids were produced in hexane solvent at reaction temperatures reaching 55 °C with a molar triacylglycerol conversion over than 75 ± 5% in 24 h showing excellent hydrolysis characteristics. The favorable conditions for the hydrolysis reaction allowed fats with higher melting points to be analyzed facilitating the coupling of the hydrolysis reaction to the later steps in the analytical protocol. The lipid composition was elucidated for the first time by employing a highly efficient UHPLC-MS method with a novel embedded linear retention index approach. MAGs and DAGs obtained during the enzymatic hydrolysis could be used for the production of glycerol based emulsifiers of nutritional interest.
Asunto(s)
Diglicéridos/química , Ácidos Grasos Omega-3/análisis , Aceites de Pescado/química , Lipasa/metabolismo , Monoglicéridos/química , Animales , Biocatálisis , Cromatografía Líquida de Alta Presión , Diglicéridos/metabolismo , Enzimas Inmovilizadas/metabolismo , Ácidos Grasos Insaturados/análisis , Aceites de Pescado/metabolismo , Hidrólisis , Espectrometría de Masas , Monoglicéridos/metabolismo , Serratia/enzimología , Tiburones , Triglicéridos/metabolismoRESUMEN
In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for the formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5-methyl-4-pentyl-3,4-dihydro-2H-pyrrole (5 a, resulting from transamination of the aldehyde of 3-acetyloctanal) and 2-methyl-3-pentyl-3,4-dihydro-2H-pyrrole (6, resulting from transamination of the ketone), were synthesised. Only 5 a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colourimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5 a were synthesised, varying in the C2 and C3 side chains, and tested as substrates of HapB inâ vitro and for restoration of pigment production in Serratia ΔpigD inâ vivo. All lengths of side chain tested at C3 were accepted, but only short side chains at C2 were accepted. The knowledge that 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/química , Gammaproteobacteria/enzimología , Monoaminooxidasa/química , Prodigiosina/biosíntesis , Serratia/enzimología , Especificidad por SustratoRESUMEN
Protealysin, a metalloprotease of Serratia proteamaculans, is the prototype of a subgroup of the M4 peptidase family. Protealysin-like proteases (PLPs) are widely spread in bacteria but also occur in fungi and certain archaea. The interest in PLPs is primarily due to their putative involvement in the bacterial pathogenesis in animals and plants. Studying PLPs requires an efficient quantitative assay for their activity; however, no such assay has been reported so far. Here, we used the autoprocessing site sequence of the protealysin precursor to construct an internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. Protealysin and thermolysin, the prototype of the M4 family, proved to hydrolyze only the Ser-Val bond of the substrate. The substrate exhibited a KM = 35 ± 4 µM and kcat = 21 ± 1 s-1 for protealysin as well as a KM = 33 ± 8 µM and kcat = 7 ± 1 s-1 for thermolysin at 37 °C. Comparison of the effect of different enzymes (thermolysin, trypsin, chymotrypsin, savinase, and pronase E) on the substrate has demonstrated that it is not strictly specific for protealysin; however, this enzyme has higher molar activity even compared to the closely related thermolysin. Thus, the proposed substrate can be advantageous for quantitative studies of protealysin as well as for activity assays of other M4 peptidases.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pruebas de Enzimas/métodos , Péptidos/metabolismo , Termolisina/metabolismo , Fluorescencia , Hidrólisis , Péptidos/química , Serratia/enzimología , Especificidad por SustratoRESUMEN
Nine bacterial strains were previously isolated in association with pinewood nematode (PWN) from wilted pine trees. They proved to be nematicidal in vitro, and one of the highest activities, with potential to control PWN, was showed by Serratia sp. M24T3. Its ecology in association with plants remains unclear. This study aimed to evaluate the ability of strain M24T3 to colonize the internal tissues of the model plant Arabidopsis thaliana using confocal microscopy. Plant growth-promoting bacteria (PGPB) functional traits were tested and retrieved in the genome of strain M24T3. In greenhouse conditions, the bacterial effects of all nematicidal strains were also evaluated, co-inoculated or not with Bradyrhizobium sp. 3267, on Vigna unguiculata fitness. Inoculation of strain M24T3 increased the number of A. thaliana lateral roots and the confocal analysis confirmed effective bacterial colonization in the plant. Strain M24T3 showed cellulolytic activity, siderophores production, phosphate and zinc solubilization ability, and indole acetic acid production independent of supplementation with L-tryptophan. In the genome of strain M24T3, genes involved in the interaction with the plants such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinolytic activity, and quorum sensing were also detected. The genomic organization showed ACC deaminase and its leucine-responsive transcriptional regulator, and the activity of ACC deaminase was 594.6 nmol α-ketobutyrate µg protein-1 µl-1. Strain M24T3 in co-inoculation with Bradyrhizobium sp. 3267 promoted the growth of V. unguiculata. In conclusion, this study demonstrated the ability of strain M24T3 to colonize other plants besides pine trees as an endophyte and displays PGPB traits that probably increased plant tolerance to stresses.
Asunto(s)
Arabidopsis/microbiología , Nematodos/microbiología , Serratia/fisiología , Animales , Antibiosis , Arabidopsis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Carbono/genética , Liasas de Carbono-Carbono/metabolismo , Pinus/parasitología , Enfermedades de las Plantas/parasitología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Percepción de Quorum , Serratia/enzimología , Serratia/genética , Serratia/aislamiento & purificación , Vigna/crecimiento & desarrollo , Vigna/microbiologíaRESUMEN
A new thermophilic non-induced lipase producer named Serratia rubidaea strain Nehal-mou was isolated from oil waste in Tissemsilat, Algeria. The most influential lipase production parameters were screened by the Plackett-Burman design for enhancing enzyme yield. An optimum condition of a 1.5% of glucose, a 0.01% of potassium, and a 0.025% of manganese contents resulted in a 41.13 U/mL. This yield was 6.29 times higher than the one achieved before the application of the Box-Behnken Design. Lipase activity showed a high organic solvent tolerance following its exposure to hexane, ethanol, methanol, and acetone. Lipase was also perfectly stable in the presence of 10 mM Fe2+, K+, and Na+ ions with more than 75% of the retaining activity. The enzyme half-life times were 22 h, 90 min, and 25 min at 50, 60, and 70 °C respectively. Polyvinyl alcohol (PVA)/boric acid/Starch/CaCO3 were utilized as a carrier for lipase covalent immobilization in order to be used efficiently. The Scanning Electron Microscopy (SEM) Technique and the Fourier Transform Infrared Spectroscopy (FTIR) Method confirmed the covalent bonding success and the excellent carrier characteristics. Thus, the immobilization yield reached 73.5% and the optimum temperature was shifted from 40 to 65 °C. The immobilized lipase kept 80% of its total activity after 10 cycles and had 3 and 3.2-fold half-lives at 70, and 80 °C respectively compared to the free enzyme.