RESUMEN
Some microorganisms can produce cyclodextrin glycosyltransferases, which degrades starch by catalyzing cyclization and giving rise to cyclodextrin. Thus, to fully degrade starch, microorganisms can also synthesize cyclodextrinases, which hydrolyze cyclodextrins. In this work, a truncated gene, without the signal peptide coding sequence, encoding a cyclodextrinase from Massilia timonae was PCR amplified, cloned, and expressed in E. coli. The histidine-tagged recombinant enzyme was purified by immobilized metal ion affinity chromatography. The purified protein was found to be a tetramer of about 260â¯kDa, with monomers of about 65â¯kDa, as estimated by gel filtration and SDS-PAGE, respectively. The enzyme presented an optimum temperature of 40⯰C, optimum pH of 7.0, and remained stable after 30â¯min of incubation at 45⯰C, with a T50 of 48.45⯰C. The enzyme showed a higher activity toward ß-cyclodextrin compared to that for maltodextrin and starch. KM for ß-cyclodextrin was 2.1â¯mM, Vmax was 0.084⯵mol/min, kcat was 8326 min-1, and kcat/KM was 4.1â¯×â¯106â¯M-1min-1. Calcium acted as an activator and SDS, CTAB, several cations, and EDTA acted as strong inhibitors. The purified cyclodextrinase produced glucose and maltose as final products by hydrolysis of ß-cyclodextrin, maltotetraose, and maltoheptaose. This novel cyclodextrinase could be a promising alternative for the enzymatic hydrolysis of starch.
Asunto(s)
Proteínas Bacterianas , Expresión Génica , Glicósido Hidrolasas , Oxalobacteraceae , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/enzimología , Escherichia coli/genética , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/aislamiento & purificación , Oxalobacteraceae/enzimología , Oxalobacteraceae/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
ABSTRACT The presence of mycotoxins or related fungi in animal feed is a major problem for animal and human health. Silage and concentrated feed samples were collected from 21 dairy farms in the Western part of Paraná state in Southern Brazil. Water activity and pH of all samples were measured, and each sample was analyzed to check for the presence of aflatoxigenic Aspergillus. Water activity was observed to be lower in the concentrated feed samples. The pH was lower in the silage samples, indicating fermentation processes. Two silage samples and four concentrated feed samples were contaminated with Aspergillus spp. Seven isolates of Aspergillus spp. were obtained and their potential to produce aflatoxins was evaluated. Four of the isolates, two from the silage samples and two from the concentrated feed samples, produced the aflatoxins B1, B2, G1, and G2 in culture media. These isolates were identified as Aspergillus parasiticus and Aspergillus nomius. The presence of aflatoxigenic isolates of Aspergillus spp. in silage and concentrated feed samples is a matter of concern, because of the risk of aflatoxin production and contamination of the animal feed.
Asunto(s)
Animales , Bovinos , Aspergillus/aislamiento & purificación , Contaminación de Alimentos/análisis , Aflatoxinas/metabolismo , Alimentación Animal/microbiología , Aspergillus/clasificación , Aspergillus/genética , Aspergillus/metabolismo , Ensilaje/clasificación , Ensilaje/microbiología , Brasil , Alimentación Animal/análisisRESUMEN
The presence of mycotoxins or related fungi in animal feed is a major problem for animal and human health. Silage and concentrated feed samples were collected from 21 dairy farms in the Western part of Paraná state in Southern Brazil. Water activity and pH of all samples were measured, and each sample was analyzed to check for the presence of aflatoxigenic Aspergillus. Water activity was observed to be lower in the concentrated feed samples. The pH was lower in the silage samples, indicating fermentation processes. Two silage samples and four concentrated feed samples were contaminated with Aspergillus spp. Seven isolates of Aspergillus spp. were obtained and their potential to produce aflatoxins was evaluated. Four of the isolates, two from the silage samples and two from the concentrated feed samples, produced the aflatoxins B1, B2, G1, and G2 in culture media. These isolates were identified as Aspergillus parasiticus and Aspergillus nomius. The presence of aflatoxigenic isolates of Aspergillus spp. in silage and concentrated feed samples is a matter of concern, because of the risk of aflatoxin production and contamination of the animal feed.
Asunto(s)
Aflatoxinas/metabolismo , Alimentación Animal/microbiología , Aspergillus/aislamiento & purificación , Contaminación de Alimentos/análisis , Alimentación Animal/análisis , Animales , Aspergillus/clasificación , Aspergillus/genética , Aspergillus/metabolismo , Brasil , Bovinos , Ensilaje/análisis , Ensilaje/microbiologíaRESUMEN
Abstract Aflatoxins are mutagenic, carcinogenic, and teratogenic mycotoxins. The objective of this work was to study the presence of aflatoxigenic Aspergillus in commercial Bulgur wheat in the city of Maringá, Paraná, Brazil. Thirty samples of commercial Bulgur wheat, acquired in the period of August 2011 to January 2012, were evaluated. The enumeration analysis showed that samples had up to 273.3 CFU of molds and 133.3 CFU of aflatoxigenic Aspergillus per gram of wheat. Forty-two monosporic isolates were obtained and identified as Aspergillus flavus. The isolates were analyzed regarding their aflatoxigenic potential by culture in coconut milk agar; hydroxide vapor exposure; chromatography; and polymerase chain reaction (PCR) targeting genes that code enzymes of the aflatoxins synthesis pathway. Some of the isolates were confirmed to be aflatoxin producers and several of them presented a genetic profile of aflatoxin synthesis. The obtained results demonstrated that Bulgur wheat A. flavus contamination is concerning.