Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using β-galactosidase as a model

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using β-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant β-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized β-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant β-galactosidase, showing its potential application in other high-molecular-weight enzymes

Construção de uma biblioteca de fragmentos de lambda em Escherichia coli

18

Construção de uma biblioteca de fragmentos de lambda em Escherichia coli

18

Isolamento e caracterização de BliAI, um isoesquisômero de ClaI de Bacillus licheniformis

The restriction endonuclease BliAI, an isoschizomer of ClaI, which recognizes the sequence 5'- AT¯CGAT - 3', was purified from a natural isolate identified as Bacillus licheniformis. The restriction endonuclease was isolated from cell extracts using single-step purification by phosphocellulose column chromatography. The restriction endonuclease is active at 37ºC and over a wide range of pH and salt concentration. The molecular weight of the purified restriction enzyme is consistent with a value of 39 kDa.A endonuclease de restrição BliAI, um isoesquisômero de ClaI, que reconhece a seqüência 5'- AT¯CGAT - 3', foi purificada de um isolado natural identificado como Bacillus licheniformis. A endonuclease de restrição em questão foi isolada a partir de um extrato celular em um único passo cromatográfico utilizando uma coluna contendo a resina fosfocelulose. A endonuclease de restrição é ativa à 37ºC e em uma ampla escala de pH e concentração de sais. O peso molecular da enzima purificada corresponde a um valor de kDa 39

Human interferon B1 ser17: Coding DNA synthesis, expression, purification and characterization of bioactive recombinant protein

A protocol to produce large amounts of bioactive homogeneous human interferon β1 expressed in Escherichia coli was developed. Human interferon β1 ser17 gene was constructed, cloned and subcloned, and the recombinant protein expressed in E. coli cells. Solubilization of recombinant human interferon β1 ser17 (rhIFN-β1 ser17) was accomplished by employing a brief shift to high alkaline pH in the presence of non-ionic detergent. The recombinant protein was purifi ed by three chromatographic steps. N-terminal amino acid sequencing and mass spectrometry analysis provided experimental evidence for the identity of the recombinant protein. Reverse phase liquid chromatography demonstrated that the content of deamidates and sulphoxides was similar to a commercial standard. Size exclusion chromatography demonstrated the absence of high molecular mass aggregates and dimers. The protocol represents an effi cient and high-yield method to obtain bioactive homogeneous monomeric rhIFN-β1 ser17 protein. It may thus represent an important step towards scaling up for rhIFN-β1 ser17 large-scale production. © 2010 Villela AD, et al

One-step purification of a recombinant beta-galactosidase using magnetic cellulose as a support : rapid immobilization and high thermal stability

For the first time, this work reported the one-step purification and targeted immobilization process of a β-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after β-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between β-galactosidase and the substrate 1.2 × higher in the lactose hydrolysis of milk. β-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 × . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of β-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries

Transmissão transovariana de Babesia bovis em Boophilus microplus: obtenção de cepa de carrapato livre de Babesia spp. Babesia bovis transovarian transmission in Boophilus microplus: obtention of a Babesia free tick strain

O presente trabalho objetivou o estudo de parte do ciclo da Babesia bovis no seu hospedeiro invertebrado, o carrapato Boophilus microplus. Analisou-se a capacidade de infeccção e transmissão transovariana de B. bovis em partenóginas de B. microplus, alimentadas em bovinos portadores e enfermos por esse protozoário. No 18º dia após a infestação, coletaram-se partenóginas diretamente do corpo dos bovinos e teleóginas após o desprendimento natural, a partir do 21º dia. Todos os grupos foram incubados a 27ºC e umidade relativa superior a 70%. No 5º dia após o início da postura, realizou-se o exame de hemolinfa a fim de diagnosticar a infecção dos ínstares por B. bovis. A ausência de infecção detectada no exame de hemolinfa foi confirmada posteriormente com o teste biológico, revelando que partenóginas não transmitem B. bovis transovarianamente. Esses resultados oferecem uma técnica simplificada para a obtenção de cepas de carrapatos livres de B. bovis.<br>In this experiment part of the life cycle of Babesia bovis in its invertebrate host, the tick Boophilus microplus was studied. In order to evaluate the capacity of infection and transmission of B. bovis were collected semi-engorged females of B. microplus fed on carrier and ill bovines. In the 18th day after infestation, semi-engorged females were collected directly from bovine bodies and after 21st day engorged females dropped on the ground. All the collected groups were incubated at 27°C and relative humidity greater than 70%. At the 5th day of oviposition the diagnosis was made by direct examination of haemolymph smears. The biological test reveled that B. bovis transovarial transmission doesn't happer in semi-engorged females. The results offer a simple techique to obtain strains of ticks free of B. bovis