RESUMO
We have recently shown that SmbP, the small metal-binding protein of Nitrosomonas europaea, can be employed as a fusion protein to express and purify recombinant proteins and peptides in Escherichia coli. SmbP increases solubility, allows simple, one-step purification through affinity chromatography, and provides superior final yields due to its low molecular weight. In this work, we report for the first time the use of SmbP to produce a recombinant peptide with anticancer activity: the antitumor-analgesic peptide (BmK-AGAP), a neurotoxin isolated from the venom of the Chinese scorpion Buthus martensii Karsch. This peptide was expressed in Escherichia coli SHuffle for correct, cytoplasmic, disulfide bond formation and tagged with SmbP at the N-terminus to improve its solubility and allow purification using immobilized metal affinity chromatography. SmbP_BmK-AGAP was found in the soluble fraction of the cell lysate. After purification and removal of SmbP by digestion with enterokinase, 1.8 mg of pure and highly active rBmK-AGAP was obtained per liter of cell culture. rBmK-AGAP exhibited antiproliferative activity on the MCF-7 cancer cell line, with a half-maximal inhibitory concentration value of 7.24 µM. Based on these results, we considered SmbP to be a suitable carrier protein for the production of recombinant, biologically active BmK-AGAP. We propose that SmbP should be an attractive fusion protein for the expression and purification of additional recombinant proteins or peptides that display anticancer activities.
RESUMO
A novel pigmented bacterium, initially identified as 11E, was isolated from a site historically known to have various iron-related ores. Phylogenetic analysis of this bacterial strain showed that it belongs to Serratia marcescens. This pigmented S. marcescens 11E cultured individually with glucose, acetate, and glycerol as electron donors along with the soluble electron acceptor iron (Fe) (III) citrate offered a large reduction extent (45.3 %, 31.4 %, and 13.5 %, respectively). On the other hand, when iron oxide (Fe2O3) is used as electron acceptor, the pigmented strain produced a null reduction extent. Surprisingly, the absence of prodigiosin on the bacterial surface (non-pigmented strain) resulted in a large reduction extent of the non-soluble iron form (20-49%). All these extents were comparable and, in some cases, superior to those presented in the literature. Additionally, in the present study, it was found that anthraquinone sulfonate (AQS) stimulated Fe(III) reduction of soluble and non-soluble Fe species only with pigmented S. marcescens. In contrast, in the culture media with the non-pigmented strain, the presence of AQS did not stimulate the Fe(III) reduction. These results suggest that the pigmented phenotype of S. marcescens 11E may perform non-soluble Fe(III) reduction by electron shuttling. In contrast, for the non-pigmented phenotype of this bacterium, non-soluble Fe(III) reduction seems to proceed by direct contact. Our study demonstrates that this bacterium may be used in bioreduction process of heavy metals or as a biocatalyst in bioelectrochemical devices.
Assuntos
Compostos Férricos/metabolismo , Prodigiosina/metabolismo , Serratia marcescens , Enzimas , Filogenia , RNA Ribossômico 16S/genética , Serratia marcescens/genética , Serratia marcescens/isolamento & purificação , Serratia marcescens/metabolismoRESUMO
There is a current need to develop low-cost strategies to degrade and eliminate industrially used colorants discharged into the environment. Colorants discharged into natural water streams pose various threats, including: toxicity, degradation of aesthetics and inhibiting sunlight penetration into aquatic ecosystems. Dyes and colorants usually have complex aromatic molecular structures, which make them very stable and difficult to degrade and eliminate by conventional water treatment systems. The results in this work demonstrated that heavy metal-resistant Rhodotorula mucilaginosa strain UANL-001L isolated from the northeast region of Mexico produce an exopolysaccharide (EPS), during growth, which has colorant adsorption potential. The EPS produced was purified by precipitation and dialysis and was then physically and chemically characterized by Scanning Electron Microscopy, Fourier Transform Infrared Spectroscopy, and chemical elemental analysis. Here, the ability of the purified EPS produced to adsorb methylene blue (MB), which served as a model colorant, is studied. MB adsorption by the EPS is found to follow Langmuir Adsorption Isotherm kinetics at 25°C. Further, by calculating the Langmuir constant the adsorption capabilities of the EPS produced by the Rhodotorula mucilaginosa strain UANL-001L is compared to that of other adsorbents, both, microbially produced and from agroindustrial waste. The total adsorption capacity of the EPS, from the Rhodotorula mucilaginosa strain UANL-001L, was found to be two-fold greater than the best bioadsorbents reported in the literature. Finally, apart from determining which heavy metals stimulated EPS production in the strain, the optimal conditions of pH, heavy metal concentration, and rate of agitation of the growing culture for EPS production, was determined. The EPS reported here has the potential of aiding in the efficient removal of colorants both in water treatment plants and in situ in natural water streams.