Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.FAPESPCNP

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (μ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.FAPESPCNP

Cross-Domain Deep Face Matching for Real Banking Security Systems

Ensuring the security of transactions is currently one of the major challenges that banking systems deal with. The usage of face for biometric authentication of users is attracting large investments from banks worldwide due to its convenience and acceptability by people, especially in cross-domain scenarios, in which facial images from ID documents are compared with digital self-portraits (selfies) for the automated opening of new checking accounts, e.g, or financial transactions authorization. Actually, the comparison of selfies and IDs has also been applied in another wide variety of tasks nowadays, such as automated immigration control. The major difficulty in such process consists in attenuating the differences between the facial images compared given their different domains. In this work, in addition to collecting a large cross-domain face dataset, with 27,002 real facial images of selfies and ID documents (13,501 subjects) captured from the databases of the major public Brazilian bank, we propose a novel architecture for such cross-domain matching problem based on deep features extracted by two well-referenced Convolutional Neural Networks (CNN). Results obtained on the dataset collected, called FaceBank, with accuracy rates higher than 93%, demonstrate the robustness of the proposed approach to the cross-domain face matching problem and its feasible application in real banking security systems

Wavelet-based Texture Model for Crowd Dynamic Analysis

Crowd event detection techniques aim at solving real-world surveillance problems, such as detecting crowd anomaly and tracking specific person in a highly dynamic crowd scene. In this paper, we proposed an innovate texture-based analysis method to model crowd dynamics and us it to distinguish the crowd behaviours. To describe complicated crowd scenes, homogeneous random features have been deployed in the research for behavioural template matching. Experiment results have shown that the anomaly appearing in crowd scenes can be effectively and efficiently identified by using the devised methods

Classificação não-supervisionada de dados hiperespectrais usando análise em componentes independentes

No passado recente foram desenvolvidas v árias t écnicas para classi ca ção de dados hiperspectrais. Uma abordagem tí pica consiste em considerar que cada pixel e uma mistura linear das reflectancias espectrais dos elementos presentes na c élula de resolu ção, adicionada de ru ído. Para classifi car e estimar os elementos presentes numa imagem hiperespectral, v ários problemas se colocam: Dimensionalidade dos dados, desconhecimento dos elementos presentes e a variabilidade da reflectância destes. Recentemente foi proposta a An álise em Componentes Independentes,para separa ção de misturas lineares. Nesta comunica ção apresenta-se uma metodologia baseada na An álise em Componentes Independentes para detec ção dos elementos presentes em imagens hiperespectrais e estima ção das suas quantidades. Apresentam-se resultados desta metodologia com dados simulados e com dados hiperespectrais reais, ilustrando a potencialidade da t écnica