RESUMO
We reported an on-demand type of metalloprotease from Exiguobacterium undae Su-1. Although this species of bacterium is known to inhabit the permafrost, there are no reports on either strong proteases or peptidases. We found that Su-1 protease is superior to commercially available proteases in proteolytic activity in a lower to normal range of temperature (10-50 °C) as well as in rapid inactivation heat-dependently on the Ca2+ concentration. These characteristics meet well with the demands from food processing and manufacturing. Biochemical investigations of the purified enzyme and protein structural analysis after gene cloning confirmed that Su-1 protease conserved high identity in its primary sequence with thermophilic proteases of the M4 family. On the other hand, its flexibility was enhanced when one Ca2+ binding site was lost and by replacement for proline and isoleucine residues.
Assuntos
Bacillaceae/enzimologia , Proteínas de Bactérias/metabolismo , Cálcio/farmacologia , Metaloproteases/metabolismo , Sequência de Aminoácidos , Bacillaceae/efeitos dos fármacos , Bacillaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Clonagem Molecular , Temperatura Baixa , Estabilidade Enzimática , Escherichia coli , Tecnologia de Alimentos , Concentração de Íons de Hidrogênio , Isoleucina/química , Isoleucina/genética , Metaloproteases/genética , Metaloproteases/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Prolina/química , Prolina/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
BACKGROUND: Few studies have examined the relationship between dyspnea perception and bronchial hyperresponsiveness (BHR) in asthmatic children. OBJECTIVE: To test the hypothesis that severe BHR is associated with poor perception of the severity of airway obstruction. METHODS: One hundred one asthmatic children (mean [SD] age, 11.1 [2.3] years) were evaluated using acetylcholine chloride (Ach) challenge. The BHR was assessed as the provocative concentration of Ach causing a 20% decrease in forced expiratory volume in 1 second (FEV1) (PC20). Perception of dyspnea was scored using a modified Borg scale after each dose of Ach and bronchodilator. The dyspnea threshold was defined as the point at which the Borg scale score became higher than 0. We evaluated the dyspnea perception score at a 20% decrease in FEV1 relative to baseline (PS20) and after bronchodilator administration (PS(BD)). RESULTS: The mean (SD) PS20 and PS(BD) were significantly lower in the severe vs the mild BHR group (PS20: 2.1 [1.9] vs 4.2 [2.4], P < .001; PS(BD): 0.5 [1.0] vs 1.0 [1.3], P = .048). The mean (SD) % decrease in FEV1 at the dyspnea threshold was significantly greater in the severe vs the mild BHR group (14.1% [11%] vs 5.4% [11%], P < .001). The PS20 was correlated positively with the PC20 (r2 = 0.25, P < .001), and the decrease in FEV1 at the dyspnea threshold was correlated negatively with the PC20 (r2 = 0.18, P < .001). CONCLUSIONS: Moderate to severe asthmatic children with severe BHR perceive dyspnea only after the stage of mild bronchoconstriction has passed. This relative insensitivity to dyspnea in asthmatic children with severe BHR may lead to undertreatment of asthma.