RESUMO
AIMS: Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria. METHOD AND RESULTS: A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34.6 kDa active cold-adapted protease with a maximum activity at 25-35°C and optimum pH of 8.0-9.0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis. CONCLUSIONS: A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind.
Assuntos
Aclimatação , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Subtilisina/metabolismo , Regiões Antárticas , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Estabilidade Enzimática , Mutagênese Sítio-Dirigida , Análise de Sequência de Proteína , Especificidade por Substrato , Subtilisina/química , Subtilisina/genéticaRESUMO
ABSTRACT The use of different substances for the storage of the entomopathogenic nematodes Steinernemacarpocapsae A11 and Heterorhabditis sp. JPM4 was evaluated. The nematodes were kept in distilled water (control) and the other treatments were made with water with the addition of: Tween 80® (0.1%), ethylene glycol (0.1%), glycerin (1%), glucose (1%), CaCO3 (0.1%), Triton® (0.1%), KMnO4 (0.01%) and NaOCl (0.1%). The evaluations were made at 30, 60, 90, 120, 150 and 180 days, counting the number of infective juveniles (IJ) and determining their survival and infectivity. It was found that glycerin acted as a preserving substance at the temperature of 28º C for both nematodes and also in 16º C for S. carpocapsae A11. The other substances tested, even when they kept the nematodes alive, did not show this effect in relation to infectivity.
RESUMO Foi avaliado o uso de diferentes substâncias com potencial conservante no armazenamento dos nematóides entomopatogênicos Steinernema carpocapsae A11 e Heterorhabditis sp. JPM4. Os nematóides foram mantidos em água destilada (testemunha), sendo os demais tratamentos compostos por água adicionada de: Tween 80® (0,1%), etileno glicol (0,1%), glicerina (1%), glicose (1%), CaCO3 (0,1%), Triton® (0,1%), KMnO4 (0,01%) e NaOCl (0,1%), ambos armazenados em diferentes temperaturas. As avaliações foram realizadas aos 30, 60, 90, 120, 150 e 180 dias, por meio de contagens dos juvenis infectantes (JI), sendo determinadas a sua viabilidade e infectividade. Constatou-se que a glicerina agiu como substância conservante na temperatura de 28º C para os dois nematóides testados e também a 16º C para o nematóide S. carpocapsae A11. As demais substâncias usadas, mesmo quando mantiveram os nematóides vivos, não preservaram a infectividade.