Isolation and characterization of a newly isolated Pseudomonas mutant for protease production

Abstract

A potent bacterium for extracellular protease production was isolated from local soil and identified as Pseudomonas sp. RAJR 044. A mutant of this strain JNGR 242 with protease productivity 2.5 fold higher was obtained by ultraviolet irradiation under experimentally optimized conditions of pH 7.0, temperature of 34ºC, inoculum volume of 1.0 mL and incubation time of 24 hours. Comparative analysis of the chemical characteristics i.e. assimilation of carbon and nitrogen sources were also carried out. Maximum growth of the mutant strain in 2% gelatin agar plate was obtained in presence of dextrose (2%), maltose (2%), ammonium sulfate (2%) and potassium nitrate (2%) whereas, that of the parent strain was found in sucrose (2%) and ammonium nitrate (2%). The purified proteases from both the strains (parent and mutant) appeared as single homogeneous bands corresponding to 14.4 kDa molecular weight on SDS-PAGE. On studying the kinetic properties of both strains it was observed that the rate of casein hydrolysis was maximum at pH 8.0 and 7.0 and temperatures 45º C and 60º C for the parent and mutant strains respectively. It was also observed that both the extracellular proteases were inhibited by a serine protease inhibitor i.e. PMSF at 2mM concentration.Uma bactéria isolada do solo e identificada como Pseudomonas sp. RAJR 044 demonstrou ser uma potencial produtora de protéase extracelular. Um mutante JNGR 242 dessa espécie foi obtido mediante radiação ultravioleta sob condições experimentalmente otimizadas de pH 7,0, temperatura de 34ºC, volume de inóculo de 1,0 mL e tempo de incubação de 24 hora produziu 2,5 mais protease. Também foram realizadas análises comparativas das características química quanto assimilação de diferentes fontes de carbono e de nitrogênio. O crescimento máximo desse mutante na placa de ágar gelatina (2%) foi obtido na presença de sacarose (2%), maltose (2%), sulfato de amônia (2%) e nitrato de potássio (2%), ao passo que no caso da linhagem original a melhor fonte de carbono foi a sacarose (2%) e o nitrato de amônia (2%) como a melhor fonte de nitrogênio. As protéases purificadas de ambas as espécies (original e mutante) mostraram faixas homogêneas que correspondem ao peso molecular 14,4 kDa na SDS-PAGE. Ao se estudar as propriedades cinéticas de ambas as espécies, observou-se que a taxa de hidrólise da caseína era máxima em pH 7,0 e 8,0, , temperaturas de 45ºC e 60ºC para a espécie original e a mutante, respectivamente. Observou-se também que ambas as protéases extracelulares foram inibidas pela serina, isto é, PMSF à concentração de 2mM.

Keywords

• Extracellular protease
• Purification
• Kinetic characterization
• Ultraviolet mutagenesis
• Pseudomonas sp.
• Physicochemical