ABSTRACT
Alkaline proteases are of industrial importance, mainly in the detergent industry. In this study, the extracellular alkaline serine protease gene, aprE, from Bacillus clausii was amplified by PCR and further cloned and expressed in B. subtilis WB600 using the pWB980 expression vector. Protease activity of the recombinant B. subtilis WB600 harboring the plasmid pWB980/aprE reached up to 1020 U/ml, approximately 3-folds higher than the native B. clausii strain. Characterization of the recombinant alkaline protease by SDS-PAGE and zymogram analyses indicated a molecular weight of 31 kDa. DNA sequence analysis and the deduced amino acid sequence revealed 98% homology with the extracellular alkaline serine protease from B. clausii KSM-K16
ABSTRACT
The effect of Triton X-100, Na cholate and Tween 80 on the solubilization of integral membrane proteins in intact cells, spheroplasts and inner-membrane fragments of Salmonella typhimurium was studied. The detergents were used in various concentrations [1.6 to 64 mM] and cytochromes b and d were used as marker to monitor the solubilization of membrane-bound proteins. Results showed that no inner-membrane protein solubilization was detected after the treatment of intact cells with detergents. The effect of Na cholate and Tween 80 on spheroplasts and inner-membrane fragments was also negligible in comparison to Triton X-100. Triton X-100 solubilized cytochromes from inner-membrane fragments more efficiently than from spheroplasts. The ratio of total protein solubilization to solubilize cytochromes showed that in spheroplasts this ratio was maximum at 1.6 mM Triton X-100 while it was maximum at 16-32 mM Triton X-100 in inner-membrane fragments. This difference between spheroplasts and inner-membrane fragments may be due to the orientation of the inner- membrane in spheroplasts [right side out] and inner-membrane fragments [in-side out as well as right side out], and to the presence of peripheral proteins attached to cytoplasmic membrane in spheroplasts