Screening Of A Alkaline Protease Producing Strain And Cloning And Expression Of Its Apr Gene

Alkaline proteases(or subtilisins, E.C.3.4.21.14)are a physiologically and commercially important group of enzymes, it has been apply universally to industrial of detergents, leather processing and silk.Besides these application, it has a wonderful prospect of application potential in medical purpose, food processing, feeds, silver recovery, chemical industries and environment protection, as well as basic research.A alkaline protease producing strain, named A-109, was isolated from samples of the soil of Cattle farm by tradition microbiology method. On the basis of observing of modality and the analysis of the physiological biochemistry reaction, A-109 was identified as Bacillus subtilis primarily. A 1.2 kb fragment which homologous to aprE(GenBank No:AJ539133) gene encoding alkaline proteases of Bacillus subtilis, was amplified by touchdown PCR, using conservative sequence of aprE gene as primers. The 1.2 kb fragment was sequenced. A open reading frame(ORF) encoded 363 amino acids was identified from this fragment and was shared 99% homology with reported aprE gene. A recombinant plasmids, named PETA-109, was obtained by ligatied the 1.2 kb fragment with pET-30a(+) vector and transported into E.coli strains DH5 α . It was shown that this ap gene can express in E.coli strains DH5a.